Application of nano-Amy@NPs-MM / PL1 in treatment of endometriosis

Abstract

This invention relates to the field of nanomedicine technology and discloses the application of nano-Amy@NPs-MM / PL1 in the treatment of endometriosis. The nano-Amy@NPs-MM / PL1 comprises a nanocarrier PEG-PLGA, a nanomedicine Amy, a nano-coated macrophage membrane, and a targeting peptide PL1. The steps are: S1, macrophage membrane extraction: using a cell membrane extraction kit, macrophage membranes are separated; S2, targeting peptide insertion into the macrophage membrane: the targeting peptide PL1 is inserted into the macrophage membrane to obtain the targeting macrophage membrane MM / PL1; S3, preparation of PEG-PLGA-loaded Amy. This invention demonstrates that Amy@NPs-MM / PL1 inhibits the activation of the TNF-α / TNFR1 / NF-κB signaling pathway, blocks the downstream caspase-1 / GSDMD-mediated pyroptosis cascade, reduces the release of inflammatory factors, and inhibits angiogenesis and the expression of invasive factors. It achieves multi-level intervention from anti-oxidation and anti-pyroptosis to neuroprotection and metabolic regulation, providing a new strategy with both theoretical innovation and clinical translational potential for the comprehensive treatment of endometriosis.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and more particularly to the application of a nano-Amy@NPs-MM / PL1 in the treatment of endometriosis. Background Technology

[0002] Endometriosis (EM) is a chronic gynecological disease characterized by the growth of endometrial-like tissue outside the uterine cavity, causing serious health impacts on women of reproductive age. More than 60% of patients suffer from persistent pelvic or abdominal pain, often accompanied by extrapelvic symptoms such as chronic back pain, fibromyalgia, vulvar pain, and migraines, significantly reducing their daily functional status and quality of life. Current treatments mainly include hormone therapy and surgery, but these methods typically provide only short-term symptom relief and are often accompanied by significant side effects and high recurrence rates. Therefore, elucidating the biological mechanisms underlying the persistence of lesions and chronic pain is crucial for developing more precise, mechanism-guided treatment strategies.

[0003] Recent studies have shown that abnormal inflammatory responses and oxidative stress are core factors driving the progression of endometriosis. In the microenvironment of ectopic lesions, the accumulation of excessive reactive oxygen species (ROS) can lead to redox imbalance, further activating inflammasomes and inducing pyroptosis. Pyroptosis is an inflammatory programmed cell death mechanism mediated by caspase-1 and Gasdermin D (GSDMD) through pore formation. Pyroptosis not only promotes lesion development by inducing a strong inflammatory response but also enhances cell invasion and fibrosis. Notably, recent studies have found that pyroptosis is not limited to a single-cell process. After GSDMD-mediated pore formation, pyroptotic cells release large amounts of IL-1β, IL-18, and various damage-associated molecular patterns (DAMPs), thereby activating inflammatory signaling pathways in neighboring cells. Furthermore, pyroptotic cells can release pro-inflammatory extracellular vesicles, propagating pyroptotic signals to neighboring and even distant cells, further amplifying the inflammatory response at the tissue level. Based on this finding, it is speculated that in endometriosis, pyroptotic endometrial stromal cells may transmit inflammatory signals to nearby neurons, thereby inducing neuronal pyroptosis and neuroinflammatory responses. This "pyroptosis-neuritis" axis may be an important pathological mechanism leading to chronic pain in endometriosis.

[0004] Natural active ingredients with multi-target regulatory properties have received extensive attention because they can regulate complex inflammatory networks. In the process of exploring effective therapeutic drugs, traditional Chinese medicine compound prescriptions provide valuable resources. Guizhi Fuling Wan (GZFL), which originated from Synopsis of the Golden Chamber written by Zhang Zhongjing in the Eastern Han Dynasty, consists of five herbs, namely Ramulus Cinnamomi, Poria, Cortex Moutan, Radix Paeoniae Rubra, and Semen Persicae. It has the effects of promoting blood circulation to remove blood stasis and gradually dissipating mass. For thousands of years, it has been widely used in the treatment of gynecological "Zhengjia" (equivalent to pelvic mass) and "dysmenorrhea". Modern pharmacological studies have confirmed that GZFL has anti-inflammatory, analgesic, immunomodulatory, and inhibitory effects on the growth of ectopic endometrium. Its clinical use in the adjuvant treatment of endometriosis shows certain efficacy. However, as a compound traditional Chinese medicine, problems such as its complex composition, unclear mechanism of action, and low bioavailability limit its further application and development in the era of precision medicine. How to screen out key active ingredients from traditional compound prescriptions, overcome their pharmacokinetic defects, and achieve precise targeted therapy is an important topic in the modernization research of traditional Chinese medicine. Based on the previous systematic screening of GZFL, it was found that amygdalin (Amy) is one of its core active ingredients. Amygdalin is a natural cyanogenic glycoside compound derived from bitter almonds and has been proven to have antioxidant, anti-inflammatory, and anti-fibrotic effects in various disease models such as breast cancer, cervical cancer, and neuroinflammation. Previous studies have shown that Amy can reduce the level of oxidative stress and inhibit the expression of multiple key pyroptosis-related molecules (such as NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD domain (ASC), and interleukin-1β (IL-1β)). These research results suggest that Amy may be a potential candidate drug for targeting the "pyroptosis-neuroinflammation" axis in the treatment of endometriosis. However, the clinical application of Amy is still limited by its inherent defects, including cyanide-related toxicity, low bioavailability, and rapid in vivo metabolic degradation. Therefore, a safer and more efficient delivery strategy is urgently needed.

[0005] Nanomedicine delivery systems (NDDS) have become a hot topic in research on the treatment of chronic inflammatory diseases due to their precise targeting capabilities and programmed controlled release characteristics. A novel amphiphilic block copolymer nanoplatform, PEG-PLGA nanoparticles (PEG-PLGA NPs), can be constructed by coupling polyethylene glycol (PEG) with polylactic-co-glycolic acid (PLGA). Studies have shown that PEG-PLGA hybrid nanoparticles exhibit superior efficacy in tumor treatment and have the potential to improve immune tolerance compared to unpolyethylene glycol-modified PLGA particles; they also significantly reduce nanoparticle fusion during storage, enhancing physical stability and internalization efficiency in cellular uptake experiments. Despite the excellent delivery characteristics of nanosystems, they still face the challenge of being recognized and phagocytosed by macrophages in the in vivo environment, leading to insufficient effective accumulation of drugs at the lesion site. Macrophages, as key phagocytic cells in the immune system, are a key obstacle to the in vivo stability and efficacy of drug delivery systems due to their uptake of exogenous nanoparticles. Therefore, how to prolong the blood circulation time of nanoparticles and reduce clearance by the mononuclear phagocytic system has become a key scientific issue in the design of nanomedicine systems.

[0006] In this study, a multifunctional nanomedicine platform loaded with amygdalin was constructed for targeted intervention in endometriosis. The nanosystem, named Amy@NPs-MM / PL1, has a core composed of polyethylene glycol-polylactic acid-glycolic acid copolymer (PEG-PLGA) for encapsulating amygdalin. It achieves dual targeting functionality through biomimetic macrophage membrane coating and PL1-targeting peptide modification (PPRRRGLIKLKTS). The PEG-PLGA matrix ensures drug stability and controlled release, overcoming the low bioavailability of naturally occurring amygdalin. In endometriosis lesions, the barrier formed by inflammatory cells hinders drug penetration. Macrophage membrane coating endows nanoparticles with inflammatory homing ability, membrane fusion ability, and immune escape properties, enabling them to cross the inflammatory barrier and enter the lesion area while avoiding phagocytosis and clearance. PL1 peptide modification further enhances delivery specificity by targeting tenascin-C (TNC), a protein significantly overexpressed in endometriosis lesions. PL1 can specifically bind to the C-terminal domain of TNC (TNC-C), thereby achieving precise delivery to lesion tissue.

[0007] The results indicate that this nanomedicine can significantly inhibit the oxidative stress-driven caspase-1 / GSDMD pyroptosis cascade and reduce secondary neuroinflammatory responses, thereby inhibiting lesion development and relieving pain. Amy@NPs-MM / PL1 effectively disrupts this pathological axis by simultaneously clearing lesion-origin ROS, blocking pyroptosis activation, and reducing neuroinflammatory damage, ultimately achieving lesion inhibition and analgesia. These results further demonstrate the therapeutic potential of this biomimetic nanoplatform and emphasize its application value in mechanism-guided, microenvironment-targeted therapy for endometriosis. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention provides an application of nano-Amy@NPs-MM / PL1 in the treatment of endometriosis.

[0009] The present invention is achieved by the following technical solution: the application of nano-Amy@NPs-MM / PL1 in the preparation of a drug for treating endometriosis, comprising nano-Amy@NPs-MM / PL1, wherein the nano-Amy@NPs-MM / PL1 comprises nanocarrier PEG-PLGA, nanodrug Amy, nano-coated macrophage membrane and targeting peptide PL1.

[0010] As a further improvement to the above scheme, the preparation steps are as follows: Step S1: Macrophage membrane extraction: Macrophage cell membranes were isolated using a cell membrane extraction kit; Step S2: Insertion of the targeting peptide into the macrophage membrane: The targeting peptide PL1 was inserted into the macrophage membrane to obtain the macrophage membrane MM / PL1; Step S3, Preparation of PEG-PLGA loaded with Amy: Amygdalin, PLGA, and DSPE-PEG were dissolved in acetone as the organic phase; polyvinyl alcohol aqueous solution was used as the aqueous phase. The organic and aqueous phases were mixed using microfluidic technology, and the effluent was collected. The effluent was then dialyzed, filtered, and concentrated to obtain amygdalin-loaded nanoparticles Amy@NPs. Step S4, Preparation of Amy@NPs-MM / PL1: The obtained targeted macrophage membrane MM / PL1 was extruded to form uniform membrane vesicles. The obtained amygdalin-loaded nanoparticles Amy@NPs were mixed with the membrane vesicles, incubated, and extruded to form a mixture. The mixture was centrifuged, filtered, and concentrated to obtain Amy@NPs-MM / PL1.

[0011] As a further improvement to the above scheme, Amy@NPs-MM / PL1 inhibits the activation of the TNF-α / TNFR1 / NF-κB signaling pathway, blocks the downstream caspase-1 / GSDMD-mediated pyroptosis cascade, reduces the release of inflammatory factors, and inhibits angiogenesis and the expression of invasive factors.

[0012] As a further improvement to the above scheme, the targeting peptide PL1 in Amy@NPs-MM / PL1 specifically recognizes tendinin-C, which is highly expressed in endometriosis lesions, to achieve active targeting and anchoring.

[0013] As a further improvement to the above scheme, the nano-Amy@NPs-MM / PL1 has antioxidant activity, clears excessive ROS accumulation in the lesion microenvironment, and inhibits pyroptosis activation driven by oxidative stress.

[0014] As a further improvement to the above scheme, the nano-Amy@NPs-MM / PL1 protects neurons from pyroptosis damage by inhibiting pyroptosis in hEMSCs and blocking the propagation of pyroptosis signals to neurons.

[0015] As a further improvement to the above scheme, the nano-Amy@NPs-MM / PL1 is used to prepare bioproducts.

[0016] As a further improvement to the above scheme, the biological product is a reagent or kit.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through UPLC-HRMS combined with network pharmacology and in vitro pharmacodynamics comparisons, has for the first time systematically screened and confirmed amygdalin as the core active ingredient for treating endometriosis from the traditional Chinese medicine compound Guizhi Fuling Wan. This ingredient can effectively inhibit the proliferation, migration, and invasion of ectopic endometrial stromal cells, induce apoptosis, and downregulate the expression of pain and inflammation-related genes, providing a new research strategy for the modern development and precise application of traditional Chinese medicine compound formulas.

[0018] This invention successfully constructed a dual-targeting nanodelivery system, Amy@NPs-MM / PL1, which combines macrophage membrane biomimicry with PL1-targeting peptide modification. This nanosystem possesses a uniform core-shell structure, suitable particle size, pH-responsive drug release characteristics, and fully preserves the functional proteins of the macrophage membrane, achieving both immune evasion and active targeting. In vitro and in vivo experiments confirmed that this system significantly improves the accumulation efficiency of amygdalin at lesion sites, prolongs its in vivo retention time, and reduces non-specific organ distribution.

[0019] This invention is the first to propose and verify the crucial role of the "lesion microenvironment-pyroptosis propagation-neuroinflammation" axis in endometriosis-related pain. Studies have shown that pyroptotic ectopic endometrial stromal cells can propagate inflammatory signals to neurons, inducing neuronal pyroptosis and neuroinflammation. Amy@NPs-MM / PL1, through its excellent antioxidant activity, effectively scavenges excess reactive oxygen species accumulated in the lesion microenvironment, inhibiting oxidative stress-driven pyroptosis activation at its source and blocking the propagation of pyroptosis signals to neurons, thereby alleviating neuroinflammation and pain at their root.

