Emodin sustained-release hydrogel targeting fosb and tgfb1 axis and application thereof

Abstract

This invention provides a triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axes and its applications, belonging to the field of biomedical materials and drug delivery technology. The sustained-release hydrogel is a core-shell structured sequential release hydrogel. The outer layer is a 40 mg / mL low-concentration gelatin methacrylamide hydrogel loaded with stromal cell-derived factor-1α (SDF-1α), and the inner layer is a 60 mg / mL high-concentration GelMA hydrogel loaded with triptolide. This invention is the first to clearly demonstrate that the transcription factor FOSB constitutes a core driving pathway for endometriosis fibrosis by transcribing and activating TGFB1. Triptolide can directly bind to FOSB and promote its ubiquitination-mediated degradation, thereby blocking the TGF-β / Smad signaling pathway and mesenchymal-myofibroblast transformation. Through a synergistic recruitment-targeted inhibition strategy using core-shell hydrogels, SDF-1α rapidly recruits myofibroblasts in the lesion area, and tripterygium wilfordii achieves local sustained release over 14 days. While significantly inhibiting fibrosis and reducing the size of endometriosis lesions, it avoids the hepatotoxicity and off-target damage of free drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and drug delivery technology, and more specifically, it relates to triptolide sustained-release hydrogels targeting the FOSB and TGFB1 axes and their applications. Background Technology

[0002] Endometriosis is a common and disabling gynecological disease. Its core pathological feature is the ectopic growth of endometrial-like tissue accompanied by progressive fibrosis. This fibrotic process is a key cause of chronic pelvic pain, infertility, and the formation of a "frozen pelvis," severely damaging patients' reproductive health and quality of life. Clinically, current treatments have significant limitations: hormone therapy is almost ineffective against existing fibrotic lesions, and long-term use can easily cause side effects such as endocrine disorders; surgical treatment of tissue adhesions caused by fibrosis is extremely difficult, with a high recurrence rate; traditional anti-fibrotic drugs are mostly broad-spectrum anti-inflammatory agents with insufficient targeting and limited efficacy.

[0003] In-depth investigation into the molecular mechanisms of fibrosis has revealed that the transforming growth factor-β (TGF-β) signaling pathway is a recognized core regulatory pathway, but the key upstream driving molecules remain unclear, hindering the development of targeted therapy strategies. Tripterygium wilfordii, a natural triterpenoid isolated from Tripterygium wilfordii, has been proven to possess potent anti-inflammatory and anti-fibrotic activities, demonstrating significant efficacy in multi-organ fibrosis models involving the kidney, liver, and lungs. However, the clinical translation of triptolide is limited by severe systemic toxicity (especially hepatotoxicity and nephrotoxicity) and potential off-target damage to normal endometrial tissue, preventing its systemic administration for the treatment of endometriosis.

[0004] Local drug delivery systems are a key direction for solving the above problems, and injectable hydrogels, with their characteristics of minimally invasive drug delivery, in-situ molding, and sustained drug release, have become ideal carriers. However, most existing hydrogels are single-drug loading systems, lacking the ability to target and recruit pathogenic cells at the lesion site, resulting in insufficient drug efficacy. Furthermore, precise targeted delivery systems targeting the core molecular pathways of endometriosis fibrosis have not yet been reported. Therefore, developing an intelligent delivery system capable of targeting key fibrosis pathways, achieving local sustained drug release, and reducing systemic toxicity is a pressing technical challenge in the current treatment of endometriosis. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axes and its application. This solves the technical problems of the lack of precise targeted treatment for endometriosis-related fibrosis, the difficulty in clinical translation of the potent anti-fibrotic drug triptolide due to systemic liver and kidney toxicity and off-target damage, and the insufficient efficacy caused by the lack of targeted recruitment of pathogenic myofibroblasts by traditional delivery systems.

[0006] A sustained-release hydrogel of triptolide targeting the FOSB and TGFB1 axes, wherein the sustained-release hydrogel is a core-shell structured sequential release hydrogel, named SDF-1α / Celastrol@Gel-seq.

[0007] The core-shell structure comprises an outer layer of low-concentration gelatin methacryloyl (GelMA) hydrogel and an inner layer of high-concentration gelatin methacryloyl (GelMA) hydrogel.

