Use of forsythoside a in the preparation of a drug for preventing or treating sepsis-related encephalopathy

Abstract

The application belongs to the technical field of biological medicine, and relates to application of forrestin in preparation of a medicine for preventing or treating sepsis-related encephalopathy. The active ingredient combines with STAT3 protein and inhibits phosphorylation of the STAT3 protein, thereby blocking pyroptosis of microglial cells mediated by an NLRP3 / Caspase-1 / GSDMD signal path, effectively inhibiting a hippocampal neuroinflammatory response, reducing levels of proinflammatory factors such as IL-6, TNF-alpha, IL-1beta and IL-18, protecting synaptic ultrastructure integrity of neurons, and significantly improving cognitive dysfunction and improving survival rate of model animals. The application provides a clear action target and a mechanism-clear candidate drug for prevention or treatment of sepsis-related encephalopathy, and has important clinical application value and development prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of forsythoside A in the preparation of drugs for the prevention or treatment of sepsis-related encephalopathy. Background Technology

[0002] Sepsis is a critical clinical syndrome characterized by an uncontrolled inflammatory response triggered by pathogen infection, leading to life-threatening multi-organ dysfunction. In the progression of sepsis-related multi-organ damage, the central nervous system is a key target organ that is highly susceptible to early involvement, with the corresponding characteristic lesion being sepsis-associated encephalopathy (SAE). SAE is a diffuse brain dysfunction secondary to systemic infection without direct intracranial pathogen invasion, exhibiting significant clinical heterogeneity: mild cases are characterized by delirium and short-term cognitive impairment, while severe cases can rapidly progress to persistent deep coma. This complication occurs in approximately 30%–50% of sepsis patients. SAE is not only closely associated with increased acute-phase mortality but is also a major cause of long-term cognitive impairment and decreased quality of life in survivors. However, the pathophysiological mechanisms of SAE are complex, involving cross-system interaction and regulation of peripheral inflammatory cascade activation and central brain parenchymal damage. Current technologies lack specific therapeutic drugs targeting the core pathogenesis pathways, and clinical treatment mainly relies on supportive therapies, which are insufficient to effectively intervene in the core pathological processes. Therefore, developing novel treatment strategies that can target and intervene in the pathological process of SAE is a pressing technical challenge that needs to be overcome in this field. Summary of the Invention

[0003] This invention provides the application of forsythoside A in the preparation of drugs for the prevention or treatment of sepsis-related encephalopathy, in order to solve the problem of the lack of specific therapeutic drugs targeting the core pathogenesis pathway of sepsis-related encephalopathy in the prior art.

[0004] This invention, through the integration of bioinformatics prediction and surface plasmon resonance (SPR) validation, reveals that phillygenin (PHI) can directly and physically bind to STAT3 protein with high affinity and inhibit its phosphorylation. Clinically relevant multispecies sepsis (CLP) models have confirmed that PHI not only significantly improves the survival rate of SAE model mice, but also effectively protects neuronal ultrastructure and reverses cognitive deficits by inhibiting STAT3 phosphorylation in microglia of the hippocampus (especially the CA3 subregion) and activation of the NLRP3 / GSDMD pyroptosis pathway. This invention clarifies the mechanism of action of phillygenin in treating SAE, providing new candidate compounds and theoretical basis for the development of specific targeted drugs for this disease.

[0005] In summary, the technical solution provided by this invention is as follows: In a first aspect, the present invention provides the use of forsythoside A in the preparation of drugs for the prevention or treatment of sepsis-related encephalopathy.

[0006] In conjunction with the first aspect of the invention, in some embodiments, the forsythoside A works by inhibiting NLRP3 inflammasome activation and / or inhibiting the STAT3 / NLRP3 signaling pathway.

[0007] Furthermore, the forsythoside A inhibits NLRP3 inflammasome activation and / or Caspase-1 / GSDMD-mediated pyroptosis by binding to STAT3 protein and inhibiting its phosphorylation.

[0008] In conjunction with the first aspect of the invention, in some embodiments, the sepsis-associated encephalopathy is accompanied by one or more of the following: cognitive impairment, hippocampal neuronal ultrastructural damage, or elevated levels of inflammatory factors.

[0009] In conjunction with the first aspect of the present invention, in some embodiments, the pathological manifestations of the sepsis-associated encephalopathy include one or more of the following: loss of neurons in the CA3 region of the hippocampus, Nissl body dissolution, thinning of the postsynaptic compact area, and widening of the synaptic cleft.

[0010] In conjunction with the first aspect of the invention, in some embodiments, the drug is used in at least one of the following applications: (1) Inhibits STAT3 phosphorylation in hippocampal microglia; (2) Reduce the levels of inflammatory factors IL-6, TNF-α, IL-1β or IL-18 in hippocampal tissue; (3) Protect the integrity of the ultrastructure of neuronal synapses; (4) Improve the survival rate of subjects.