[0020] The Amy@NPs-MM / PL1 of this invention exhibits multidimensional therapeutic effects in vivo: significantly inhibiting the growth of ectopic lesions and improving histopathological features; effectively alleviating mechanical hyperalgesia and anxiety- and depression-like behaviors; reducing microglia activation in the hippocampus and central nervous system inflammation; and systematically reshaping systemic metabolic homeostasis by regulating metabolic-signaling axes such as indole / AHR and taurine / NLRP3. This nanoplatform achieves multi-level intervention from anti-oxidation and anti-pyroptosis to neuroprotection and metabolic regulation, providing a new strategy with both theoretical innovation and clinical translational potential for the comprehensive treatment of endometriosis. Attached Figure Description

[0021] Figure 1 The image shows the effects of Guizhi Fuling Wan extract on hEMSC proliferation, migration, invasion, and related gene expression; where: Figure 1 A is an analysis diagram of the cell viability of hEMSCs after treatment with different concentrations of GZFL extract using the CCK-8 assay. Figure 1 B is a graph showing the effect of GZFL extract on the migration and invasion ability of hEMSCs as detected by Transwell assay. Figure 1 C is a quantitative analysis diagram of the ability of GZFL extract to inhibit the migration and invasion of hEMSCs as detected by Transwell assay; Figure 1 D is the expression diagram of the effect of GZFL extract on the horizontal migration ability of hEMSCs as detected by the scratch healing assay; Figure 1 ERT-qPCR detection of mRNA expression levels of NGF, TUBB3, NINJ1, UCHL1, TNF-α and IL-10 in hEMSCs after treatment with GZFL extract; Figure 2 This is a BPC diagram for Chinese medicine (drugname); where: Figure 2 A is the BPC diagram in positive ion mode for the traditional Chinese medicine (drugname); Figure 3 B represents the BPC diagram under negative ion mode for traditional Chinese medicine (drugname); Figure 3 This is a network pharmacological analysis diagram of the active ingredients; where: Figure 3 A is a Venn diagram showing the relationship between traditional Chinese medicine and disease targets; Figure 3 B is a statistical chart of GO annotations for the core targets; Figure 3 C represents the KEGG signal path diagram; Figure 3 D is the Compound-Target-Pathway (Top 20) network diagram; Figure 4 This is a chromatogram showing the in vitro screening analysis of candidate components; where: Figure 4 A is an analytical graph showing the effects of three candidate components on hEMSCs activity as detected by the CCK-8 assay; Figure 4 B is an analysis diagram of the effects of the three components on the migration and invasion ability of hEMSCs as assessed by Transwell migration and invasion assays and cell scratch assays. Figure 4 C is a flow cytometry analysis of the effects of the three components on the induction of apoptosis in hEMSCs; Figure 4 Figure D shows the analysis of the effects of three components on the expression of pain and inflammation-related gene mRNAs in hEMSCs detected by qRT-PCR. Figure 5 A schematic diagram illustrating the construction of the Amy@NPs-MM / PL1 dual-targeting biomimetic nanodelivery system; Figure 6 The physicochemical characterization analysis results for Amy@NPs-MM / PL1 are shown below; where: Figure 6 A is a transmission electron microscope image of Amy@NPs-MM / PL1; Figure 6 B is a particle size characterization diagram of Amy@NPs and Amy@NPs-MM / PL1 under transmission electron microscopy particle size measurement; Figure 6 C represents the dynamic light scattering particle size distribution of Amy@NPs and Amy@NPs-MM / PL1; Figure 6 D is a graph showing the Zeta potential and colloidal stability of Amy@NPs and Amy@NPs-MM / PL1; Figure 7 Analysis of drug release behavior and membrane protein retention of Amy@NPs-MM / PL1; where: Figure 7 A shows the in vitro drug release curves under different pH conditions; Figure 7 B is a diagram showing the retention analysis of membrane proteins detected by Coomassie Brilliant Blue staining. Figure 7 C is a diagram showing the expression of functional membrane proteins CD47, CD68, and CD11b detected by Western blot. Figure 7 D is a semi-quantitative analysis diagram of membrane protein expression; Figure 8 The expression diagram of the reactive oxygen species scavenging ability of Amy@NPs-MM / PL1; Figure 9 A graph showing the distribution of different nano-formulations in the Transwell co-culture model; Figure 10 The analytical spectrum shows the inhibitory effect of Amy@NPs-MM / PL1 on hEMSCs and its reactive oxygen species scavenging effect; where: Figure 10 A is an expression diagram showing the survival status of hEMSCs after drug treatment in each group, as detected by live / dead staining. Figure 10 B is a graph showing the expression of reactive oxygen species levels in hEMSCs after drug treatment in each group, detected by fluorescent probes. Figure 10 C is an expression diagram showing the quantitative detection of the scavenging effects of each group of drugs on H2O2, ·OH and DPPH free radicals in hEMSCs; Figure 11 Analytical spectra validating the ability of Amy@NPs-MM / PL1 to inhibit pyroptosis in hEMSCs; where: Figure 11 A is a diagram showing the morphological changes of pyroptosis in hEMSCs after treatment observed by scanning electron microscopy in each group. Figure 11 B shows the expression of cleaved caspase-1, GSDMD-N, and Ki-67 in hEMSCs after treatment by immunofluorescence assay. Figure 11 C is a graph showing the expression of key proteins in the pyroptosis pathway in hEMSCs after treatment by Western blot. Figure 11 D is a graph showing the secretion levels of IL-1β and IL-18 in the cell supernatant after treatment in each group, as detected by ELISA. Figure 12 An analytical spectrum validating the ability of Amy@NPs-MM / PL1 to inhibit the propagation of pyroptosis into neurons; wherein: Figure 12A is a diagram showing the morphological changes of neurons after treatment in each group as observed by scanning electron microscopy; Figure 12 B is a graph showing the expression of cleaved caspase-1, GSDMD-N, BDNF and NGF in neurons as detected by immunofluorescence. Figure 12 C is a diagram showing the expression of key proteins and neurotrophic factors in the pyroptosis pathway in neurons as detected by Western blot. Figure 12 D is the expression diagram of the secretion levels of BDNF and NGF in the supernatant of the co-culture system detected by ELISA; Figure 13 The analytical spectrum for in vivo targeting validation of Amy@NPs-MM / PL1; where: Figure 13 A is a graph showing the distribution and retention of different Cy5.5-labeled formulations in model mice as observed by in vivo fluorescence imaging. Figure 13 B is a graph showing the distribution of different preparations in ectopic lesions and major organs in ex vivo organ fluorescence imaging; Figure 14 The analytical spectrum for evaluating the in vivo therapeutic effect of Amy@NPs-MM / PL1; where: Figure 14 A shows representative photographs of ectopic lesions in the peritoneal cavity of mice in each group, along with a quantitative statistical chart of the number and weight of the lesions. Figure 14 B shows representative HE and Masson staining images of ectopic lesions in each group; Figure 15 Analytical spectra for in vivo mechanism validation of Amy@NPs-MM / PL1; where: Figure 15 A shows the expression of Ki-67 / CD31, Vimentin / MMP9, cleaved caspase-1 / GSDMD-N, and IL-1β / IL-18 in lesion tissues of each group using immunofluorescence detection. Figure 15 B is a diagram showing the expression of key proteins in pyroptosis and inflammatory pathways detected by Western blot. Figure 15 C is a statistical graph showing the concentrations of TNF-α, IL-18, IL-1β and PGE2 in the serum of mice in each group as detected by ELISA; Figure 16 The analytical spectrum for biosafety assay of Amy@NPs-MM / PL1; where: Figure 16 A is a distribution graph of the weight change curves of mice in each group during the treatment period; Figure 16 B shows representative HE staining images of major organs (uterus, heart, liver, spleen, lungs, and kidneys); Figure 16 C shows the blood routine, blood biochemistry and hormone level detection results of mice in each group; Figure 17 Analysis profiles of behavioral tests for each group of mice; Figure 18 The analysis profile of Amy@NPs-MM / PL1 in the assessment of central nervous system inflammation; where: Figure 18 A shows the expression of IBA-1 and Ki-67 in the hippocampus of mice in each group, as detected by immunohistochemistry. Figure 18 B is a graph showing the concentrations of TNF-α, IL-1β, 3-NT and NSE in the cerebrospinal fluid of mice in each group as detected by ELISA; Figure 19 Analytical profiles for whole-body metabolic reprogramming analysis; where: Figure 19 A shows the principal component analysis (PCA) plot of serum metabolomics in each group of mice; Figure 19 B is a heatmap of representative differentially expressed metabolites; Figure 19 C represents the Western blot analysis of changes in key metabolite levels and corresponding pathway proteins. Detailed Implementation

[0022] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0023] Example 1:

[0024] The application of nano-Amy@NPs-MM / PL1 in the preparation of a drug for treating endometriosis in this embodiment includes nano-Amy@NPs-MM / PL1, wherein the nano-Amy@NPs-MM / PL1 comprises a nanocarrier PEG-PLGA, a nanodrug Amy, a nano-coated macrophage membrane, and a targeting peptide PL1.

[0025] The preparation steps are as follows: Step S1: Macrophage membrane extraction: Macrophage cell membranes were isolated using a cell membrane extraction kit; Step S2: Insertion of the targeting peptide into the macrophage membrane: The targeting peptide PL1 was inserted into the macrophage membrane to obtain the macrophage membrane MM / PL1; Step S3, Preparation of PEG-PLGA loaded with Amy: Amygdalin, PLGA, and DSPE-PEG were dissolved in acetone as the organic phase; polyvinyl alcohol aqueous solution was used as the aqueous phase. The organic and aqueous phases were mixed using microfluidic technology, and the effluent was collected. The effluent was then dialyzed, filtered, and concentrated to obtain amygdalin-loaded nanoparticles Amy@NPs. Step S4, Preparation of Amy@NPs-MM / PL1: The obtained targeted macrophage membrane MM / PL1 was extruded to form uniform membrane vesicles. The obtained amygdalin-loaded nanoparticles Amy@NPs were mixed with the membrane vesicles, incubated, and extruded to form a mixture. The mixture was centrifuged, filtered, and concentrated to obtain Amy@NPs-MM / PL1. Amy@NPs-MM / PL1 inhibits the activation of the TNF-α / TNFR1 / NF-κB signaling pathway, blocks the downstream caspase-1 / GSDMD-mediated pyroptosis cascade, reduces the release of inflammatory factors, and inhibits angiogenesis and the expression of invasive factors. The targeting peptide PL1 in nano-Amy@NPs-MM / PL1 specifically recognizes tendinin-C, which is highly expressed in endometriosis lesions, to achieve active targeting and anchoring. Nano-Amy@NPs-MM / PL1 possesses antioxidant activity, clearing excessively accumulated ROS in the lesion microenvironment and inhibiting pyroptosis activation driven by oxidative stress; Nanoparticles Amy@NPs-MM / PL1 protect neurons from pyroptosis damage by inhibiting pyroptosis in hEMSCs and blocking the propagation of pyroptosis signals to neurons. Nano-Amy@NPs-MM / PL1 is used to prepare bioproducts; Biological products are reagents or kits.

[0026] Example 2:

[0027] Preparation of nano-Amy@NPs-MM / PL1: Step S1: Macrophage membrane extraction: Macrophage membranes were isolated using a cell membrane extraction kit, following the manufacturer's instructions. The simplified steps are as follows: Take 5-10 × 10⁶ cells... 6RAW264.7 cells were centrifuged at 4°C and 800×g for 10 min, the supernatant was discarded, and the culture medium was aspirated as dry as possible. The cells were washed twice with cold PBS, and the supernatant was aspirated as dry as possible after each wash. Pre-prepared chilled extraction buffer A and washing buffer D were prepared in advance, with 2 μL of protease inhibitor mixture and 2 μL of phosphatase inhibitor mixture added to each 1 mL of solution, mixed well, and placed on ice. 500–1000 μL of chilled extraction buffer A was added to the cell pellet, mixed thoroughly, and incubated on ice for 30 min, gently pipetting to mix every 10 min. After incubation, the cells were centrifuged at 4°C and 1000×g for 10 min, and the supernatant was transferred to a pre-chilled centrifuge tube. The supernatant was then centrifuged again at 4°C and 20000×g for 30 min, and the supernatant was discarded, retaining the pellet. 400 μL of chilled washing buffer D was added to the pellet, mixed thoroughly, and centrifuged again at 4°C and 20000×g for 30 min. Discard the supernatant, and the resulting precipitate is the cell membrane component. Resuspend the membrane precipitate in membrane preservation solution (containing 10 mM Tris-HCl pH 7.4 and 1 mM EDTA), determine the protein concentration by BCA method, aliquot and store at -80℃ for later use. Step S2: Insertion of the targeting peptide into the macrophage membrane: PL1 targeting peptide (sequence: PPRRGLIKLKTS, purity >95%) was synthesized by Xi'an Ruixi Biotechnology Co., Ltd. Macrophage membranes extracted in step S1 (protein content 1 mg) were added to PL1 peptide (final concentration 50 μg / mL), and incubated with gentle shaking at 37°C for 1 h to allow the PL1 peptide to insert into the cell membrane. The membrane suspension with inserted PL1 peptide was centrifuged at 4°C, 100,000 × g for 30 min to remove uninserted free peptides. The precipitate was the PL1 peptide-modified macrophage membrane (MM / PL1), which was resuspended in membrane preservation solution for later use. Step S3, Preparation of PEG-PLGA loaded with Amy: Drug-loaded nanoparticles were prepared using microfluidic technology. Amygdalin (10 mg), PLGA (50 mg), and DSPE-PEG (10 mg) were dissolved in 5 mL of acetone as the organic phase. A 2% (w / v) aqueous solution of polyvinyl alcohol (PVA) was used as the aqueous phase. The organic and aqueous phases were mixed at a flow rate of 3:9 mL / min using a microfluidic mixer (NanoAssemblr, PrecisionNanosystems, Canada), and the eluent was collected. The eluent was transferred to a dialysis bag (MWCO 300 kDa), dialyzed against deionized water for 12 h to remove the organic solvent, filtered through a 0.22 μm membrane, and concentrated using an ultrafiltration tube (MWCO 100 kDa) to obtain amygdalin-loaded nanoparticles (Amy@NPs). Step S4, Preparation of Amy@NPs-MM / PL1: The separated cell membranes were repeatedly extruded (11 times) through a 200 nm polycarbonate membrane to form homogeneous membrane vesicles. The prepared PEG-PLGA-AMY nanoparticles were concentrated to 8.0 mL and mixed with the membrane vesicles at a protein-to-nanoparticle mass ratio of 1:1 (w / w). The mixture was gently incubated on ice for 30 min to promote spontaneous adsorption. Subsequently, it was extruded 11 times through a 200 nm polycarbonate membrane to promote membrane-nanoparticle fusion, forming Amy@NPs-MM / PL1. The resulting mixture was centrifuged at 4°C and 4000×g for 5 min to remove uncoated membranes. The supernatant was collected, filtered through a 0.22 μm filter, and then concentrated by centrifugation using a 100 kDa ultrafiltration tube and brought to a final volume of 8.0 mL. 500 μL of the sample was used for characterization. The preparation method for fluorescently labeled nanoparticles was the same as above, except that Cy5,5NHS ester (0.5 mg) was added to the organic phase.

[0028] Example 3:

[0029] Screening and validation of active ingredients in Guizhi Fuling Pill 1.1 Basic efficacy evaluation of Guizhi Fuling Pill extract To evaluate the potential of the traditional Chinese medicine Guizhi Fuling Wan (GZFL) in treating endometriosis (EMs), a systematic in vitro pharmacodynamic evaluation of its ethanol extract was first conducted. Using the CCK-8 assay, it was found that the GZFL extract significantly inhibited the activity of human endometriosis stromal cells (hEMSCs) in a dose-dependent manner. Figure 1 As shown in Figure A, the Transwell chamber invasion and scratch wound healing assays indicated that GZFL treatment effectively inhibited the invasion and migration abilities of hEMSCs. Figure 1 As shown in BD. Further qRT-PCR analysis revealed that GZFL extract significantly downregulated the expression of a series of genes associated with neuropathic pain and inflammation in hEMSCs, including nerve growth factor (NGF), tubulin β3 chain (TUBB3), damage-inducing protein 1 (NINJ1), ubiquitin C-terminal hydrolase L1 (UCHL1), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10), such as Figure 1 As shown in Figure E. These results collectively suggest that GZFL has the potential to inhibit the proliferation, invasion, and migration of ectopic endometrial cells and alleviate related inflammatory and pain signals.