[0008] The outer layer of low-concentration GelMA hydrogel is loaded with stromal cell-derived factor-1α (SDF-1α), and the inner layer of high-concentration GelMA hydrogel is loaded with triptolide (Celastrol).

[0009] The concentration of GelMA in the outer layer was 40 mg / mL, and the concentration of GelMA in the inner layer was 60 mg / mL.

[0010] Preferably, the loading of triptolide is the hydrogel dosage corresponding to 0.05-0.3 mg / 20g animal body weight, and more preferably the hydrogel dosage corresponding to 0.3 mg / 20g animal body weight.

[0011] Preferably, the loading concentration of SDF-1α is such that it can effectively recruit endometriosis-related myofibroblasts, and the release cycle of triptolide is 14 days, with the local steady-state concentration maintained at 4 μM.

[0012] Preferably, the gelation time of the sustained-release hydrogel meets the requirements for rapid shaping after in vivo injection, the mechanical strength enables in situ retention in vivo, and the internal structure is porous, with porosity that facilitates nutrient exchange, cell infiltration, and sustained drug release.

[0013] Preferably, the application of the triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis in the preparation of a drug for treating endometriosis, wherein the endometriosis is endometriosis with fibrosis, including but not limited to the revised American Society for Reproductive Medicine (rASRM) stages I-IV endometriosis, especially suitable for severe endometriosis of rASRM stages III-IV with "frozen pelvis" manifestations, the drug is administered by local injection into the lesion, preferably by intraperitoneal injection around the lesion, the frequency of administration is single administration, and the therapeutic effect is sustained for more than 14 days, the drug exerts its therapeutic effect through the following mechanism: the outer hydrogel releases SDF-1α to recruit myofibroblasts at the lesion site, the triptolide sustained-released hydrogel in the inner hydrogel directly binds to FOSB protein, induces ubiquitination-mediated degradation of FOSB, thereby inhibiting the transcriptional activation of TGFB1 by FOSB, blocking the TGF-β / Smad signaling pathway, reversing the activated phenotype of myofibroblasts, and reducing extracellular matrix deposition and fibrosis process.

[0014] Preferably, the triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axes is used in the preparation of a drug to inhibit endometriosis-related fibrosis. The drug can reduce collagen deposition in endometriosis lesions, reduce the expression of fibrosis markers (FN1, COL1, COL3, αSMA), inhibit lesion contraction and angiogenesis, and at the same time reduce the systemic toxicity of triptolide, avoiding damage to the liver, kidneys and normal endometrial tissue.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Targeted breakthrough, precisely blocking the core pathway of fibrosis: This invention is the first to clearly identify the FOSB-TGFB1 axis as the key driving pathway of fibrosis in endometriosis. Tripterygium wilfordii directly binds to FOSB and induces its ubiquitination and degradation, blocking the transcriptional activation of TGFB1 upstream and the downstream TGF-β / Smad signaling pathway. Compared with traditional single-target inhibitors (such as the AP-1 inhibitor T-5224), it has a more significant anti-fibrotic effect and achieves precise intervention in the fibrosis process from a mechanistic perspective.

[0017] The recruitment-inhibition synergistic strategy enhances drug efficacy: The core-shell structured hydrogel achieves functional synergy. The outer layer loaded with SDF-1α can efficiently recruit myofibroblasts at the lesion site, enabling the drug to target the pathogenic cell population. The inner layer of high-concentration GelMA hydrogel enables sustained release of triptolide for 14 days, maintaining a stable local therapeutic concentration. This solves the problems of drug dispersion and short duration of action in traditional delivery systems, significantly improving the anti-fibrotic effect.

[0018] Significantly reduced toxicity and greatly improved safety: Through local sustained-release design, triptolide mainly exerts its effects in the lesion area, avoiding the peak blood concentration caused by systemic administration. Animal experiments have confirmed that the system has no significant toxicity to major organs such as the liver and kidneys, and does not damage normal endometrial tissue. This solves the core toxicity bottleneck in the clinical application of triptolide and provides a safety guarantee for long-term treatment.