[0011] In conjunction with the first aspect of the present invention, in some embodiments, the dosage form of the drug is an injection or an oral preparation.

[0012] In a second aspect, the present invention provides the use of a combination of forsythoside A and at least one other drug for treating sepsis in the preparation of a drug for the prevention or treatment of sepsis-related encephalopathy.

[0013] In conjunction with a second aspect of the invention, in some embodiments, the other drugs for treating sepsis include one or more of antibiotics, vasoactive drugs, or NLRP3 inhibitors.

[0014] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of forsythoside A and a pharmaceutically acceptable carrier, said pharmaceutical composition for the prevention or treatment of sepsis-associated encephalopathy.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a novel use of forsythoside in the preparation of drugs for treating sepsis-related encephalopathy. Surface plasmon resonance studies have confirmed that forsythoside can bind to STAT3 protein and inhibit its phosphorylation, thereby blocking the NLRP3 / GSDMD-mediated microglial pyroptosis pathway. In vitro and in vivo experiments show that this compound can significantly improve the survival rate of sepsis model mice, effectively protect the ultrastructural integrity of hippocampal neurons and reverse cognitive dysfunction, and has low cytotoxicity. It provides a novel, safe, and effective treatment strategy and candidate drug with a clear mechanism of action for sepsis-related encephalopathy, which lacks specific targeted drugs in clinical practice, and has significant neuroprotective value and industrialization prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Target binding verification. A represents the molecular docking binding mode between PHI and STAT3; B represents SPR verification of the physical binding between PHI and STAT3.

[0018] Figure 2 In vivo pharmacodynamic evaluation. A shows that PHI improved the survival rate of CLP-induced sepsis mice; B shows that PHI intervention reduced the sepsis severity score in CLP mice; C shows representative swimming trajectories in the Morris water maze spatial exploration experiment for each group of mice; D shows that PHI intervention shortened the escape latency in the Morris water maze; E shows the time spent in the target quadrant of the Morris water maze spatial exploration for each group of mice; F shows the number of times each group of mice crossed the original platform in the Morris water maze; G shows the heatmap of the Y-maze exploration activity trajectory for each group of mice; H shows the spontaneous alternation rate in the Y-maze for each group of mice.

[0019] Figure 3Hippocampal pathology and ultrastructural protection. A shows PHI-induced improvement of CLP-induced hippocampal tissue pathological morphological damage in mice; B shows PHI-reduced Nissl body damage in the hippocampus of CLP-induced mice; C shows quantitative analysis of the number of surviving neurons in the CA3 region of Figure A; D shows quantitative analysis of the number of Nissl bodies in the CA3 region of the hippocampus in Figure B; E shows that PHI intervention can improve hippocampal synaptic ultrastructural damage, where SC: Synaptic cleft; PSD: Postsynaptic density; AZ: Active zone; SV: Synaptic vesicle; F shows quantitative statistics of the thickness of the postsynaptic density (PSD) in the hippocampus of each group of mice; G shows quantitative statistics of the width of the hippocampal synaptic cleft in each group of mice.

[0020] Figure 4 In vivo mechanism of action verification. A shows that PHI reduces IL-6 protein levels in the hippocampus of CLP mice; B shows that PHI reduces TNF-α protein levels in the hippocampus of CLP mice; C shows that PHI reduces IL-18 protein levels in the hippocampus of CLP mice; D shows that PHI reduces IL-1β protein levels in the hippocampus of CLP mice; EF shows the Western blot detection of STAT3 phosphorylation levels in the hippocampus, where E is a representative image of STAT3 and p-STAT3 protein levels detected by Western blot, and F is the quantitative analysis result of p-STAT3 protein levels; G shows the p-STAT3 protein level in mouse hippocampal microglia. STAT3 immunofluorescence representative image; H represents the quantitative analysis of p-STAT3 fluorescence intensity in Figure G; I represents the stat3 mRNA expression level in mouse hippocampus tissue detected by qRT-PCR; JM shows the expression levels of NLRP3 / Caspase-1 / GSDMD pyroptosis-related proteins in mouse hippocampus tissue detected by Western blot in different treatment groups, where K represents the quantitative analysis of relative NLRP3 expression, L represents the quantitative analysis of relative Caspase-1 p20 expression, and M represents the quantitative analysis of relative GSDMD-NT expression; N represents the Nlrp3 mRNA expression level in mouse hippocampus tissue detected by qRT-PCR in different treatment groups; OP represents the immunofluorescence detection of GSDMD-NT expression in microglia in the CA3 region of mouse hippocampus, where O is the representative immunofluorescence image of GSDMD-NT and P represents the quantitative analysis of GSDMD-NT fluorescence intensity.