[0030] 1.2 Chemical Composition Analysis of Guizhi Fuling Pill To clarify the material basis of GZFL, ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to systematically analyze its chemical components. The obtained mass spectrometry data was compared with the local traditional Chinese medicine high-resolution mass spectrometry database for compound identification. The identification criteria were set as follows: the first-order accurate mass error was less than 25 ppm, and the matching degree score (score) of the second-order mass spectrometry fragmentation spectrum was greater than 0.7. The analysis results showed that the extract of Guizhi Fuling Pills presented a highly complex chromatographic peak distribution in both positive and negative ion detection modes, indicating that it contains rich and diverse chemical components, such as Figure 2 shown in A-B. Through database comparison and spectrum analysis, a total of 3,547 compounds were identified, including 1,827 identified in the positive ion mode (POS) and 1,720 identified in the negative ion mode (NEG). This comprehensive chemical component identification provided a solid material basis and data support for subsequent network pharmacology analysis and screening of key active components.

[0031] 1.3 Network pharmacology analysis of active ingredients To further predict the potential active ingredients and their mechanisms of action of Guizhi Fuling Pills in the treatment of endometriosis (EMs), based on the full-component identification of UPLC-HRMS, combined with literature reports and absorption, distribution, metabolism, excretion (ADME) parameters (oral bioavailability OB≥30%, drug-likeness DL≥0.18) of the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, BATMAN-TCM), 16 representative compounds with good pharmacokinetic properties and high abundance were selected for subsequent network pharmacology analysis. These 17 components cover structural types such as flavonoids, terpenoids, organic acids, and glycosides, including Albiflorin, Amygdalin, Gallic acid, Benzoylpaeoniflorin, etc., which can comprehensively reflect the chemical composition characteristics of Guizhi Fuling Pills; Table 1 shows the list of traditional Chinese medicine chemical components for network pharmacological analysis:

[0032]

[0033] After collection, screening, and duplicate removal, 254 corresponding potential targets of traditional Chinese medicine were obtained. A total of 18,122 genes related to endometriosis were collected in the CTD database. Taking the intersection of the component targets and the disease targets, 234 common targets were obtained. These 234 common targets were regarded as the possible potential targets of traditional Chinese medicine in the treatment of endometriosis, such as Figure 3 shown in A.

[0034] To further elucidate the biological functions of these core targets, GO function enrichment analysis was performed using the GO database. The results showed that, for example... Figure 3 As shown in Figure B, the core targets were enriched in 2,259 biological processes (BP), 81 cellular components (CC), and 137 molecular functions (MF). Significantly enriched items in biological processes included "negative regulation of apoptosis," "inflammatory response," "regulation of neuronal death," "regulation of neuronal apoptosis," "oxidative stress response," and "regulation of reactive oxygen species metabolism." Cellular components were mainly enriched in "membrane rafts," "exosomes," and "cytoplasm." Molecular functions were mainly enriched in "cytokine receptor binding," "enzyme binding," and "cytokine activity." The enrichment analysis results showed two clear directions: first, the significant enrichment of items related to neuronal death and apoptosis regulation suggests that the potential targets of Guizhi Fuling Wan may participate in the balance regulation of neuronal damage and protection; second, the significant enrichment of items related to oxidative stress response and reactive oxygen species metabolism regulation suggests that anti-oxidative stress may be an important link in its protective effect. It is worth noting that "membrane rafts," as microdomains on the plasma membrane rich in cholesterol and sphingolipids, are assembly platforms for various death receptors, inflammatory signaling complexes, and ion channels. The significant enrichment of this entry further suggests that the active ingredients of Guizhi Fuling Pill may intervene in the activation of oxidative stress signal transduction and neuronal death-related pathways by acting on membrane microstructural domains.

[0035] KEGG pathway enrichment analysis showed that, for example Figure 3 As shown in Figure C, the core targets were significantly enriched in the TNF signaling pathway, NF-κB signaling pathway, CASP signaling pathway, and apoptosis signaling pathway. These pathways are all closely associated with oxidative stress, amplified inflammation, and cell death programs, further supporting the findings of the GO enrichment analysis.

[0036] To visually illustrate the complex regulatory relationships between compounds, targets, and pathways, a compound-target-pathway network diagram containing the top 20 significantly enriched pathways was constructed, as shown below. Figure 3 As shown in D. Network topology analysis revealed that components such as amygdalin, gallic acid, and benzoylpaeoniflorin exhibited high connectivity, suggesting that these compounds may regulate multiple pathways by acting on multiple targets, and are key nodes for Guizhi Fuling Wan to exert its synergistic therapeutic effect.

[0037] In summary, the network pharmacology analysis system predicted the potential active ingredient profile and molecular network of Guizhi Fuling Wan in treating EMs. GO and KEGG enrichment results both pointed to two major functional modules: regulation of oxidative stress-related cell death and neuronal damage. Based on network topological characteristics (such as connectivity) and the relative abundance of compounds in the extract, six core candidate components were initially screened from 16 representative components: penta-O-galloyl-α-D-glucopyranose, gallic acid (GA), amygdalin (Amy), benzoylpaeoniflorin, riboflavin, and citric acid. These six components all exhibit high node connectivity in the network and can simultaneously associate with multiple key targets and pathways related to oxidative stress, neuronal death, and inflammation regulation, suggesting that they may be the main material basis for the multi-target synergistic therapeutic effect of Guizhi Fuling Wan. However, the actual contributions of each component in inhibiting malignant phenotypes in EMs cells and regulating related molecular events are still unclear and need to be verified and confirmed one by one through systematic in vitro pharmacodynamic comparative studies.

[0038] 1.4 In vitro screening of candidate components To further screen the six core components predicted by network pharmacology to identify the most therapeutically promising active monomers, three representative components—gallic acid, amygdalin, and benzoylpaeoniflorin—were selected based on their pharmacological properties, compound abundance, and literature reports for a systematic comparative in vitro pharmacodynamic study.

[0039] First, the effects of the three components on the viability of human endometriosis stromal cells (hEMSCs) were examined using the CCK-8 assay. The results showed that all three components inhibited hEMSC proliferation in a dose-dependent manner, but the inhibition rates differed. Figure 4 As shown in Figure A, the half-maximal inhibitory concentration (IC50) was calculated using a four-parameter logistic model. 50 The concentrations were: gallic acid 745.5 µmol / L, amygdalin 1096 µmol / L, and benzoylpaeoniflorin 1282 µmol / L. Although the IC50 of gallic acid... 50 While it has the lowest value, it exhibits some cytotoxicity at high concentrations, whereas amygdalin has a flatter dose-response curve, suggesting that it may have a better safety window.

[0040] The effects of the three components on the migration and invasion abilities of hEMSCs were further evaluated using a Transwell assay. The results showed that, compared with the control group, the number of cells crossing the basement membrane was significantly reduced after treatment with all three components, but the degree of inhibition differed. Figure 4 As shown in Figure B, the amygdalin treatment group showed the most significant reduction in the number of migrating and invading cells, with inhibition rates reaching 75% and 86%, respectively; gallic acid was the second most effective, while benzoylpaeoniflorin showed relatively weaker inhibitory effects. The cell scratch assay also yielded consistent results: after 24 hours of amygdalin treatment, the percentage of scratch healing area was significantly lower than the other two groups, demonstrating the strongest migration inhibition ability. Figure 4 As shown in B.

[0041] To investigate the effects of the three components on cell death pathways, the apoptosis rate of hEMSCs was detected using Annexin V-FITC / PI double staining combined with flow cytometry. The results showed that amygdalin treatment increased the total apoptosis rate by approximately 30% compared to the control group, while gallic acid treatment only increased it by approximately 10%, and the apoptosis rate in the benzoylpaeoniflorin treatment group remained almost unchanged. Figure 4 As shown in C. This indicates that amygdalin not only inhibits cell proliferation but also effectively induces apoptosis in hEMSCs, while benzoylpaeoniflorin has limited effect in this regard.

[0042] At the molecular level, the effects of three components on the mRNA expression of pain-related genes (NGF, NINJ1, TUBB3, and UCHL1) and inflammatory factors (TNF-α and IL-1β) in hEMSCs were detected by qRT-PCR. The results showed that amygdalin significantly downregulated the expression levels of all detected genes, with a downregulation range of 50%–70%; gallic acid also showed some downregulation, but the effect was weaker than that of amygdalin; benzoylpaeoniflorin only had a slight inhibitory effect on NGF and IL-1β, and had no significant effect on NINJ1, TUBB3, UCHL1, and TNF-α. Figure 4 As shown in D.

[0043] Based on the above in vitro functional comparisons, amygdalin showed the best overall effect in inhibiting hEMSC migration and invasion, inducing apoptosis, and downregulating the expression of pain / inflammation-related genes. Although gallic acid had a lower IC50 value in inhibiting cell viability... 50 While its activity is high, its efficacy in regulating cellular function and molecular events is inferior to that of amygdalin. Benzoylpaeoniflorin also exhibits relatively weak overall activity. Therefore, amygdalin was ultimately identified as the core active ingredient in Guizhi Fuling Wan for treating endometriosis, and it was chosen as a candidate drug for subsequent nanodelivery system research.

[0044] Example 4:

[0045] Successful construction and functional characterization of a dual-target nanodelivery system 2.1 Schematic diagram of preparation: Construction process of Amy@NPs-MM / PL1 To achieve precise targeted delivery to endometriosis lesions and enhance the therapeutic effect of amygdalin, a dual-targeting nanodelivery system based on macrophage membrane biomimicry and PL1-targeting peptide modification was designed and constructed, named Amy@NPs-MM / PL1. The system's structural composition is as follows: Figure 5 As shown, it mainly consists of three parts: (i) a drug-loaded core: amygdalin (Amy) is encapsulated with biodegradable PEG-PLGA copolymer to form a stable nanocore (Amy@NPs), achieving efficient drug loading and controlled release; (ii) a biomimetic shell: the cell membrane of RAW264.7 macrophages is extracted, and its surface functional proteins (such as CD47, CD11b, etc.) are completely preserved as an outer coating material, endowing the nanoparticles with immune escape and inflammatory chemotaxis capabilities; (iii) a targeting ligand: the PL1 targeting peptide (amino acid sequence: PPRRGLIKLKTS) is inserted into the macrophage membrane. This peptide can specifically recognize the highly expressed tenosynovin-C (TNC) in endometriosis lesions, achieving active targeting and anchoring. By uniformly encapsulating the functional macrophage membrane (MM / PL1) on the surface of the Amy@NPs core, a dual-targeting biomimetic nanoparticle Amy@NPs-MM / PL1 with a "core-shell" structure is finally formed. This design, through the synergistic effect of a biomimetic shell and an active targeting ligand, is expected to significantly improve the efficiency of drug accumulation at the lesion site.

[0046] 2.2 Drug loading and encapsulation efficiency To evaluate the loading efficiency of amygdalin on Amy@NPs-MM / PL1, high-performance liquid chromatography (HPLC) was used to determine the drug loading and encapsulation efficiency of the nanoparticles. The results showed that the drug loading of Amy@NPs-MM / PL1 was 18.45 ± 0.48%, and the encapsulation efficiency was 48.39 ± 1.26% (Table 2). This result indicates that the constructed PEG-PLGA nanocore can effectively load amygdalin with a high encapsulation efficiency, providing sufficient drug dosage for subsequent in vitro and in vivo experiments.

[0047] Table 2 shows the statistical table of amygdalin loading and encapsulation efficiency in Amy@NPs-MM / PL1:

[0048] 2.3 Physicochemical Characterization To verify the successful construction of Amy@NPs-MM / PL1, it was first systematically characterized by physicochemical methods.

[0049] Transmission electron microscopy (TEM) revealed that uncoated Amy@NPs exhibited a uniform and regular spherical shape; while coated Amy@NPs-MM / PL1 displayed a clear "core-shell" structure, with an outer film layer approximately 10-15 nm thick. Figure 6 As shown in Figure A, the cell membrane was successfully coated onto the surface of the nanoparticles.

[0050] Particle size analysis results showed that the diameter of uncoated nanoparticles measured under transmission electron microscopy was approximately 200 nm, which increased to approximately 220 nm after coating. Figure 6 As shown in Figure B, the dynamic light scattering (DLS) results are consistent with the electron microscopy observations. The average hydrated particle size of Amy@NPs is 198.6 ± 5.2 nm, and the polydispersity index (PDI) is 0.09 ± 0.02; the average hydrated particle size of Amy@NPs-MM / PL1 increases to 221.3 ± 4.7 nm, and the PDI is 0.10 ± 0.03. Figure 6 As shown in Figure C, the particle size increases slightly after coating, but the particle still maintains a high degree of uniform monodispersity.

[0051] Zeta potential measurements showed that the surface potential of Amy@NPs was -21.5 ± 1.2 mV, while the potential of Amy@NPs-MM / PL1 dropped to -30.2 ± 0.9 mV. Figure 6 As shown in Figure D, the membrane potential is highly similar to that of the extracted macrophages, further confirming successful cell membrane encapsulation. Furthermore, the suspension of Amy@NPs-MM / PL1 exhibits a significant Tyndall effect, such as... Figure 6 As shown in E, it has good colloidal stability, which is beneficial for subsequent in vitro and in vivo applications.

[0052] 2.3 Drug Release and Membrane Protein Retention Further evaluation was conducted on the drug release behavior and membrane protein retention of Amy@NPs-MM / PL1. Drug release experiments showed that under simulated physiological conditions (pH 7.4), drug release from Amy@NPs-MM / PL1 was relatively slow, with a cumulative release rate of only 18.5% after 48 hours. However, under weakly acidic conditions simulating the microenvironment of an inflammatory lesion (pH 5.5), drug release was significantly accelerated, with a cumulative release rate reaching 65.3% after 48 hours. Figure 7 As shown in Figure A. This pH-responsive release characteristic helps reduce premature drug leakage during circulation, while simultaneously achieving targeted drug release in the acidic lesion microenvironment. To verify whether the membrane protein was successfully transferred to the nanoparticle surface, Coomassie brilliant blue staining and Western blot analysis were performed. The Coomassie brilliant blue staining results showed that the protein band pattern of Amy@NPs-MM / PL1 was highly consistent with that of native macrophage membranes, while no obvious protein bands were observed in Amy@NPs. Figure 7As shown in B. Western blot further confirmed that Amy@NPs-MM / PL1 successfully retained key functional proteins of the macrophage membrane, including the immune escape-related protein CD47, the macrophage marker CD68, and the integrin CD11b, as shown in Figure B. Figure 7 As shown in C and D, the retention of these membrane proteins is crucial for nanoparticles to achieve immune evasion and inflammation targeting.