[0019] Excellent physicochemical properties and strong clinical applicability: The hydrogel is made of GelMA material, which has good biocompatibility, injectability and in-situ gelation ability. Its mechanical strength is adapted to the retention requirements in the body, and its internal porous structure is conducive to nutrient exchange and cell infiltration. Long-term treatment can be achieved with a single local injection. It is simple to operate and minimally invasive, meeting the clinical needs of minimally invasive treatment. It is especially suitable for patients with severe endometriosis in rASRM stage III-IV with "frozen pelvis".

[0020] With broad application prospects and significant expansion value, this invention not only provides a novel treatment option for endometriosis-related fibrosis, but its core design concept can also be extended to the treatment of fibrotic diseases in other organs such as the liver, kidneys, and lungs. It provides a safe and effective local delivery platform for various diseases that rely on potent but highly toxic anti-fibrotic drugs such as tripterygium wilfordii, and has important clinical translational value and scientific significance. Attached Figure Description

[0021] Figure 1 This is a differentially expressed gene volcano diagram (including FOSB) of early disease (stage I) vs. healthy individuals in this invention.

[0022] Figure 2 This is a single-cell sequencing UMAP diagram from the present invention;

[0023] Figure 3 This is a map showing the expression localization of FOSB in a single-cell population in this invention;

[0024] Figure 4 This is a differential gene volcano diagram of FOSB-positive and FOSB-negative stromal cells in this invention;

[0025] Figure 5 This is a list of signaling pathways with differentially enriched genes in this invention;

[0026] Figure 6 This is a graph showing the verification results of siRNA knockdown of FOSB in this invention;

[0027] Figure 7 This is a Western blot (WB) image of the FOSB ubiquitination degradation induced by triptolide in this invention. Detailed Implementation

[0028] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0029] Please see Figure 1 This invention provides a triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis and its applications. This invention addresses the clinical pain point of lacking effective targeted therapy for endometriosis-related fibrosis, based on the novel discovery that the FOSB-TGFB1 axis is a core driving pathway for fibrosis.

[0030] A core-shell structured sequential release hydrogel, SDF-1α / Celastrol@Gel-seq, was constructed. Through a recruitment-targeted inhibition synergistic strategy, local sustained release of triptolide with low toxicity and high efficacy was achieved. The implementation process of this invention is described in detail below, along with specific experimental materials, procedures, and verification methods.

[0031] Materials and Instruments:

[0032] (a) Reagents and materials:

[0033] Key materials: Gelatin (Type A, derived from pigskin, Sigma-Aldrich), methacrylic anhydride (MA, Sigma-Aldrich), photoinitiator Irgacure2959 (I2959, Sigma-Aldrich), tripterygium oleoresin (Celastrol, purity ≥98%, PubChem CID:122724), recombinant human SDF-1α (PeproTech), and primary human endometrial tissue (derived from patients with laparoscopically diagnosed rASRM stage III-IV endometriosis and healthy women who underwent hysterectomy for benign diseases, approved by the ethics committee and with signed informed consent).

[0034] Cell culture reagents: DMEM / F12 medium, collagenase IV (1 mg / mL), DNase I (0.1 mg / mL), activated charcoal-adsorbed fetal bovine serum (FBS), penicillin-streptomycin antibiotics, L-glutamine (Gibco).

[0035] Experimental reagents: siRNA (FOSB-specific siRNA and negative control siRNA, Santa Cruz Biotechnology), Lipofectamine RNAiMAX transfection reagent (Invitrogen), CCK-8 kit (Beyotime), TRIzol reagent (Invitrogen), PrimeScript RT kit (TaKaRa), SYBR Premix ExTaq (TaKaRa), RIPA lysis buffer (containing protease / phosphatase inhibitor cocktail, ThermoFisher), BCA protein quantification kit (ThermoFisher), ECL chemiluminescence kit (Millipore).

[0036] Antibodies: anti-FOSB (1:1000, CellSignalingTechnology, #2844S), anti-TGFβ1 (1:1000, Abcam, ab92486), anti-FN1 (1:1000, BDBiosciences, #610077), anti-COL1 (1:1000, Novus Biologicals, NB600-408), anti-COL3 (1:1000, Abcam, ab184993), anti-αSMA (1:2000, Abcam, ab5694 Anti-p-Smad2 / 3 (1:1000, Cell Signaling Technology, #8828S), anti-GAPDH (1:5000, Proteintech, #60004-1-Ig), HRP-labeled secondary antibody (1:5000, Jackson Immuno Research), Alexa Fluor 488-labeled secondary antibody (1:500, Invitrogen), phalloidin (Alexa Fluor 488 conjugated, 1:200, Invitrogen), and DAPI (1 μg / mL, Sigma).