[0021] Figure 5In vitro cell safety and inflammation suppression. A shows the effect of PHI on BV2 microglia viability; B shows the effect of PHI combined with inflammatory stimulation on BV2 microglia viability; C and D show Western blot detection of synapse-related protein expression, with C being a representative Western blot image, D being quantitative analysis of PSD95 relative protein expression, and E being quantitative analysis of SYN relative protein expression; F and G show ELISA detection of inflammatory factor levels in BV2 microglia culture supernatant, with F showing IL-6 expression level in cell culture supernatant; G shows TNF expression in cell culture supernatant. α represents the expression level; H represents the expression level of IL-1β in the cell culture supernatant; I represents the expression level of IL-18 in the cell culture supernatant.

[0022] Figure 6 In vitro cell mechanism validation. A and B show the phosphorylation level of STAT3 in BV2 microglia detected by Western blot, where A is a representative image of STAT3 and p-STAT3 protein levels detected by Western blot, and B is the quantitative analysis of p-STAT3 protein levels; C and D show the expression of p-STAT3 in BV2 microglia detected by immunofluorescence, where C is a representative image of p-STAT3 immunofluorescence; D is the quantitative analysis of p-STAT3 fluorescence intensity; E shows the expression level of Stat3 mRNA in BV2 microglia detected by qRT-PCR; FI shows the expression of NLRP3 / Caspase-1 / GSDMD pyroptosis-related proteins in BV2 microglia detected by Western blot, where F is a representative image of Western blot, G is the quantitative analysis of relative expression of NLRP3, H is the quantitative analysis of relative expression of Caspase-1 p20, and I is the quantitative analysis of relative expression of GSDMD-NT; J shows the detection of Nlrp3 in BV2 microglia by qRT-PCR. mRNA expression level; KL shows the immunofluorescence detection of GSDMD-NT expression in BV2 microglia, where K is a representative immunofluorescence image of GSDMD-NT and L is the quantitative analysis of GSDMD-NT fluorescence intensity. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] In particular, in the cell grouping of this invention, the LPS model group is equivalent to the LPS / Nig model group, the LPS + PHI-L group is equivalent to the LPS / Nig + PHI-L group, the LPS + PHI-M group is equivalent to the LPS / Nig + PHI-M group, and the LPS + PHI-H group is equivalent to the LPS / Nig + PHI-H group, where Nig represents Nigericin.

[0025] Neuroinflammation is a core pathological link driving the occurrence and development of SAE, with abnormal immune responses mediated by microglia being particularly crucial. In sepsis, excessive microglia activation leads to pyroptosis via the NLRP3 / Caspase-1 / GSDMD pathway, releasing large amounts of pro-inflammatory mediators, thereby disrupting synaptic homeostasis and causing cognitive impairment. STAT3, as an upstream transcriptional hub, not only drives the neurotoxic activation of microglia but is also a necessary prerequisite for the initiation of the NLRP3 inflammasome and subsequent pyroptosis cascade. Therefore, targeting and regulating the STAT3 / NLRP3 signaling axis to inhibit microglia pyroptosis constitutes a potential strategy for treating SAE.

[0026] While forsythoside A (PHI) is known to possess broad-spectrum anti-inflammatory activity, its specific role and mechanism in SAE (subarachnoid hemorrhage) have remained unclear. In this study, the inventors discovered that PHI can act as a direct ligand for STAT3, physically inhibiting its phosphorylation activation and thus blocking the downstream pyroptosis pathway. To confirm the effectiveness and scientific validity of this technique, a multi-level experimental system was used for systematic verification: First, molecular docking prediction and surface plasmon resonance (SPR) techniques were used to confirm the direct binding ability of PHI to STAT3; second, a CLP-induced SAE mouse model was constructed, and the effects of PHI on survival rate, cognitive function, and hippocampal pathological structure were evaluated through preoperative administration, and its regulatory mechanism on the STAT3 / NLRP3 / GSDMD pathway was analyzed at the tissue level; finally, in vitro microglia and neuronal models were used to further eliminate interference from complex in vivo factors and verify the cellular safety and mechanism specificity of the drug.

[0027] Based on this, the technical solution provided by the present invention is as follows: In a first aspect, the present invention provides the use of forsythoside A in the preparation of drugs for the prevention or treatment of sepsis-related encephalopathy.

[0028] In conjunction with the first aspect of the invention, in some embodiments, the forsythoside A exerts its effect by inhibiting NLRP3 inflammasome activation and / or inhibiting the STAT3 / NLRP3 signaling pathway.

[0029] Furthermore, the forsythoside A inhibits NLRP3 inflammasome activation and / or Caspase-1 / GSDMD-mediated pyroptosis by binding to STAT3 protein and inhibiting its phosphorylation.