[0053] 2.4 Evaluation of the reactive oxygen species scavenging capacity of Amy@NPs-MM / PL1 Given the crucial driving role of oxidative stress in the activation of pyroptosis in endometriosis, the reactive oxygen species (ROS) scavenging ability of Amy@NPs-MM / PL1 was further evaluated. The scavenging effects of the nanoparticles on hydrogen peroxide (H2O2), superoxide anion (O2⁻), hydroxyl radicals (·OH), and DPPH radicals were determined using various ROS detection systems. The results showed that Amy@NPs-MM / PL1 effectively scavenged the above four ROS, such as… Figure 8 As shown in the figure. This result indicates that Amy@NPs-MM / PL1 not only serves as a drug delivery carrier, but also possesses excellent antioxidant activity. It can inhibit oxidative stress-driven pyroptosis activation at its source by clearing excessive ROS accumulation in the lesion microenvironment.

[0054] 2.5 Validation of in vitro targeting and immune escape functions To evaluate the immune escape capability and targeted delivery efficiency of Amy@NPs-MM / PL1, a Transwell co-culture model was constructed to simulate the in vivo inflammatory microenvironment. In this model, macrophages were seeded in the upper chamber, and hEMSCs were cultured in the lower plate. Cy5.5-labeled Amy@NPs or Amy@NPs-MM / PL1 were added to the upper chamber, respectively. After co-culturing for 24 hours, the distribution of nanoparticles was observed using a fluorescence microscope.

[0055] The results showed that in the Amy@NPs group, strong red fluorescence signals were observed in the upper layer of macrophages, indicating that the uncoated nanoparticles were largely phagocytosed by macrophages; while only weak fluorescence signals were detected in the lower layer of hEMSCs. Conversely, in the Amy@NPs-MM / PL1 group, the fluorescence signal in the upper layer of macrophages was significantly weakened, while strong red fluorescence was observed in the lower layer of hEMSCs. Figure 9 As shown in the figure. This result indicates that macrophage membrane coating endows nanoparticles with immune escape capabilities, effectively preventing them from being cleared by the mononuclear phagocytic system; at the same time, the modification of retained membrane proteins and PL1 targeting peptides promotes the interaction between nanoparticles and hEMSCs, enhancing the uptake of nanoparticles by target cells.

[0056] Example 5:

[0057] In vitro efficacy verification and inhibition of pyroptosis and its spread To verify the inhibitory effect of Amy@NPs-MM / PL1 on endometriotic stromal cells and its ability to block pyroptosis signaling, a transwell co-culture model was first established. LPS-induced pyroptosis-inducing hEMSCs cells were placed in the upper layer, and neuronal cells were cultured in the lower layer. Five groups were set up for in vitro experiments: control group, blank nanoparticle group (PPNPs), free amygdalin group (Amy), uncoated nanomedicine group (Amy@NPs), and dual-targeting nanomedicine group (Amy@NPs-MM / PL1).

[0058] 3.1 Inhibitory effect of Amy@NPs-MM / PL1 on hEMSCs and reactive oxygen species scavenging effect First, the killing effect of each drug on hEMSCs was evaluated using live / dead staining. For example... Figure 10 As shown in Figure A, cells in both the Control and PPNPs groups exhibited uniform green fluorescence (live cells) with almost no red fluorescence (dead cells), indicating that the blank nanocarrier itself has no cytotoxicity. The Amy group showed a small amount of red fluorescence, while the Amy@NPs group showed increased red fluorescence. The Amy@NPs-MM / PL1 group exhibited the most significant red fluorescence and a large number of cell deaths, indicating that the dual-targeted nanodelivery system significantly enhanced the killing effect of amygdalin on hEMSCs.

[0059] Reactive oxygen species (ROS) play a key driving role in pyroptosis activation; therefore, the ROS levels in hEMSCs after each treatment group were further investigated. Fluorescent probes were used to label ROS, and the results are shown below. Figure 10 As shown in Figure B, the Control group and the PPNPs group exhibited bright green fluorescence, indicating a high level of intracellular ROS. The fluorescence intensity of the Amy group and the Amy@NPs group was reduced. The Amy@NPs-MM / PL1 group had the weakest fluorescence, almost invisible, indicating that this group could most effectively scavenge intracellular ROS.

[0060] To quantitatively assess the antioxidant capacity of each drug group, the levels of intracellular hydrogen peroxide (H2O2), hydroxyl radicals (·OH), and DPPH radicals were measured. The results are as follows: Figure 10 As shown in Figure C, compared with the Control group, the Amy@NPs-MM / PL1 group showed the most significant scavenging effect on the three free radicals, significantly better than the Amy group and the Amy@NPs group. This result indicates that the dual-targeting strategy of macrophage membrane biomimicry and PL1 peptide modification not only enhances cellular drug uptake but also synergistically improves the antioxidant activity of amygdalin, laying the foundation for subsequent inhibition of pyroptosis.

[0061] 3.2 Verification of the ability of Amy@NPs-MM / PL1 to inhibit pyroptosis in hEMSCs The inhibitory effects of different treatments on pyroptosis of hEMSCs were further evaluated using various experimental methods.

[0062] First, scanning electron microscopy was used to observe the cell morphology of hEMSCs after treatment in each group. For example... Figure 11 As shown in Figure A, cells induced by LPS (pyroptosis model control group) exhibited typical pyroptosis morphological features, including cell swelling, numerous bubble-like protrusions on the membrane surface, and membrane pore formation. The pyroptosis morphology in the Amy group and Amy@NPs group was improved, but some cell membrane damage was still visible. In contrast, the cell morphology in the Amy@NPs-MM / PL1 group was basically restored to normal, with a smooth and intact membrane surface and almost no pyroptosis features, indicating that this group could most effectively inhibit pyroptosis.

[0063] Immunofluorescence double staining was used to detect the expression and localization of pyroptosis executive proteins cleaved caspase-1 and GSDMD-N, while the expression of the proliferation marker Ki-67 was also detected. Figure 11 As shown in Figure B, the fluorescence signals of cleaved caspase-1 (green) and GSDMD-N (red) were weak in the Control and PPNPs groups, while Ki-67 (red) positive cells were more numerous. After LPS induction, the fluorescence signals of the two pyroptosis proteins were significantly enhanced, and the number of Ki-67 positive cells decreased. In the Amy and Amy@NPs groups, the pyroptosis protein signals were somewhat weakened, and Ki-67 was partially restored. In the Amy@NPs-MM / PL1 group, the fluorescence signals of cleaved caspase-1 and GSDMD-N were the weakest, almost invisible, while the number of Ki-67 positive cells was significantly increased, indicating that this group could not only inhibit pyroptosis but also restore cell proliferation activity.

[0064] Western blot analysis was used to detect the expression levels of key proteins in the pyroptosis pathway. For example... Figure 11 As shown in C, after LPS induction, there was no significant change in pro-caspase 1 in each group, but the expression of cleavedcaspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 proteins were significantly upregulated; the expression of the above proteins decreased in the Amy group and Amy@NPs group; while the expression levels of cleavedcaspase-1, GSDMD-N, IL-1β and IL-18 were the lowest in the Amy@NPs-MM / PL1 group, which was significantly better than other treatment groups.

[0065] LISA was used to detect the secretion levels of the inflammatory factors IL-1β and IL-18 in cell supernatant. For example... Figure 11As shown in Figure D, the concentrations of IL-1β and IL-18 in the supernatant were significantly increased after LPS induction; the concentrations of both factors decreased in the Amy group and the Amy@NPs group; while the concentrations of IL-1β and IL-18 were lowest in the Amy@NPs-MM / PL1 group, with no significant difference from the Control group, further confirming that this group can effectively inhibit the release of pyroptosis-related inflammatory factors.

[0066] In summary, Amy@NPs-MM / PL1 enhances intracellular uptake of amygdalin through a dual-target delivery system, significantly inhibits caspase-1 / GSDMD-mediated pyroptosis activation in hEMSCs, reduces the release of inflammatory factors, and restores cell proliferation activity, laying a solid foundation for subsequent in vivo experiments.

[0067] 3.3 Verification of the ability of Amy@NPs-MM / PL1 to inhibit the propagation of pyroptosis to neurons To investigate whether pyroptosis signals from hEMSCs can propagate to neurons and induce neuroinflammation, and whether Amy@NPs-MM / PL1 can block this propagation process, we will conduct different tests on neurons co-cultured with hEMSCs from different groups to observe morphological changes and pyroptosis-related indicators.

[0068] The morphology of neurons was observed using scanning electron microscopy. For example... Figure 12 As shown in Figure A, after co-culturing with the LPS-induced hEMSCs control group, neurons exhibited typical pyroptosis morphological features, including cell body swelling, bubble-like protrusions on the membrane surface, and protrusion breakage. Neuronal morphology improved in the Amy group and Amy@NPs group, but some damage was still visible. In contrast, the neuronal morphology of the Amy@NPs-MM / PL1 group was basically restored to normal, with full cell bodies and intact protrusions, indicating that this group could most effectively block the propagation of pyroptosis signals to neurons.

[0069] Immunofluorescence double staining was used to detect the expression of cleaved caspase-1 and GSDMD-N in neurons, while the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) was also detected. Figure 12 As shown in Figure B, the fluorescence signals of cleaved caspase-1 (green) and GSDMD-N (red) were significantly enhanced in the model group neurons, NGF (red) expression was increased, while BDNF (green) expression was weakened; in the Amy group and Amy@NPs group, the pyroptosis protein signal was weakened, NGF was partially decreased, and BDNF was partially restored; while in the Amy@NPs-MM / PL1 group, the fluorescence signals of cleaved caspase-1 and GSDMD-N were the weakest, NGF expression was significantly decreased, and BDNF expression was significantly increased, indicating that this group can not only inhibit neuronal pyroptosis, but also restore neurotrophic balance.

[0070] Western blot analysis was used to detect the expression of key proteins and neurotrophic factors in the pyroptosis pathway in neurons. For example... Figure 12 As shown in Figure C, the expression of cleaved caspase-1, GSDMD-N, IL-1β, IL-18, and NGF was significantly upregulated in the model group, while the expression of neuronal markers β-3-tubulin and BDNF was significantly downregulated. The above abnormal expressions were improved in the Amy group and the Amy@NPs group. In the Amy@NPs-MM / PL1 group, the expression levels of cleaved caspase-1, GSDMD-N, IL-1β, IL-18, and NGF were the lowest, and the expression of β-3-tubulin and BDNF recovered to levels close to those of the Control group, which was significantly better than other treatment groups.

[0071] ELISA was used to detect the secretion levels of BDNF and NGF in the supernatant of the co-culture system. For example... Figure 12 As shown in Figure D, the concentration of BDNF in the supernatant of the model group was significantly reduced, while the concentration of NGF was significantly increased; in the Amy group and the Amy@NPs group, BDNF increased and NGF decreased; while the Amy@NPs-MM / PL1 group had the highest concentration of BDNF and the lowest concentration of NGF, further confirming that this group can effectively restore neurotrophic balance.

[0072] In summary, Amy@NPs-MM / PL1 inhibits pyroptosis in hEMSCs, blocks the propagation of pyroptosis signals to neurons, protects neurons from pyroptosis damage, and restores the balance of neurotrophic factors, providing important evidence for elucidating the mechanism of its analgesic effect in vivo.

[0073] Example 6:

[0074] In vivo treatment efficacy evaluation and pyroptosis inhibition effect To evaluate the in vivo efficacy of Amy@NPs-MM / PL1 in treating endometriosis and its inhibitory effect on pyroptosis in lesions, a mouse model of endometriosis was established using allogeneic mouse endometrial intraperitoneal injection. Female BALB / c mice were used as recipients. Donor mice were induced to proliferate by estrogen, and after sacrifice, the uterus was harvested, fragmented into tissue pieces <1 mm³, suspended in sterile PBS, and injected intraperitoneally into the recipient mice to establish the endometriosis model. One week after modeling, a portion of the mice were randomly selected and sacrificed to verify successful modeling. The remaining mice were randomly divided into six groups (n=6): PBS group (negative control), GnRH-a group (positive drug control, commonly used clinical treatment drug), PPNPs group (blank nanocarrier), Amy group (free amygdalin), Amy@NPs group (uncoated nanomedicine), and Amy@NPs-MM / PL1 group (dual-targeting nanomedicine). All groups received intraperitoneal injection every two days for three consecutive weeks.

[0075] 4.1 In vivo targeting validation To verify the lesion-targeting ability of Amy@NPs-MM / PL1 in vivo, in vivo fluorescence imaging was used to observe the distribution of different Cy5.5-labeled formulations in model mice. In vivo imaging was performed at different time points after intraperitoneal injection, and the results showed... Figure 13 As shown in Figure A: the fluorescence signal in the free Cy5.5 group rapidly weakened 24 hours after injection and basically disappeared 72 hours later; the Amy@NPs-Cy5.5 group still showed a weak fluorescence signal 7 days after injection, but the signal was almost invisible by 14 days; while the Amy@NPs-MM / PL1-Cy5.5 group still showed a strong fluorescence signal in the abdominal lesion area 14 days after injection, demonstrating a significantly prolonged in vivo retention time.

[0076] To further clarify the tissue distribution of the nanoparticles, mice were sacrificed 7 days after injection, and ectopic lesions and major organs (heart, liver, spleen, lung, kidney, uterus, and ovary) were collected for in vitro fluorescence imaging. The results are as follows: Figure 13 As shown in Figure B, the Amy@NPs-Cy5.5 group showed significant fluorescence signals not only in the lesions but also in the uterus and liver; while the Amy@NPs-MM / PL1-Cy5.5 group showed highly concentrated fluorescence signals in the ectopic lesions, with almost no fluorescence distribution in the major organs. This result indicates that the dual-targeting strategy of macrophage membrane biomimicry and PL1 peptide modification significantly improves the lesion-targeting specificity of nanoparticles and reduces non-specific organ distribution.

[0077] 4.2 Evaluation of in vivo therapeutic effect Three weeks after treatment, mice in each group were sacrificed, and ectopic lesions in the abdominal cavity were isolated for macroscopic and histological evaluation.