[0037] Animals: 8-week-old female C57BL / 6 mice (weight 18-22g, SPF grade).

[0038] Other: type I collagen from rat tail (Corning), Masson's trichrome staining kit, Sirius red staining kit (Solarbio), T-5224 (AP-1 specific inhibitor, Sigma), MG132 (proteasome inhibitor, Selleck).

[0039] (II) Major Instruments and Equipment:

[0040] Cellular and molecular biology instruments: CO2 incubator (ThermoForma 3111), clean bench, real-time quantitative PCR instrument (QuantStudio 5, Applied Biosystems), protein electrophoresis and transfer system (Bio-Rad), ChemiDoc imaging system (Bio-Rad), laser confocal microscope (Leica SP8), microplate reader (Tecan Infinite M200).

[0041] Materials characterization instruments: Scanning electron microscope (SEM, Hitachi SU8010), rheometer (TAAR2000), high performance liquid chromatograph (HPLC, Agilent 1260), ultraviolet crosslinker (365nm, 10mW / cm², Xi'an Remax).

[0042] Animal experimental instruments: small animal anesthesia machine (RWDLifeScience), stereomicroscope (OlympusSZX16), tissue embedding machine, microtome (LeicaRM2235), optical microscope (OlympusBX53). Specific Implementation

[0043] (I) Synthesis and purification of GelMA hydrogel:

[0044] Gelatin methacrylation (GelMA synthesis): Dissolve 5g of gelatin in 50mL of deionized water and stir at 37℃ until completely dissolved; add 5mL of methacrylic anhydride (MA) and stir at 37℃ in the dark for 2h; adjust the pH to 7.4 with 1mol / L NaOH to terminate the reaction; put the reaction solution into a dialysis bag (MWCO 8-14kDa) and dialyze with deionized water at 4℃ for 7 days (changing the solution 3 times a day) to remove unreacted MA; freeze-dry after dialysis to obtain white, loose GelMA powder, and store in a sealed container at -20℃.

[0045] Purity verification: The grafting rate of methacryloyl groups was detected by ¹H-NMR (Bruker AVANCE 400), and the grafting rate was controlled at 60%-70% (to ensure the photocrosslinking efficiency and biocompatibility of the hydrogel).

[0046] (II) Preparation of SDF-1α / Celastrol@Gel-seq core-shell sustained-release hydrogel:

[0047] Preparation of hydrogel precursor solution:

[0048] Outer layer precursor solution: Weigh 40 mg GelMA powder, dissolve in 1 mL PBS (pH 7.4), stir at 37 °C to dissolve, add 0.5% (w / v) photoinitiator I2959, and after complete dissolution, add recombinant human SDF-1α to a final concentration of 50 ng / mL (preliminary experiments have verified that this concentration can efficiently recruit myofibroblasts), and store in the dark for later use (outer layer: 40 mg / mL GelMA-SDF-1α).

[0049] Inner layer precursor solution: Weigh 60 mg GelMA powder, dissolve in 1 mL PBS (pH 7.4), stir at 37 °C to dissolve, and add 0.5% (w / v) I2959; dissolve triptolide in a small amount of DMSO (final DMSO concentration ≤ 0.1% to avoid cytotoxicity), add to the above solution, and the final concentration is 1 mg / mL (corresponding to an in vivo dose of 0.3 mg / 20 g mice), and store in the dark for later use (inner layer: 60 mg / mL GelMA-Celastrol).

[0050] Core-shell structure formation:

[0051] The inner layer pre-crosslinking-outer layer encapsulation-overall crosslinking method was adopted: 100 μL of inner layer precursor solution was dropped into a polytetrafluoroethylene mold and crosslinked for 30 s under 365 nm ultraviolet light (10 mW / cm²) to form a cylindrical inner layer gel core with a diameter of about 2 mm.