[0078] like Figure 14 As shown in Figure A, multiple large cystic lesions with abundant surface blood vessels were observed in the peritoneal cavity of mice in the PBS and PPNPs groups. The number and size of lesions decreased in the GnRH-a group. The number and size of lesions further decreased in the Amy and Amy@NPs groups. The Amy@NPs-MM / PL1 group had the fewest lesions and the smallest lesions, with some mice showing almost no obvious lesions in their peritoneal cavity. Statistical analysis of the number and weight of lesions in each group showed that the number and average weight of lesions in the Amy@NPs-MM / PL1 group were significantly lower than in the other groups, indicating superior efficacy compared to the clinical positive control drug GnRH-a.

[0079] Ectopic lesions from each group were paraffin-embedded and sectioned, and subjected to HE staining and Masson staining to assess histological changes and the degree of fibrosis. Figure 14As shown in Figure B, HE staining results revealed that the ectopic lesions in the PBS and PPNPs groups exhibited typical endometrial-like structures, abundant glands, densely packed stromal cells, and significant inflammatory cell infiltration. The GnRH-a group showed a decrease in glandular number and looser stroma. The Amy and Amy@NPs groups showed improved lesion tissue structure, but some glands and inflammatory infiltration were still visible. The Amy@NPs-MM / PL1 group showed significantly improved lesion tissue structure, with glandular atrophy, sparse stroma, and a significant reduction in inflammatory cell infiltration. Masson staining results showed abundant blue collagen fiber deposition in the stroma of the lesions in the PBS and PPNPs groups, indicating significant fibrosis. Collagen deposition was reduced in the GnRH-a group. Collagen fibers were reduced in the Amy and Amy@NPs groups, but some blue-stained areas were still visible. The Amy@NPs-MM / PL1 group showed the least blue collagen fiber deposition and a significantly reduced degree of stromal fibrosis.

[0080] The above results indicate that Amy@NPs-MM / PL1 can effectively inhibit the growth of ectopic lesions, improve the pathological structure of lesion tissue, reduce inflammatory infiltration and fibrosis, and has better efficacy than free Amy and unencapsulated nanomedicines, and is also superior to the clinically commonly used drug GnRH-a.

[0081] 4.3 In vivo mechanism verification: Amy@NPs-MM / PL1 inhibits pyroptosis and inflammatory pathways in lesions. To investigate the molecular mechanism by which Amy@NPs-MM / PL1 inhibits the progression of endometriosis in vivo, immunofluorescence staining, Western blot analysis, and serum inflammatory factor analysis were performed on ectopic lesion tissues.

[0082] The expression levels of Ki-67 / CD31, Vimentin / MMP9, cleaved caspase-1 / GSDMD-N, and IL-1β / IL-18 in lesion tissues of each group were detected using immunofluorescence double staining. Figure 15As shown in Figure A, Ki-67 (a proliferation marker) and CD31 (a vascular marker) co-localized significantly in the lesions of the control group and PPNPs group, exhibiting strong fluorescence signals, indicating active cell proliferation and abundant angiogenesis in the lesions. Vimentin and MMP9 co-localized significantly, suggesting that the mesenchymal cells highly express matrix metalloproteinases and have strong invasive ability. Cleaved caspase-1 and GSDMD-N co-localized significantly, indicating activation of the pyroptosis pathway. IL-1β and IL-18 also showed strong positive expression. In the GnRH-a group, the above indicators decreased, but positive signals were still visible. The fluorescence intensity of each indicator was weakened in the Amy group and Amy@NPs group. In the Amy@NPs-MM / PL1 group, the fluorescence signals of the above indicators were the weakest, and the number of Ki-67 / CD31 positive cells was significantly reduced, indicating that proliferation and angiogenesis were inhibited; Vimentin / MMP9 colocalization was almost invisible, indicating a decrease in invasive ability; the number of cleaved caspase-1 / GSDMD-N and IL-1β / IL-18 positive cells was significantly reduced, confirming that pyroptosis activation was effectively blocked and the local inflammatory response was alleviated.

[0083] Further extraction of proteins from ectopic lesion tissues in each group was performed, and the expression levels of TNF-α, TNFR1, NF-κB, Caspase-1, cleaved Caspase-1, GSDMD, GSDMD-N, IL-18, IL-1β, COX-2, MMP-9, MMP-2, and VEGF were detected by Western blot. Figure 15 As shown in Figure B, compared with normal endometrium, the expression of all the above-mentioned proteins in the lesion tissues of the PBS group and PPNPs group was significantly upregulated, with the most significant increases in pyroptosis executive proteins cleavedCaspase-1 and GSDMD-N, and downstream inflammatory factors IL-1β and IL-18. The expression of upstream signaling molecules TNF-α, TNFR1, and NF-κB was also significantly increased, indicating that the pyroptosis pathway was abnormally activated. The expression of the above-mentioned proteins decreased in the Amy group and Amy@NPs group, but was still higher than normal levels. In the Amy@NPs-MM / PL1 group, the expression of all detected proteins was significantly downregulated to near normal endometrial levels, with the most significant decreases in cleavedCaspase-1, GSDMD-N, IL-1β, and IL-18. At the same time, the expression of angiogenesis-related factor VEGF, invasion-related factor MMP-2 / MMP-9, and inflammatory mediator COX-2 was also significantly downregulated, consistent with the immunofluorescence results and the histological results of inhibited lesion growth.

[0084] Serum was collected from each group of mice, and the levels of systemic inflammatory factors TNF-α, IL-18, IL-1β, and PGE2 were detected by ELISA. Figure 15As shown in Figure C, the serum concentrations of the four inflammatory factors in the control group and the PPNPs group were significantly increased; the levels of inflammatory factors in the Amy group and the Amy@NPs group were further decreased; while the concentrations of TNF-α, IL-18, IL-1β and PGE2 in the Amy@NPs-MM / PL1 group were the lowest, close to the levels in normal mice, indicating that this nanomedicine can not only inhibit local lesion inflammation, but also effectively reduce systemic inflammatory response.

[0085] In summary, Amy@NPs-MM / PL1 achieves multidimensional and multi-targeted anti-endometriosis efficacy in vivo by targeting and delivering amygdalin to the lesion site, inhibiting the activation of the TNF-α / TNFR1 / NF-κB signaling pathway, blocking the downstream caspase-1 / GSDMD-mediated pyroptosis cascade, reducing the release of inflammatory factors, and inhibiting the expression of angiogenesis and invasion-related factors.

[0086] 4.4 Biosafety Testing To assess the biosafety of Amy@NPs-MM / PL1 in vivo, weight monitoring, major organ pathological examinations, and hematological index analysis were performed on mice in each group during and after treatment.

[0087] During the treatment period, the body weight of mice in all groups steadily increased, with no significant weight loss or abnormal fluctuations observed. Figure 16 As shown in Figure A, the body weight of mice in the PBS group, PPNPs group, Amy group, Amy@NPs group, and Amy@NPs-MM / PL1 group showed a steady upward trend over time, with no significant differences between the groups, indicating that each treatment agent had no significant toxic effects on the overall condition of the mice.

[0088] Mice were euthanized after treatment, and major organs such as the uterus, heart, liver, spleen, lungs, and kidneys were collected for HE staining and compared with healthy mice to assess histopathological changes. Figure 16 As shown in Figure B, the organs and tissues of the healthy mice showed normal structures with no obvious pathological changes. The morphology of the uterus, heart, liver, spleen, lungs, and kidneys of the Amy@NPs-MM / PL1 group mice was basically the same as that of the healthy group, with no obvious pathological changes such as necrosis, fibrosis, or inflammatory cell infiltration, indicating that the nanomedicine had no significant toxic effects on the major organs.

[0089] Whole blood and serum were collected from each group of mice for complete blood count, blood biochemistry, and hormone level testing. The tested indicators included: liver function-related indicators (AST, ALP, ALT), kidney function-related indicators (BUN, Cr), complete blood count indicators (WBC, RBC, PLT), and sex hormone levels (E2, PG). Figure 17As shown, compared with the healthy group, there were no significant differences in the above indicators in the Amy@NPs-MM / PL1 group, and all fluctuated within the normal physiological range, indicating that the nanomedicine did not cause significant liver and kidney function damage, hematopoietic system abnormalities, or sex hormone level disorders.

[0090] In summary, Amy@NPs-MM / PL1 exhibits good in vivo biocompatibility at therapeutic doses, with no significant adverse effects on the overall condition, major organ morphology, or hematological parameters in mice, providing important safety evidence for its subsequent clinical translation and application.

[0091] Example 7:

[0092] Central therapeutic effects and systemic metabolic reprogramming To comprehensively evaluate the effect of Amy@NPs-MM / PL1 on improving endometriosis-related pain and its influence on the central nervous system and systemic metabolism, behavioral tests, central nervous system inflammation assessments, and serum metabolomics analyses were further performed on mice after treatment.

[0093] 5.1 Animal behavioral evaluation Patients with endometriosis often experience chronic pain and psychological symptoms such as anxiety and depression. To evaluate the effect of Amy@NPs-MM / PL1 on improving endometriosis-related pain and mood disorders, healthy mice, model mice (PBS group), and mice in the Amy@NPs-MM / PL1 treatment group were selected for mechanical pain threshold measurement and a series of behavioral tests. Figure 17 As shown, compared with the healthy group, the 50% paw-withdrawal threshold of the model mice was significantly reduced, indicating significant mechanosensitive hypersensitivity; the central region dwell time in the open field test was significantly shortened, suggesting anxiety-like behavior; the immobility time in the forced swimming and tail suspension tests was significantly prolonged, suggesting depressive-like behavior; and the percentage of sucrose preference was significantly reduced, suggesting anhedonia. After treatment with Amy@NPs-MM / PL1, the mice showed a significant increase in pain threshold, a prolonged central region dwell time, a shortened immobility time, and an increased percentage of sucrose preference. All behavioral indicators were significantly improved, approaching the levels of the healthy group. These results indicate that Amy@NPs-MM / PL1 can not only effectively alleviate EMs-related mechanosensitive pain but also significantly improve accompanying anxiety, depressive-like behavior, and anhedonia, demonstrating its comprehensive therapeutic potential for pain-emotional comorbidities.

[0094] 5.2 Assessment of Central Nervous System Inflammation To investigate the central mechanism by which Amy@NPs-MM / PL1 improves pain and mood disorders, the neuroinflammation status in the hippocampus and the levels of related factors in the cerebrospinal fluid of healthy mice, model mice, and mice treated with Amy@NPs-MM / PL1 were further examined.

[0095] Immunohistochemical staining was used to detect the expression of IBA-1, a marker of microglia in the hippocampus, and Ki-67, a marker of cell proliferation. Figure 18 As shown in Figure A, in the healthy group of mice, IBA-1 positive microglia in the hippocampus exhibited a branched, resting morphology, and the number of Ki-67 positive cells was moderate. In the model group of mice, the number of IBA-1 positive cells in the hippocampus increased, and their morphology showed an amoeboid activated state, while the number of Ki-67 positive cells decreased. In the Amy@NPs-MM / PL1 treatment group, the morphology of IBA-1 positive cells in the hippocampus returned to a branched, resting state, and the number was close to that of the healthy group. The number of Ki-67 positive cells also returned to normal levels, indicating that this nanomedicine can effectively inhibit central nervous system inflammation and promote nerve cell proliferation.

[0096] Cerebrospinal fluid was collected from each group of mice, and the levels of inflammatory factors and neuronal damage markers were detected by ELISA. Figure 18 As shown in Figure B, compared with the healthy group, the concentrations of TNF-α and IL-1β in the cerebrospinal fluid of the model group were significantly increased, and the levels of neuronal injury markers 3-nitrotyrosine (3-NT) and neuron-specific enolase (NSE) were also significantly increased; while the concentrations of TNF-α, IL-1β, 3-NT and NSE in the Amy@NPs-MM / PL1 treatment group were significantly reduced, close to the levels of the healthy group, indicating that the nanomedicine can effectively reduce central inflammatory response and neuronal damage.

[0097] 5.3 Whole-body metabolic reprogramming analysis To investigate the effects of Amy@NPs-MM / PL1 treatment on systemic metabolism, serum samples from mice in each group were collected for non-targeted metabolomics analysis.

[0098] Principal component analysis (PCA) results showed that the serum metabolic profiles of the healthy group, the model group (PBS group), and the Amy@NPs-MM / PL1 treatment group exhibited a clear separation trend, such as... Figure 19 As shown in Figure A, the EMs model mice exhibit significant metabolic disorders, and Amy@NPs-MM / PL1 treatment can reverse this abnormal metabolic state to some extent.

[0099] Through differential metabolite screening and analysis, heatmaps were created for 14 representative differential metabolites associated with EM progression, pain, neuroinflammation, and immune regulation, such as... Figure 19 As shown in B. The results showed that, compared with the PBS group, the Amy@NPs-MM / PL1 treatment group had increased levels of multiple beneficial metabolites (such as taurine and thiamine) and decreased levels of multiple harmful metabolites (such as indole, linoleoylcarnitine, oleoylcarnitine, and N-arachidonicylglycine).

[0100] Four key metabolites were further selected for quantitative analysis, and changes in the expression of related pathway proteins were detected. For example... Figure 19 As shown in Figure C, compared with the healthy group, the model group showed significantly increased indole levels, accompanied by upregulation of its receptor, the aryl hydrocarbon receptor (AHR), protein expression; significantly increased linoleoylcarnitine levels; significantly decreased taurine levels, accompanied by increased NLRP3 inflammasome-related protein expression; and significantly increased N-arachidonicylglycine levels, accompanied by upregulation of its receptor, the GPR18, protein expression. After treatment with Amy@NPs-MM / PL1, indole and linoleoylcarnitine levels significantly decreased, AHR and AMPKα1 protein expression tended to normalize; taurine levels significantly rebounded, NLRP3 expression was downregulated; N-arachidonicylglycine levels decreased, and GPR18 expression recovered to levels close to those of the healthy group. These results indicate that Amy@NPs-MM / PL1 exerts anti-inflammatory and neuroprotective effects by regulating the levels of key metabolites, thereby affecting the activity of related signaling pathways.

[0101] In summary, Amy@NPs-MM / PL1 can not only effectively inhibit lesion growth and local pyroptosis, but also reduce central nervous system inflammation, improve pain and mood disorders by regulating systemic metabolic reprogramming, thus achieving a multi-dimensional therapeutic effect from peripheral lesions to the central nervous system.