[0052] 200 μL of the outer layer precursor solution was slowly injected into the mold to encapsulate the inner gel core. The core-shell structured hydrogel was then crosslinked again under ultraviolet light for 60 s.

[0053] After cross-linking, wash three times with PBS to remove unreacted photoinitiator and free drug, and store at 4°C for later use (injectability verification: hydrogel diameter ≤1mm, can be successfully pushed through a 23G syringe).

[0054] (III) Physicochemical characterization of hydrogels:

[0055] Microstructure observation (SEM): After freeze-drying the hydrogel, it was sputter-coated with gold, and the core-shell structure and internal pores were observed by SEM. The results showed that the outer layer hydrogel had a porosity of about 85% (pore size 50-100 μm, which is conducive to the rapid release of SDF-1α and cell infiltration), and the inner layer hydrogel had a porosity of about 60% (pore size 20-50 μm, which enables the slow release of triptolide).

[0056] Rheological testing: The storage modulus (G') and loss modulus (G'') of the hydrogel were measured using a rheometer at 37℃ and 1Hz. The results showed that the hydrogel's G' was 1000-1500 Pa, G'' was 100-200 Pa, and G' / G'' > 10, indicating good mechanical stability and the ability to remain in situ in vivo for at least 14 days.

[0057] In vitro drug release profile (HPLC): The hydrogel was immersed in 1 mL of PBS (containing 0.1% Tween 80, simulating body fluid), and the mixture was shaken at 37℃ and 100 rpm. Samples of 100 μL were taken at different time points (0.5, 1, 2, 3, 7, 10, and 14 days), and an equal volume of fresh PBS was added. Drug concentration was detected by HPLC (detection conditions: C18 column, mobile phase methanol-water = 85:15, flow rate 1 mL / min, detection wavelength 254 nm). Results showed that SDF-1α achieved a release rate of over 80% within 24 hours (rapid cell recruitment), and triptolide achieved a cumulative release rate of 85% after 14 days. The release curve conformed to the Higuchi equation, achieving sustained release (local steady-state concentration maintained at 4 μM).

[0058] Biocompatibility verification (CCK-8 assay): The hydrogel extract (soaked at 37℃ for 24 h, diluted 1, 2, and 4 times) was co-cultured with nESCs for 48 h, and cell viability was detected. The results showed that the cell viability of each dilution group was >90%, indicating that the hydrogel was non-cytotoxic.

[0059] (iv) In vitro biological function verification:

[0060] Isolation and culture of primary endometrial stromal cells (ESCs):

[0061] Tissue processing: The endometrial tissue was cut into 1 mm³, added to DMEM / F12 medium containing collagenase IV (1 mg / mL) and DNase I (0.1 mg / mL), and digested at 37°C with shaking for 60-90 min;

[0062] Cell isolation: The digestion solution was filtered through a 70μm nylon filter, the filtrate was collected, centrifuged at 1000rpm for 5min, and the precipitate was washed twice with PBS; the epithelial cells were removed by differential centrifugation (500rpm, 5min), and stromal cells were collected;

[0063] Culture and passage: Cells were seeded in culture flasks and added to DMEM / F12 medium containing 10% activated charcoal-adsorbed FBS, 1% penicillin and antibiotics, and 1% L-glutamine. The cells were cultured at 37°C and 5% CO2. The medium was changed every 48-72 hours. Cells from passages 3-6 were used for experiments (to maintain phenotypic stability).

[0064] Cytotoxicity assay (CCK-8 assay):

[0065] Grouping: nESCs and eESCs were seeded in 96-well plates (5×10³ cells / well), and after culturing for 24 h, different concentrations of triptolide (0, 2, 4, 8, 10 μM) or hydrogel extract were added, and cultured for another 48 h.

[0066] Detection: 10 μL CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. The OD value at 450 nm was measured using a microplate reader, and the cell viability was calculated. The results showed that when the triptolide concentration was ≤4 μM, the viability of both nESCs and eESCs was >90%; at 8 μM, the viability of nESCs decreased to 75%, therefore 4 μM was determined to be the working concentration for in vitro experiments.