[0102] This invention, through UPLC-HRMS combined with network pharmacology and in vitro pharmacodynamics comparisons, has for the first time systematically screened and confirmed amygdalin as the core active ingredient for treating endometriosis from the traditional Chinese medicine compound Guizhi Fuling Wan. This ingredient can effectively inhibit the proliferation, migration, and invasion of ectopic endometrial stromal cells, induce apoptosis, and downregulate the expression of pain and inflammation-related genes, providing a new research strategy for the modern development and precise application of traditional Chinese medicine compound formulas.

[0103] This invention successfully constructed a dual-targeting nanodelivery system, Amy@NPs-MM / PL1, which combines macrophage membrane biomimicry with PL1-targeting peptide modification. This nanosystem possesses a uniform core-shell structure, suitable particle size, pH-responsive drug release characteristics, and fully preserves the functional proteins of the macrophage membrane, achieving both immune evasion and active targeting. In vitro and in vivo experiments confirmed that this system significantly improves the accumulation efficiency of amygdalin at lesion sites, prolongs its in vivo retention time, and reduces non-specific organ distribution.

[0104] This invention is the first to propose and verify the crucial role of the "lesion microenvironment-pyroptosis propagation-neuroinflammation" axis in endometriosis-related pain. Studies have shown that pyroptotic ectopic endometrial stromal cells can propagate inflammatory signals to neurons, inducing neuronal pyroptosis and neuroinflammation. Amy@NPs-MM / PL1, through its excellent antioxidant activity, effectively scavenges excess reactive oxygen species accumulated in the lesion microenvironment, inhibiting oxidative stress-driven pyroptosis activation at its source and blocking the propagation of pyroptosis signals to neurons, thereby alleviating neuroinflammation and pain at their root.

[0105] The Amy@NPs-MM / PL1 of this invention exhibits multidimensional therapeutic effects in vivo: significantly inhibiting the growth of ectopic lesions and improving histopathological features; effectively alleviating mechanical hyperalgesia and anxiety- and depression-like behaviors; reducing microglia activation in the hippocampus and central nervous system inflammation; and systematically reshaping systemic metabolic homeostasis by regulating metabolic-signaling axes such as indole / AHR and taurine / NLRP3. This nanoplatform achieves multi-level intervention from anti-oxidation and anti-pyroptosis to neuroprotection and metabolic regulation, providing a new strategy with both theoretical innovation and clinical translational potential for the comprehensive treatment of endometriosis.

[0106] Example 8:

[0107] Research Materials and Methods 1. Reagents and Instruments 1.1 Main reagents and consumables Table 3 is a detailed list of relevant experimental reagents:

[0108]

[0109] Table 4 is a detailed list of the relevant experimental reagent kits:

[0110] 1.2 Main Experimental Instruments Table 5 is a detailed list of relevant experimental instruments:

[0111]

[0112] 1.3 Main experimental cells Table 6 is a detailed list of the relevant experimental cells:

[0113] 1.4 Major Antibodies Table 7 is a list of antibodies used:

[0114]

[0115] 2. Drug screening experiments 2.1 Preparation of Guizhi Fuling Pill Extract Grind the Guizhi Fuling Pill sample into powder, take an appropriate amount and place it in a centrifuge tube (1.5 mL), add 1 mL of methanol aqueous solution, vortex mix, sonicate in a water bath for 30 min, centrifuge at 16000 g at 4℃ for 10 min, take the supernatant and freeze dry under vacuum, add 40% methanol aqueous solution to the freeze-dried sample, vortex mix, centrifuge at 16000 g at 4℃ for 15 min, take the supernatant, and you have the product.

[0116] 2.2 Ultra-high performance liquid chromatography-high resolution mass spectrometry analysis 2.2.1 Chromatographic conditions Samples were separated using a Vanquish UHPLC (Thermo Scientific, Waltham, MA) system combined with an ACQUITY UPLC HSS T3 (2.1 mm x 100 mm, 1.8 µm) column; column temperature 35℃; flow rate 0.3 mL / min; mobile phase composition A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution; gradient elution was performed according to the table below; Table 8 is a detailed table of gradient elution procedures:

[0117] 2.2.2 Mass Spectrometry Conditions The primary and secondary spectra of the samples were acquired using a Q-Exactive HFX mass spectrometer.

[0118] The Q-Exactive HFX mass spectrometer was coupled with a UHPLC system, and mass spectrometry was performed using both positive and negative electrospray ionization (ESI) modes. The spray voltage was 3800V (ESI+) / 3500V (ESI-), sheath gas pressure was 45 alb, auxiliary gas pressure was 20 alb, ion transfer tube temperature was 320℃, and nebulization temperature was 350℃. The detection mode was full scan / data-dependent two-stage scan (Full-MS / dd-MS2) mode, with first-stage and second-stage resolutions of 60000 and 15000, respectively. The top 10 MS1 ions were used to obtain MS / MS spectra, and collision energies (CEs) were normalized to step levels of 20, 40, and 60. The first-stage mass-to-charge ratio scan range was 90–1300.

[0119] 2.2.3 Sample Detection and Analysis Accurately pipette 1 μL of drugname solution and analyze by LC-MS.

[0120] 2.2.4 Data Analysis Process The raw data file in .raw format was imported into ProteoWizard and converted to .mzXML format. Peak alignment, retention time correction, and peak extraction were performed using XCMS software. Compound identification was performed by searching the CAS New Life Local Traditional Chinese Medicine High-Resolution Mass Spectrometry Database. The first-order mass error was less than 25 ppm, and the second-order fragmentation spectrum matching score was greater than 0.7. The higher the score, the higher the spectrum similarity. Currently, it is generally believed that the identification results above 0.7 are reliable.

[0121] 2.3 Network Pharmacological Analysis 2.3.1 Chemical Composition Target Analysis Traditional Chinese medicine components were obtained based on our company's blood component analysis results. Potential targets for these compounds were screened using the TCMSP (https: / / old.tcmsp-e.com / tcmsp.php), TCMID (http: / / www.megabionet.org / tcmid / ), and CTD (http: / / ctdbase.com / ) databases. The targets for all components were calibrated to their official gene names using the Uniprot (https: / / www.uniprot.org / ) database.

[0122] 2.3.2 Disease Target Analysis Using the CTD (https: / / ctdbase.com / ) database, relevant target searches were conducted using keywords such as endometriosis, and gene names were standardized using the Uniprot database.

[0123] 2.3.3 Component-Target-Disease Joint Analysis By taking the intersection of potential targets of compounds with disease targets, potential targets of traditional Chinese medicine active ingredients for treating endometriosis were obtained. Venn diagrams were constructed, and targets with gene degrees greater than the median were included to identify more relevant disease targets as key targets.

[0124] 2.3.4 Network Construction and Analysis Key targets were imported into the String (https: / / cn.string-db.org / ) database to obtain protein-protein interaction relationships, and network topology analysis of components-targets-diseases was performed. The relevant network diagrams were drawn using the NetworkX software package.

[0125] 2.3.5 Functional and Pathway Analysis Gene ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed using the GO database (geneontology.org). GO enrichment analysis used three modules: biological process (BP), molecular function (MF), and cellular component (CC). Pathway analysis used KEGG. The top 20 data points for each of the BP, MF, CC, and KEGG pathways were selected based on p-values ​​(p<0.05). The results of the pathway enrichment analysis were visualized using R.

[0126] 3. Cell culture and in vitro pharmacodynamic experiments 3.1 Cell Culture hEMSCs and HT-22 cells were cultured using dedicated complete culture medium (Wuhan Baidi Biotechnology Co., Ltd.). RAW264.7 cells were cultured using DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). All cells were cultured in a 37°C, 5% CO2 incubator, with fresh medium replaced every 2-3 days. Cells were passaged when they reached 80%-90% confluence.

[0127] 3.2 Cell viability assay Logarithmically growing hEMSCs were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured overnight. Different concentrations of Guizhi Fuling Wan extract (0, 0.1, 1, 5, 10, 20 mg / mL) or candidate monomer components (amygdalin, gallic acid, benzoylpaeoniflorin, concentration gradients of 0, 200, 400, 800, 1600 μmol / L) were added to each well, with three replicates per group. After 24 h of treatment, 10 μL of LCK-8 solution was added to each well, and incubation continued for 2 h. The absorbance at 450 nm was measured using a microplate reader. The half-maximal inhibitory concentration (IC50) was calculated using GraphPadPrism software. 50 ).

[0128]

[0129] 3.3 Cell Scratch Assay hEMSCs were seeded into 6-well plates and cultured until cell confluence reached over 90%. Vertical streaks were applied to the cell monolayer using a 200 μL sterile pipette tip, and the cells were gently washed with PBS to remove floating cells. Culture media containing different treatment drugs (prepared with 1% FBS) were added, and the cells were observed and photographed under an inverted microscope at 0 h and 24 h. The streak area was measured using ImageJ software.

[0130] 3.4 Transwell Migration and Invasion Experiments 3.4.1 Transfer Experiment hEMSCs cells from each group were harvested, the culture supernatant was discarded, and the cells were washed once with PBS. They were then starved in serum-free medium for 12 hours to eliminate the influence of serum on cell migration. After starvation, cells were collected by trypsin digestion with EDTA-free enzyme, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in serum-free medium to adjust the cell density to 2 × 10⁶ cells / mL. 5 Cells / mL.

[0131] Place a Transwell chamber (8 μm pore size, 24-well plate specification, Corning Biotechnology, USA) into a matching 24-well plate. Add 600 μL of complete culture medium containing 10% FBS (as a chemokine) to the lower chamber, taking care to avoid air bubbles. Add 200 μL of the cell suspension prepared above (i.e., 4 × 10⁻⁶ cells / well) to the upper chamber. 4 (Cells / well). Incubate the culture plate at 37°C in a 5% CO2 incubator for 24 hours.

[0132] After culturing, remove the Transwell chamber and gently wipe away any unmigrated cells from the upper chamber's inner surface with a cotton swab (be careful not to apply too much force and deform the membrane). Place the chamber in wells pre-filled with 4% paraformaldehyde and fix at room temperature for 15 minutes. After fixation, place the chamber in 0.1% crystal violet staining solution and stain at room temperature for 15 minutes. Gently rinse the chamber three times with PBS to remove excess staining solution and air dry at room temperature.

[0133] Five fields of view (100× or 200×) were randomly selected under an inverted microscope for observation and photographing, and the number of cells that had perforated the membrane was counted. Each experiment was repeated three times.

[0134] 3.4.2 Invasion Experiment For invasion assays, Matrigel substrate must be pre-coated. Remove Matrigel (Corning, USA) from -20°C and allow it to thaw overnight at 4°C. Dilute Matrigel 1:8 with pre-chilled serum-free medium (all operations should be performed on ice to prevent premature solidification). Spread 50 μL of the diluted Matrigel evenly onto the upper surface of the Transwell upper chamber membrane, taking care to avoid air bubbles. Incubate the coated chamber at 37°C for 2 hours to allow the Matrigel to solidify.

[0135] The subsequent steps are the same as for the migration experiment: digest the cells, adjust the cell density, add cell suspension to the upper chamber, add complete culture medium containing 10% FBS to the lower chamber, culture for 24 hours, fix, stain, take pictures and count.

[0136] 3.5 Flow cytometry detection of apoptosis hEMSCs from each group were collected after 24 h of treatment. Cells were digested with EDTA-free trypsin and washed twice with PBS. Following the instructions of the Annexin V-FITC / PI apoptosis detection kit: cells were resuspended in 100 μL of 1× binding buffer, 5 μL of Annexin V-FITC and 5 μL of LPI were added, gently mixed, and incubated at room temperature in the dark for 15 min. Then, 400 μL of 1× binding buffer was added, and cell apoptosis was detected by flow cytometry. Data were analyzed using FlowJo software.

[0137] 3.6 Real-time quantitative PCR Total RNA was extracted from cells using the TRIzol method, and RNA concentration and purity were determined using NanoDrop 2000. 1 μg of total RNA was reverse transcribed according to the PrimeScript RT MasterMix manual to obtain cDNA. Using the cDNA as a template, amplification was performed on a QuantStudio 5 real-time quantitative PCR instrument using SYBR Green MasterMix. Reaction conditions: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 5 sec, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, for a total of 40 cycles. GAPDH was used as an internal control, and the relative expression level of the target gene was calculated using the 2⁻ΔΔCt method. Primer sequences are shown in Table 9.

[0138] Table 9 is a detailed list of primer sequences:

[0139] 4. Characterization of nanomedicines 4.4 Characterization of the physicochemical properties of nanoparticles 4.4.1 Morphological observation Take an appropriate amount of nanoparticle suspension and drop it onto a copper grid. After drying at room temperature, stain with 2% phosphotungstic acid for 2 min, dry at room temperature, observe the morphology of the nanoparticles under a transmission electron microscope and take pictures.

[0140] 4.4.2 Particle size and potential determination Take an appropriate amount of nanoparticle suspension, dilute it with deionized water to a suitable concentration, and use a dynamic light scattering particle size analyzer to determine the hydrated particle size, polydispersity index (PDI) and zeta potential of the nanoparticles. Each sample is measured three times.

[0141] 4.4.3 Determination of drug loading and encapsulation efficiency Take 100 μL of LAmy@NPs-MM / PL1 nanoparticle suspension, add 0.2 mL of acetone solution, vortex for 1 min to fully demulsify the nanoparticles, and dry in an oven at 60 °C to constant weight. Add 1 mL of methanol to reconstitute, vortex for 2 min, filter through a 0.22 μm microporous membrane, and dilute the filtrate 10 times with acetonitrile before injection for analysis.

[0142] HPLC determination conditions Chromatographic column: WondaSil C18-WR column (4.6×150mm, 5μm, Shimadzu Corporation, Japan); column temperature: 35℃; mobile phase: methanol-0.1% phosphoric acid aqueous solution (10:90, v / v); flow rate: 0.8mL / min; detection wavelength: 272nm; injection volume: 20μL; Based on the concentration-peak area standard curve established using amygdalin standards, the concentration of amygdalin in the sample was calculated, thus obtaining the actual encapsulated drug mass. The encapsulation efficiency and drug loading were calculated using the following formulas:

[0143]

[0144] In the formula, the theoretical drug mass is the total amount of amygdalin added during preparation, and the total mass of nanoparticles is the weighing mass of the nanoparticles after lyophilization. Each sample was measured three times.