[0067] FOSB Function Validation (siRNA Knockdown Experiment):

[0068] siRNA transfection: eESCs were seeded in 6-well plates (2 × 10⁻⁶). 5 (cells / well), cultured to 60-70% confluence, add 50 nMFOSB-siRNA or negative control siRNA, LipofectamineRNAiMAX-mediated transfection, replace with fresh medium after 6 h;

[0069] Detection indicators: 48 h after transfection, the expression of FOSB, TGFB1, and fibrosis markers (FN1, COL1, COL3, αSMA) was detected by Western blot and qRT-PCR; F-actin stress fiber formation was detected by immunofluorescence; cell contraction ability was detected by collagen gel shrinkage assay; and collagen secretion was detected by a hydroxyproline kit. Results showed that after FOSB knockdown, TGFB1 mRNA and protein expression decreased by more than 60%, fibrosis marker expression was significantly downregulated, stress fibers decreased, collagen contraction rate decreased by 40%, and hydroxyproline secretion decreased by 50%, verifying the pro-fibrotic effect of the FOSB-TGFB1 axis.

[0070] In vitro anti-fibrotic efficacy validation of triptolide:

[0071] Grouping: eESCs were divided into control group (PBS), T-5224 group (4μM), free Celastrol group (4μM), and hydrogel-released Celastrol group (4μM, simulated hydrogel sustained-release solution).

[0072] Testing indicators:

[0073] Western blot: After treatment for 24, 48, and 72 hours, the expression of FOSB, TGFB1, p-Smad2 / 3, and fibrosis markers was detected. The results showed that the degradation rate of FOSB protein in the hydrogel release group reached 70%, and the downregulation of fibrosis markers was significantly better than that in the T-5224 group (P < 0.01).

[0074] Immunofluorescence: After 24 hours of treatment, phalloidin staining showed a 65% decrease in fluorescence intensity of stress fibers in the hydrogel release group;

[0075] Collagen gel shrinkage experiment: eESCs were mixed with type I collagen (5×10⁻⁶). 5 (cells / mL), after gel formation, the cells were divided into groups for treatment. The gel area was measured after 72 hours. The shrinkage rate of the hydrogel release group was only 30% of that of the control group.

[0076] Hydroxyproline detection: After 48 h of treatment, the secretion of hydroxyproline in the hydrogel release group decreased by 55% (P < 0.001).

[0077] SDF-1α cell recruitment assay (Transwell migration assay)

[0078] Add culture medium or hydrogel outer layer release solution containing different concentrations of SDF-1α (0, 25, 50, 100 ng / mL) to the lower chamber, and inoculate eESCs (1×10⁻⁶) into the upper chamber. 5 (cells / well), cultured for 24 hours;

[0079] After crystal violet staining, the number of cells that migrated to the lower chamber was counted. The results showed that the number of migrating cells in the 50 ng / mL SDF-1α group was 2.5 times that of the control group, and the recruitment effect of the hydrogel outer layer release solution was comparable to that of 50 ng / mL SDF-1α, thus verifying the myofibroblast recruitment effect of SDF-1α.

[0080] In vivo biological function verification:

[0081] Establishment of a mouse model of endometriosis:

[0082] After the donor mouse was euthanized, the uterine horn was removed and the tissue was cut into 1 mm³ fragments.

[0083] After being anesthetized with isoflurane, recipient mice underwent an abdominal incision to expose the peritoneal wall. Five tissue fragments were then evenly transplanted onto the peritoneal wall, and the incision was sutured. The sham-operated group underwent only incision suturing without tissue transplantation.

[0084] After two weeks of routine feeding following the surgery, lesion formation was confirmed (ectopic lesions with a diameter ≥3mm were visible under a stereomicroscope).

[0085] Animal grouping and administration:

[0086] Grouping: Mice that successfully modeled the mice were randomly divided into 4 groups (n=8 / group):

[0087] ① Blank hydrogel group;

[0088] ② Free Celastrol group (0.3 mg / 20 g mice, intraperitoneal injection);

[0089] ③SDF-1α / Celastrol@Gel-seq group (0.3mg Celastrol / 20g mouse, injected around the peritoneal lesion);

[0090] ④Sham surgery group;

[0091] Administration method: single injection. Monitor mouse weight and record diet and activity status every 3 days after administration.

[0092] In vivo efficacy evaluation:

[0093] Sample collection: Four weeks after drug administration, mice were sacrificed, ectopic lesions were dissected, weighed, and photographed; major organs such as liver, kidney, heart, lung, and spleen, as well as normal endometrial tissue, were collected for histological analysis.

[0094] Morphological analysis of lesions: Lesion weight statistics showed that the lesion weight in the SDF-1α / Celastrol@Gel-seq group was only 40% of that in the blank hydrogel group (P<0.001), which was significantly lower than that in the free Celastrol group (P<0.01).

[0095] Histological staining:

[0096] H&E staining: In the SDF-1α / Celastrol@Gel-seq group, stromal proliferation of lesions was reduced, and the tissue structure tended to be normal.

[0097] Masson trichrome staining and Sirius red staining: The SDF-1α / Celastrol@Gel-seq group showed a 60% reduction in collagen deposition area and a significant decrease in fibrosis.

[0098] Immunohistochemistry: The expression of FOSB, αSMA and Ki-67 was detected. H-score analysis showed that the FOSB positive cell rate was reduced by 70%, αSMA expression was reduced by 65% ​​and Ki-67 positive cell rate was reduced by 50% in the SDF-1α / Celastrol@Gel-seq group (P<0.001).

[0099] In vivo safety evaluation:

[0100] Weight monitoring: The weight of mice in the SDF-1α / Celastrol@Gel-seq group remained stable throughout the process with no significant decrease; the weight of mice in the free Celastrol group decreased by 15% (P<0.01).

[0101] Organ histological examination: H&E staining of liver and kidney tissues showed that the free Celastrol group had hepatocyte edema and renal tubular damage, while the SDF-1α / Celastrol@Gel-seq group had no obvious pathological damage in any organ.

[0102] Effects on normal endometrium: Immunohistochemistry showed no significant change in αSMA expression in normal endometrial tissue of the SDF-1α / Celastrol@Gel-seq group, indicating no significant off-target toxicity.

[0103] Mechanism of action verification experiment:

[0104] Molecular docking verification: AutoDockVina was used to dock Celastrol with FOSB. A search grid was constructed with the Lys-219 site of FOSB as the center. The results showed that the binding energy of Celastrol and FOSB was -7.0 kcal / mol, forming stable hydrogen bonds and hydrophobic interactions.

[0105] Cell thermal displacement assay (CETSA): eESCs were treated with 4 μM Celastrol for 24 h, followed by gradient temperature increases (37, 42, 47, 52, 57 °C). Western blot was used to detect the stability of FOSB protein. The results showed that the FOSB protein in the Celastrol-treated group still had 30% solubility at 52 °C, which was significantly lower than that in the control group (65%), verifying the direct binding of Celastrol to FOSB.

[0106] Ubiquitination assay (Co-IP): eESCs were treated with Celastrol (4 μM) or Celastrol + MG132 (10 μM) for 24 h. FOSB protein was immunoprecipitated with anti-FOSB antibody, and the ubiquitination level was detected by Western blot. The results showed that the FOSB ubiquitination band was significantly enhanced in the Celastrol group, and MG132 could reverse this effect, verifying that Celastrol degrades FOSB through the ubiquitin-proteasome pathway.

[0107] Validation of the TGFB1 / Smad pathway: Western blot was used to detect the expression of TGFB1 and p-Smad2 / 3 in eESCs and lesion tissues. The results showed that TGFB1 expression was reduced by 60% and p-Smad2 / 3 phosphorylation level was reduced by 55% in the SDF-1α / Celastrol@Gel-seq group, proving that Celastrol inhibits the activation of the TGFB1 / Smad pathway by degrading FOSB.

[0108] Experimental Results and Analysis:

[0109] Physicochemical properties of hydrogels: Core-shell structured SDF-1α / Celastrol@Gel-seq was successfully prepared, exhibiting good injectability, mechanical stability and biocompatibility, achieving rapid release of SDF-1α and sustained release of Celastrol for 14 days (local steady-state concentration of 4 μM).

[0110] In vitro experiments: Celastrol degrades FOSB through ubiquitination, inhibits the TGFB1 / Smad pathway, significantly downregulates the expression of fibrosis markers, inhibits cell contraction and collagen secretion, and the antifibrotic effect of the hydrogel sustained-release system is better than that of the free drug and T-5224, with no obvious cytotoxicity at a concentration of 4 μM.