[0145] 4.4.4 Drug Release Assay Transfer 1 mL of Amy@NPs-MM / PL1 suspension to a dialysis bag (MWCO 10 kDa), and place it in 50 mL of PBS (pH 7.4) or acetate buffer (pH 5.5), respectively. Incubate at 37°C with shaking (100 rpm). At preset time points (0 h, 0.5 h, 2 h, 4 h, 6 h, 8 h, and 24 h), collect 1 mL of the release solution and replenish with an equal volume of fresh buffer. Analyze the peak area using high-performance liquid chromatography (HPLC) and calculate the cumulative release.

[0146] 4.5 Verification of Membrane Protein Retention Macrophage membranes, MM / PL1 membranes, Amy@NPs, and Amy@NPs-MM / PL1 samples were collected and added to 5×SDS loading buffer. The samples were boiled at 100℃ for 5 min and then subjected to SDS-PAGE electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 for 1 h, and then destained until the background was clear. Protein bands were then photographed and recorded. Separate samples were taken for Western blot analysis to detect the expression of CD47, CD68, and CD11b. The electrophoresis and transfer procedures were the same as in 5.1.

[0147] 4.6 Reactive Oxygen Scrap Capacity Test 4.6.1 Detection of hydrogen peroxide (H2O2) content Intracellular H2O2 levels were determined using the CheKine™ Hydrogen Peroxide Content Detection Kit (micro-method, catalog number: KTB1041, Abbkine, USA). After treatment, hEMSCs from each group were washed twice with pre-cooled PBS, and approximately 5 × 10⁶ cells were collected. 6 Add 1 mL of pre-cooled AssayBuffer (1×) to each cell, and sonicate on ice for 5 min (200 W, 3 s sonication, 7 s interval, repeated 30 times). Centrifuge at 10,000×g for 5 min at 4 °C, and place the supernatant on ice for analysis.

[0148] Prepare standards according to the kit instructions to establish a standard curve with concentration gradients of 0, 1, 2, 5, 10, 20, 50, and 100 μM. Add 60 μL of different concentrations of standards or samples to a 96-well plate sequentially, followed by 40 μL of reaction buffer. Mix thoroughly and incubate at 37°C for 10 min. Measure the absorbance at 580 nm using a microplate reader. Calculate the H2O2 concentration in the sample based on the standard curve, and express the results as nmol / mg protein or nmol / 10⁻¹⁰. 4 Cellular representation.

[0149] 4.6.2 Detection of hydroxyl radical (·OH) scavenging ability The hydroxyl radical scavenging ability of the samples was determined using the CheKine™ Hydroxyl Radical Scavenging Capacity Assay Kit (micro-method, catalog number: KTB1091, Abbkine, USA). Approximately 5 × 10⁶ hEMSCs were collected from each group after treatment. 6 Add 1 mL of deionized water to each cell, and sonicate on ice for 5 min (200 W, 3 s sonication, 7 s interval, repeated 30 times). Centrifuge at 10,000 × g for 10 min at 4 °C, and place the supernatant on ice for analysis.

[0150] Set up blank wells, standard wells, assay wells, and control wells in a 96-well plate according to the kit instructions. Add 40 μL of ferrous salt, 40 μL of lysicrystal acid, 40 μL of sample, and 40 μL of H₂O₂ to the assay wells; omit H₂O₂ and make up the volume with deionized water to the control wells. After mixing, incubate at 37°C for 20 min, and measure the absorbance at 520 nm using a microplate reader. Calculate the hydroxyl radical scavenging rate using the following formula:

[0151] In the formula, ΔAstandard = Astandard - Ablank, and ΔAdetermination = Adetermination - Acontrol.

[0152] 4.6.3 Detection of Superoxide Anion (O2⁻) Scavenging Capacity The superoxide anion scavenging capacity of the samples was determined using the CheKine™ Superoxide Anion Scavenging Capacity Assay Kit (micro-method, catalog number: KTB1080, Abbkine, USA). Approximately 5 × 10⁶ hEMSCs were collected from each group after treatment. 6 Add 1 mL of pre-chilled lysis buffer (50 mM potassium phosphate, 0.1 mM EDTA, 0.5% Triton X-100, pH 7.4) to each cell and incubate on ice for 10 min. Centrifuge at 12,000 × g for 5 min at 4 °C, and collect the supernatant on ice for analysis.

[0153] Prepare WorkingReagent immediately before use: 80 μL per well, mixed with 74 μL AsayBuffer, 5 μL Xanthine, 5 μL WST-8, and 1 μL Lnhancer. Prepare immediately before use. Dilute WorkingXanthine Oxidase with Sample Diluent at a ratio of 1:200. Set up blank wells, control wells, and assay wells in a 96-well plate, adding the corresponding reagents according to the instructions. Immediately after mixing, measure the absorbance at 450 nm (A0). Incubate at room temperature in the dark for 60 min, then measure the absorbance at 450 nm again (A60). Calculate the superoxide anion scavenging rate using the following formula:

[0154] In the formula, ΔA blank = A60 blank - A0 blank, ΔA control = A60 control - A0 control, ΔA measurement = A60 measurement - A0 measurement, ΔΔA control = ΔA control - ΔA blank, and ΔΔA measurement = ΔA measurement - ΔA blank.

[0155] 4.6.4 DPPH free radical scavenging ability test The DPPH radical scavenging ability of the samples was determined using the CheKine™ DPPH radical scavenging assay kit (micro-method, catalog number: KTB1092, Abbkine, USA). Approximately 5 × 10⁶ hEMSCs were collected from each group after treatment. 6 Add 1 mL of pre-cooled Extraction Buffer to each cell, and sonicate on ice for 5 min (200 W, 3 s sonication, 7 s interval, repeated 30 times). Centrifuge at 10,000 × g for 10 min at 4 °C, and place the supernatant on ice for analysis.

[0156] Prepare WorkingReagent I immediately before use: Dissolve 48T Reagent I thoroughly in 30 mL of anhydrous ethanol. Set up blank, control, and assay wells in a 96-well plate: Add 10 μL sample and 190 μL WorkingReagent I to the assay wells; add 10 μL sample and 190 μL anhydrous ethanol to the control wells; add 10 μL Extraction Buffer and 190 μL WorkingReagent I to the blank wells. Mix thoroughly and incubate at 25°C in the dark for 20 min. Measure the absorbance at 515 nm using a microplate reader. Calculate the DPPH free radical scavenging rate using the following formula:

[0157] In the formula, ΔA_determination = A_determination - A_control.

[0158] 4.7 Macrophage-Endometrial Stromal Cell Co-culture Model A model was established using a Transwell co-culture system. RAW264.7 macrophages were seeded into the upper chamber of a Transwell cell line (1 × 10⁻⁶ cells / mL). 5 hEMSCs were seeded in the lower chamber (2 × 10⁶ cells / chamber). 5 Cells / well). After overnight culture, Cy5.5-labeled Amy@NPs or Amy@NPs-MM / PL1 (final Cy5.5 concentration 10 μg / mL) were added to the upper chamber, and the cells were co-cultured for 24 h. Cells from the upper and lower chambers were collected separately, fixed with 4% paraformaldehyde, stained with DAPI, and the distribution of nanoparticles in macrophages and hEMSCs was observed under a laser confocal microscope.

[0159] 5. Molecular biological detection methods 5.1 Western blot detection (1) Protein sample preparation Protein extraction was performed on cell or tissue samples from each group after treatment. For cell samples: the culture supernatant was discarded, and the cells were washed twice with pre-chilled PBS, then the residual liquid was aspirated. An appropriate amount of RIPA lysis buffer (containing 1% PMSF, 1% protease inhibitor mixture, and 1% phosphatase inhibitor) was added, and cells were scraped off with a cell scraper and collected in 1.5 mL centrifuge tubes. The cells were lysed on ice for 30 min, with vortexing every 10 min to ensure complete lysis. For tissue samples: approximately 50 mg of tissue was taken, minced, and placed in a 1.5 mL centrifuge tube. 500 μL of RIPA lysis buffer (containing the above inhibitors) was added, and the tissue was homogenized on ice using an electric homogenizer for 10 seconds each time, with a 30-second interval, repeated 3 times until the tissue was completely lysed. The homogenate was then lysed on ice for 30 min.

[0160] After lysis, centrifuge at 12000×g for 15 min at 4℃. Carefully transfer the supernatant to a new pre-cooled centrifuge tube to obtain the total protein extract. Use a small amount of the supernatant for protein concentration determination. Add 5×SDS loading buffer to the remaining samples according to the specified ratio, boil at 100℃ for 10 min to denature the proteins, and store at -80℃ for later use.

[0161] (2) Protein concentration determination (BCA method) Protein quantification was performed using the BCA protein concentration assay kit. The BCA working solution was prepared according to the kit instructions: reagent A and reagent B were mixed at a ratio of 50:1 and thoroughly mixed before use. The protein standard (5 mg / mL BSA) was diluted with PBS to a final concentration of 0.5 mg / mL. 0, 1, 2, 4, 8, 12, 16, and 20 μL of the working solution were added to each well of a 96-well plate, and the volume was brought to 20 μL with PBS to establish a standard curve with concentration gradients of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. 2 μL of the protein sample to be tested was added to each well of the 96-well plate, and the volume was brought to 20 μL with PBS. 200 μL of BCA working solution was added to each well, gently vortexed to mix, and incubated at 37°C for 30 min. The absorbance was measured at 562 nm using a microplate reader. The protein concentration of the sample was calculated based on the standard curve.

[0162] (3) SDS-PAGE electrophoresis Prepare SDS-PAGE separating and stacking gels of appropriate concentrations according to the molecular weight of the target protein. In this study, the protein molecular weight range was 15-120 kDa, therefore an 8%-12% separating gel was used. Load the prepared gels into the electrophoresis tank and add 1×SDS-PAGE electrophoresis buffer (containing 25 mM Tris, 192 mM glycine, and 0.1% SDS). Load an equal volume of protein sample (20-40 μg) according to the calculated volume, and add 5 μL of pre-stained protein molecular weight marker to both wells of the sample. Electrophoresis at a constant voltage of 80 V for 30 min to concentrate the sample to the separating gel interface, then adjust the voltage to 120 V and continue electrophoresis for 60-90 min until the bromophenol blue indicator reaches the bottom of the gel.

[0163] (4) Transfer membrane After electrophoresis, remove the gel, trim the stacking gel and any excess, and equilibrate the separating gel in pre-chilled transfer buffer (containing 25 mM Tris, 192 mM glycine, and 20% methanol) for 10 min. Cut a PVDF membrane (0.45 μm pore size) to the same size as the gel, activate it by soaking it in methanol for 15 seconds, and then transfer it to transfer buffer to equilibrate for 5 min. Prepare a transfer "sandwich" in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge," ensuring there are no air bubbles between each layer. Place the transfer clamp in the transfer tank, add pre-chilled transfer buffer, and transfer at a constant current of 200 mA for 60-120 min (adjust the transfer time according to the molecular weight of the target protein). Place the transfer tank in an ice bath to prevent the temperature from becoming too high during the transfer process.

[0164] (5) Closed After the transfer was complete, the PVDF membrane was removed and rinsed once with TBST buffer (containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.1% Tween-20) to remove any residual transfer buffer. The membrane was then placed in protein-free rapid blocking buffer and blocked on a shaker at room temperature for 10 min.

[0165] (6) Primary antibody incubation After blocking, rinse the membrane once with TBST. Place the membrane in an incubation chamber, add the primary antibody solution diluted appropriately with the primary antibody dilution buffer, and incubate overnight (approximately 12-16 hours) at 4°C with shaking. The primary antibody dilution ratio and incubation conditions should be optimized according to the antibody's instruction manual.

[0166] (7) Secondary antibody incubation Remove the membrane, recover the primary antibody, and wash three times with TBST on a shaker for 10 min each time. Place the membrane in a new incubation chamber, add HRP-labeled secondary antibody solution diluted 1:5000, and incubate on a shaker at room temperature for 1 h. After incubation, wash three times with TBST for 5 min each time.

[0167] (8) Chemiluminescence detection Following the ECL chemiluminescence substrate kit instructions, mix solutions A and B in a 1:1 ratio to prepare the luminescent working solution (prepare fresh before use). Place the membrane in a clean Petri dish, add the luminescent working solution evenly, and react at room temperature in the dark for 1-2 minutes. Remove the membrane, blot off excess luminescent solution with absorbent paper, and place it in a chemiluminescence imaging system for development and imaging. Adjust the exposure time according to the signal strength, acquire images, and save them.

[0168] (9) Image analysis ImageJ image analysis software was used for quantitative analysis of Western blot bands by grayscale values. GAPDH was used as an internal control protein, and the relative expression levels of each target protein were calculated. Each experiment was repeated three times, and the average values ​​were used for statistical analysis.

[0169] 5.2 Immunofluorescence staining Immunofluorescence staining of cell slides and tissue sections was performed using the Immunoway rabbit dual-label three-color fluorescence detection kit (catalog number: RS0036, Immunoway, USA). The specific procedures are as follows: 5.2.1 Immunofluorescence staining of cell smears Seed hEMSCs onto confocal culture dishes or cell slides, and after appropriate treatment, aspirate the culture supernatant and gently wash three times with PBS. Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 15 min. Aspirate the fixative and wash three times with PBS for 5 min each time. Permeabilize with 0.3% Triton X-100 at room temperature for 20 min (this step can be omitted for antigens expressed on the cell membrane), and wash three times with PBS for 5 min each time.

[0170] Add 50-100 μL of reagent B (peroxidase blocking buffer) to completely cover the cell slide, incubate at room temperature for 15 min, and wash three times with PBST for 2 min each time. Draw a circle around the cell slide with an immunohistochemical pen, add diluted primary antibody (cleaved caspase-1, GSDMD-N, Ki-67, BDNF, NGF, etc., dilution ratio 1:200), and incubate overnight in a humidified chamber at 4°C. The next day, remove the slide, allow it to warm to room temperature for 30 min, and wash three times with PBST for 2 min each time.

[0171] Add 50-100 μL of reagent C (HRP polymeric anti-rabbit / mouse secondary antibody) working solution and incubate at room temperature for 30 min. Wash three times with PBST for 2 min each time. Add 50-100 μL of fluorescent dye reagent D-594 working solution (for detecting the first indicator), incubate at room temperature in the dark for 10 min, and wash three times with PBST for 2 min each time. Add reagent P (antibody stripping solution) preheated to 37℃ to cover the cells, incubate at 37℃ for 10-20 min, discard the stripping solution, and wash three times with PBST for 5 min each time.

[0172] Repeat the primary and secondary antibody incubation steps described above, changing the fluorescent dye for the second indicator to reagent D-488 working solution. After staining all indicators, add one drop of reagent G (DAPI / mounting solution) to cover the cell slide, then cover with a coverslip, taking care to avoid air bubbles. Observe and photograph under a laser confocal microscope, and perform semi-quantitative fluorescence intensity analysis using ImageJ software.

[0173] 5.2.2 Immunofluorescence staining of paraffin sections Paraffin sections (4 μm thick) of ectopic lesion tissue from each group were baked in a 60℃ oven for 1 hour. The sections were then placed in a repair cassette, and sufficient working solution of Reagent 9A (dewaxing and repair combined solution) (diluted with deionized water at a ratio of 1:19) was added. The repair cassette was then placed in a water bath containing boiling distilled water and heated for 30 minutes to perform antigen repair. After naturally cooling to room temperature, the sections were removed and rinsed 5-6 times in distilled water.

[0174] Wipe the tissue dry with filter paper, then add 50-100 μL of reagent B (peroxidase blocking buffer) to completely cover the tissue. Incubate at room temperature for 15 min, then wash three times with PBST for 2 min each time. Draw circles around the tissue with an immunohistochemical pen, then add diluted primary antibodies (Ki-67, CD31, Vimentin, MMP9, cleaved caspase-1, GSDMD-N, IL-1β, IL-18, etc., at a dilution ratio of 1:200), and incubate overnight at 4°C in a humidified chamber. The next day, remove the tissue, allow it to warm to room temperature for 30 min, and wash three times with PBST for 2 min each time.

[0175] Subsequent steps were the same as for cell slide immunofluorescence staining: secondary antibody incubation, fluorescent dye development, antibody stripping, staining for the second marker, and DAPI mounting were performed sequentially. The slides were then observed and photographed under a laser confocal microscope, and semi-quantitative fluorescence intensity analysis was performed using ImageJ software.

[0176] 5.3 Enzyme-linked immunosorbent assay (ELISA) Collect cell culture supernatant, mouse serum, or cerebrospinal fluid samples, and follow the instructions of the corresponding ELISA kit. Specific steps: Add standards and samples to pre-coated 96-well plates and incubate at 37°C for 1 hour; after washing, add biotinylated antibody and incubate at 37°C for 1 hour; after washing, add HRP-labeled streptavidin and incubate at 37°C for 30 minutes; after washing, add TMB substrate for color development and incubate at 37°C in the dark for 15 minutes; add stop solution and measure absorbance at 450 nm. Calculate the concentration of the analyte based on the standard curve.

[0177] 6. Animal Experimentation Methods 6.1 Establishment of a mouse model of endometriosis Female BALB / c donor mice were subcutaneously injected with estradiol benzoate (0.2 mg / kg) every other day for 7 consecutive days to induce endometrial proliferation. On day 8, the donor mice were sacrificed, and the uterus was removed under aseptic conditions and placed in pre-cooled PBS. The uterus was then minced with ophthalmic scissors into tissue fragments with a diameter <1 mm³. The tissue fragments were resuspended in PBS (5 mL of PBS for each donor mouse).

[0178] Female BALB / c receptor mice were intraperitoneally injected with 200 μL of the above tissue suspension per mouse. On the 7th day after surgery, 3 mice were randomly selected and sacrificed, and the ectopic lesion formation was observed through laparotomy to confirm successful modeling.

[0179] 6.2 Animal grouping and administration Mice that successfully modeled the disease were randomly divided into 6 groups of 5 mice each: (1) PBS group (negative control); (2) PPNPs group (blank nanoparticles); (3) Amy group (free amygdalin); (4) Amy@NPs group (uncoated nanomedicine); (5) Amy@NPs-MM / PL1 group (dual-targeting nanomedicine); (6) GnRH-a group (positive control). All groups were administered the drug via intraperitoneal injection once every 2 days for 3 consecutive weeks. A healthy control group (n=5) was also included, receiving no treatment. Mouse weight was recorded every 3 days during the treatment period.

[0180] 6.3 In vivo imaging of small animals and imaging of ex vivo organs Mice with endometriosis were intraperitoneally injected with Cy5.5-labeled free dye, Amy@NPs-Cy5.5, or Amy@NPs-MM / PL1-Cy5.5 (Cy5.5 dose 1 mg / kg). At 0, 3, 7, and 14 days post-injection, mice were anesthetized with isoflurane and placed in a small animal in vivo imaging system for fluorescence imaging (excitation wavelength 675 nm, emission wavelength 720 nm). Seven days post-injection, mice were sacrificed, and ectopic lesions, as well as tissues from the heart, liver, spleen, lungs, kidneys, uterus, and ovaries, were collected for in vitro fluorescence imaging to observe the tissue distribution of the nanoparticles.

[0181] 6.4 Mechanical pain threshold determination (Von Frey test) Mice were placed in an acrylic box with a metal mesh bottom and allowed to acclimatize for 30 minutes. Von Frey fibers (0.04-2.0 g) were used to vertically stimulate the mid-sole of the mouse's hind paw, causing the fibers to bend into an S-shape for 2-3 seconds. A positive response was indicated by paw lifting, licking, or paw retraction. The 50% paw withdrawal threshold (PWT) was determined using the up-down method.

[0182] 6.5 Behavioral Tests Open field test: The mouse was placed in the center of an open field test box (50×50×40cm) and allowed to explore freely for 10 minutes. The time spent in the central area (25×25cm) and the total distance traveled were recorded by a video tracking system.

[0183] Forced swimming test: The mice were placed in a transparent cylindrical water tank (25cm high, 15cm in diameter, 15cm deep, and 25±1℃). After acclimatization for 2 minutes, the immobility time of the mice in the following 4 minutes was recorded (the mice floated on the water surface and only made the minimum movements required to keep their heads above water).

[0184] Tail suspension test: The tail of the mouse was fixed to the tail suspension box 1 cm from the end with tape, so that the mouse was in an upside-down state. After acclimatization for 1 minute, the immobility time of the mouse in the following 5 minutes was recorded.

[0185] Sugar water preference test: Mice were acclimatized to sugar water for 48 hours before the experiment (two water bottles were placed in each cage, one containing 1% sucrose solution and the other containing pure water). After fasting and water restriction for 12 hours, each mouse was given one pre-weighed bottle of 1% sucrose solution and one bottle of pure water. The mice were weighed and the amount of water consumed was recorded after 1 hour.

[0186]

[0187] 6.6 Histological staining After treatment, mice in each group were euthanized, and ectopic lesion tissue and major organs (heart, liver, spleen, lung, kidney, and uterus) were isolated and fixed with 4% paraformaldehyde for more than 24 hours. The fixed tissues were dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare serial sections with a thickness of 4 μm for subsequent staining and observation.

[0188] 6.6.1 Hematoxylin-eosin (HE) staining Take paraffin sections and bake them in a 60℃ oven for 1 hour. Dewax the sections by immersing them in xylene I and II for 10 minutes each, then hydrate them by immersing them in anhydrous ethanol I and II, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each, and finally rinse them with distilled water 3 times for 2 minutes each time.

[0189] After hydration, immerse the sections in hematoxylin staining solution for 5-10 minutes, then rinse with running tap water for 5 minutes to allow them to regain their blue color. Next, immerse the sections in 1% hydrochloric acid-ethanol solution for differentiation for 3-5 seconds, then immediately rinse with running tap water for 30 minutes to allow them to regain their blue color. After observing clear cell nuclei under a microscope, immerse the sections in eosin staining solution for 1-3 minutes. After staining, immerse the sections sequentially in 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol I and II for 5 minutes each to dehydrate them, then immerse them in xylene I and II for 10 minutes each for clearing. After removing the sections, mount them with neutral resin and observe and photograph them under a light microscope. Normal cell nuclei appear blue, and cytoplasm appears red.

[0190] 6.6.2 Masson staining Masson staining kit (catalog number: G1340, Beijing Solarbio Science & Technology Co., Ltd.) was used for the procedure. The paraffin sections were baked, dewaxed, and hydrated in the same manner as for HE staining.

[0191] After hydration, immerse the sections in the mordant provided in the kit and incubate overnight at room temperature or at 56°C for 30 minutes, then rinse with running water for 10 minutes. Immerse the sections in hematoxylin staining solution for 5-10 minutes, then rinse with running water for 5 minutes. Immerse the sections in hydrochloric acid-ethanol differentiation solution for 3-5 seconds, then rinse with running water for 10 minutes to regain blue color. Immerse the sections in Ponceau S-Acid Fuchsin staining solution for 5-10 minutes, then rinse quickly with distilled water. Immerse the sections in phosphomolybdic acid solution for 5 minutes, spin dry, and counterstain directly with aniline blue staining solution for 5 minutes. Immerse the sections in 1% glacial acetic acid solution for 1 minute for differentiation. After staining, dehydrate the sections with graded ethanol, clear with xylene, and mount with neutral resin (same steps as HE staining), then observe and photograph under a light microscope. Collagen fibers appear blue, muscle fibers and cytoplasm appear red, and cell nuclei appear blue-black.

[0192] 6.7 Immunohistochemical staining Paraffin sections of mouse brain tissue were dewaxed, hydrated, antigen-retrieved, and blocked (steps as in 4.4.2). IBA-1 primary antibody (1:200) or Ki-67 primary antibody (1:200) was added, and the sections were incubated overnight at 4°C. After washing with PBS, HRP-labeled secondary antibody (1:500) was added, and the sections were incubated at room temperature for 1 hour. DAB staining was performed, followed by hematoxylin counterstaining. The sections were then dehydrated, cleared, mounted, and observed and photographed under a microscope.

[0193] 7. Metabolomics Analysis After treatment, blood was collected from the orbital cavity of mice in each group. After standing for 30 minutes, the serum was separated by centrifugation at 4°C and 3000×g for 15 minutes and stored at -80°C for later use.

[0194] Take 100 μL of serum sample, add 300 μL of methanol-acetonitrile (1:1) mixture, vortex for 1 min, and let stand at -20℃ for 30 min to precipitate protein. Centrifuge at 14,000×g for 15 min at 4℃, take the supernatant and dry it with nitrogen, redissolve it with 100 μL of 50% acetonitrile, filter it through a 0.22 μm filter membrane and then inject it for analysis.

[0195] Chromatographic separation was performed using an ACQUITY UPL CHS ST3 column (2.1 × 100 mm, 1.8 μm, Waters Corporation, USA), at a column temperature of 40 °C, a flow rate of 0.3 mL / min, and an injection volume of 5 μL. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program was: 0–2 min, 5% B; 2–15 min, 5%–95% B; 15–18 min, 95% B; 18–20 min, 95%–5% B. Mass spectrometry detection was performed using a Q-Exactive HFX mass spectrometer with electrospray ionization (ESI) in both positive and negative ion modes, with a scan range of m / z 70–1000.

[0196] Raw data underwent peak alignment, peak extraction, and normalization using ProgenesisQI software. The processed data were then imported into MetaboAnalyst 6.0 for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Differential metabolites were screened based on VIP>1, p<0.05, and |log2FC|>1. Differential metabolites were identified and pathway enrichment analyses were performed using databases such as HMDB and KEGG.

[0197] 8. Statistical Analysis All experiments were repeated at least three times. Data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPadPrism 10.1.2 software. Unpaired t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons among multiple groups; and Tukey's or Dunnett's test was used for pairwise comparisons between groups. Two-way ANOVA was used for repeated measures data. A p-value < 0.05 was considered statistically significant.

[0198] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. The application of a nano-Amy@NPs-MM / PL1 in the preparation of a drug for treating endometriosis, characterized in that, It includes nano-Amy@NPs-MM / PL1, which comprises a nanocarrier PEG-PLGA, a nanodrug Amy, a nano-coated macrophage membrane, and a targeting peptide PL1.

2. The application of the nano-Amy@NPs-MM / PL1 as described in claim 1 in the preparation of a medicament for treating endometriosis, characterized in that, The preparation steps are as follows: Step S1: Macrophage membrane extraction: Macrophage cell membranes were isolated using a cell membrane extraction kit; Step S2: Insertion of the targeting peptide into the macrophage membrane: The targeting peptide PL1 was inserted into the macrophage membrane to obtain the macrophage membrane MM / PL1; Step S3, Preparation of PEG-PLGA loaded with Amy: Amygdalin, PLGA, and DSPE-PEG were dissolved in acetone as the organic phase; polyvinyl alcohol aqueous solution was used as the aqueous phase. The organic and aqueous phases were mixed using microfluidic technology, and the effluent was collected. The effluent was then dialyzed, filtered, and concentrated to obtain amygdalin-loaded nanoparticles Amy@NPs. Step S4, Preparation of Amy@NPs-MM / PL1: The obtained targeted macrophage membrane MM / PL1 was extruded to form uniform membrane vesicles. The obtained amygdalin-loaded nanoparticles Amy@NPs were mixed with the membrane vesicles, incubated, and extruded to form a mixture. The mixture was centrifuged, filtered, and concentrated to obtain Amy@NPs-MM / PL1.

3. The application of the nano-Amy@NPs-MM / PL1 as described in claim 1 in the preparation of a medicament for treating endometriosis, characterized in that, The Amy@NPs-MM / PL1 inhibitors the activation of the TNF-α / TNFR1 / NF-κB signaling pathway, blocks the downstream caspase-1 / GSDMD-mediated pyroptosis cascade, reduces the release of inflammatory factors, and inhibits angiogenesis and the expression of invasive factors.

4. The application of the nano-Amy@NPs-MM / PL1 as described in claim 1 in the preparation of a medicament for treating endometriosis, characterized in that, The targeting peptide PL1 in the nano-Amy@NPs-MM / PL1 specifically recognizes tendinin-C, which is highly expressed in endometriosis lesions, to achieve active targeting and anchoring.

5. The application of the nano-Amy@NPs-MM / PL1 as described in claim 1 in the preparation of a medicament for treating endometriosis, characterized in that, The nano-Amy@NPs-MM / PL1 possesses antioxidant activity, clearing excessively accumulated ROS in the lesion microenvironment and inhibiting pyroptosis activation driven by oxidative stress.

6. The application of the nano-Amy@NPs-MM / PL1 as described in claim 1 in the preparation of a medicament for treating endometriosis, characterized in that, The nano-Amy@NPs-MM / PL1 protects neurons from pyroptosis damage by inhibiting pyroptosis in hEMSCs and blocking the propagation of pyroptosis signals to neurons.

7. The nano-Amy@NPs-MM / PL1 as described in any one of claims 1-6, characterized in that, The nano-Amy@NPs-MM / PL1 is used to prepare bioproducts.

8. The nano-Amy@NPs-MM / PL1 as described in claim 7, characterized in that, The biological product is a reagent or kit.

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