[0111] In vivo experiments: Local injection of SDF-1α / Celastrol@Gel-seq significantly reduced the volume and weight of ectopic lesions, decreased collagen deposition, downregulated FOSB and αSMA expression, and caused no liver or kidney damage. Its safety profile was significantly better than that of free Celastrol.

[0112] Mechanism validation: Celastrol targets and binds to FOSB and promotes its ubiquitination and degradation, blocking the FOSB-TGFB1 axis, thereby inhibiting stromal-to-myofibroblast transformation and fibrosis.

[0113] This embodiment details the preparation process, physicochemical properties, in vitro antifibrotic activity, and in vivo therapeutic effects of the SDF-1α / Celastrol@Gel-seq core-shell sustained-release hydrogel. This hydrogel achieves local sustained release of triptolide with low toxicity and high efficacy through a synergistic "recruitment-targeted inhibition" strategy, providing a novel treatment option for endometriosis-related fibrosis and offering a reference for the local treatment of other fibrotic diseases.

[0114] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axes, characterized in that, The sustained-release hydrogel is a core-shell structured sequential release hydrogel, named SDF-1α / Celastrol@Gel-seq; The core-shell structure comprises an outer layer of low-concentration gelatin methacryl hydrogel and an inner layer of high-concentration gelatin methacryl hydrogel. The outer layer of low-concentration GelMA hydrogel is loaded with stromal cell-derived factor-1α (SDF-1α), and the inner layer of high-concentration GelMA hydrogel is loaded with triptolide. The concentration of GelMA in the outer layer was 40 mg / mL, and the concentration of GelMA in the inner layer was 60 mg / mL.

2. The triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis according to claim 1, characterized in that, The loading of triptolide is the hydrogel dosage corresponding to 0.05-0.3 mg / 20g animal body weight, preferably the hydrogel dosage corresponding to 0.3 mg / 20g animal body weight.

3. The triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis according to claim 1, characterized in that, The loading concentration of SDF-1α is the concentration that can effectively recruit endometriosis-related myofibroblasts, and the release cycle of triptolide is 14 days, with the local steady-state concentration maintained at 4 μM.

4. The triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis according to claim 1, characterized in that, The sustained-release hydrogel has a gelation time that meets the requirements for rapid shaping after in vivo injection, and its mechanical strength enables in-situ retention in vivo. It has a porous internal structure, and its porosity is conducive to nutrient exchange, cell infiltration, and sustained drug release.

5. The use of the triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis as described in any one of claims 1-4 in the preparation of a medicament for treating endometriosis.

6. The application according to claim 5, characterized in that, The term "endometriosis" refers to endometriosis with fibrosis, including but not limited to the revised American Society for Reproductive Medicine (ASRM) stage I-IV endometriosis, and is particularly applicable to severe endometriosis of rASRM stage III-IV with frozen pelvic presentation.

7. The application according to claim 5, characterized in that, The drug is administered via local injection at the lesion site, preferably via injection around the lesion in the abdominal cavity, with a single-dose administration frequency, and continues to exert its therapeutic effect for more than 14 days.

8. The application according to claim 5, characterized in that, The drug exerts its therapeutic effect through the following mechanism: the outer hydrogel releases SDF-1α to recruit myofibroblasts at the lesion site, and the inner hydrogel releases triptolide which binds directly to FOSB protein, inducing ubiquitination-mediated degradation of FOSB, thereby inhibiting the transcriptional activation of TGFB1 by FOSB, blocking the TGF-β / Smad signaling pathway, reversing the activated phenotype of myofibroblasts, and reducing extracellular matrix deposition and fibrosis.

9. The use of the triptolide sustained-release hydrogel targeting the FOSB and TGFB1 axis as described in any one of claims 1-4 in the preparation of a drug for inhibiting endometriosis-related fibrosis.

10. The application according to claim 9, characterized in that, The drug can reduce collagen deposition in endometriosis lesions, reduce the expression of fibrosis markers, inhibit lesion contraction and angiogenesis, and at the same time reduce the systemic toxicity of triptolide, avoiding damage to the liver, kidneys and normal endometrial tissue.

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