[0030] Example 1 demonstrates through molecular docking simulation and surface plasmon resonance (SPR) experiments that forsythoside can directly bind to STAT3 protein with μM-level affinity. In in vivo mechanism verification (Example 4), the level of p-STAT3 (Tyr705) in the hippocampus of CLP-induced SAE model mice was significantly increased, and phosphorylated STAT3 was mainly located in the nuclei of activated microglia in the CA3 region of the hippocampus. Forsythoside (50 mg / kg) intervention significantly inhibited STAT3 phosphorylation and further downregulated the expression of NLRP3, Cleaved Caspase-1 (p20), and the pyroptosis executive protein GSDMD-NT, while also reducing the levels of downstream pro-inflammatory factors IL-6, TNF-α, IL-1β, and IL-18. In in vitro cellular mechanism verification (Example 6), LPS-stimulated BV2 microglia also showed enhanced STAT3 phosphorylation and activation of the NLRP3 / GSDMD pyroptosis pathway. Forsythoside pretreatment could inhibit these molecular events in a concentration-dependent manner, and immunofluorescence results also showed a significant reduction in p-STAT3 nuclear translocation and GSDMD-NT accumulation in microglia. The above results indicate that forsythoside A, by binding to STAT3 protein and inhibiting its phosphorylation, blocks NLRP3 inflammasome activation and Caspase-1 / GSDMD-mediated pyroptosis, thereby inhibiting the neurotoxic inflammatory response of microglia and ultimately protecting the ultrastructure of hippocampal neuronal synapses and reversing cognitive dysfunction.

[0031] In conjunction with the first aspect of the invention, in some embodiments, the sepsis-associated encephalopathy is accompanied by one or more of the following: cognitive impairment, hippocampal neuronal ultrastructural damage, or elevated levels of inflammatory factors.

[0032] In conjunction with the first aspect of the present invention, in some embodiments, the pathological manifestations of the sepsis-associated encephalopathy include one or more of the following: loss of neurons in the CA3 region of the hippocampus, Nissl body dissolution, thinning of the postsynaptic compact area, and widening of the synaptic cleft.

[0033] Morris water maze experiment in Example 2 ( Figure 2 CF showed that the escape latency of mice in the CLP model group was significantly prolonged, and the time spent in the target quadrant and the number of platform crossings were significantly reduced. The Y-maze test (…) Figure 2 GH also confirmed that its spontaneous alternation rate was significantly reduced, indicating that SAE mice have severe spatial learning and working memory impairments.

[0034] Meanwhile, the pathological examination in Example 3 ( Figure 3 Further analysis by AD revealed that the CLP group showed significant neuronal disorder, cell body shrinkage, and nuclear condensation in the CA3 region of the hippocampus. The number of surviving neurons was significantly reduced, and the density of Nissl bodies also decreased significantly, suggesting that the neuronal protein synthesis function was impaired.

[0035] At the ultrastructural level, transmission electron microscopy observation ( Figure 3 EG showed that the CLP group exhibited significantly thinner postsynaptic dense region (PSD) thickness, abnormally widened synaptic cleft, and marked structural damage including mitochondrial swelling and cristae breakage. Furthermore, the ELISA detection in Example 4 (…) Figure 4 AD confirmed that the levels of pro-inflammatory factors IL-6, TNF-α, IL-1β and pyroptosis-related factor IL-18 were significantly increased in the hippocampus of the CLP group.

[0036] In conclusion, forsythoside A can significantly improve cognitive deficits, neuronal pathological damage, synaptic ultrastructural disruption, and abnormally elevated inflammatory factors caused by SAE, confirming the application value of forsythoside A in the preparation of drugs for the prevention or treatment of SAE.

[0037] In conjunction with the first aspect of the invention, in some embodiments, the drug is used in at least one of the following applications: (1) Inhibits STAT3 phosphorylation in hippocampal microglia; (2) Reduce the levels of inflammatory factors IL-6, TNF-α, IL-1β or IL-18 in hippocampal tissue; (3) Protect the integrity of the ultrastructure of neuronal synapses; (4) Improve the survival rate of subjects.

[0038] In a CLP-induced SAE mouse model, Kaplan-Meier survival curve analysis ( Figure 2 A) showed that the survival rate of mice in the model group was only 40% 7 days after surgery, while the survival rate of the high-dose forsythia suspensa extract (50 mg / kg) pretreatment group was significantly increased to 80%, with a statistically significant difference between the two groups (P<0.05). In addition, the sepsis severity score ( Figure 2 B) also showed that the scores of the medium- and high-dose groups of forsythoside A (25 mg / kg and 50 mg / kg) were significantly lower than those of the model group, indicating that forsythoside A not only prolongs survival time but also effectively alleviates the severity of systemic sepsis. These results confirm that forsythoside A can effectively improve the survival rate of subjects by improving the overall prognosis of SAE.

[0039] In conjunction with the first aspect of the present invention, in some embodiments, the dosage form of the drug is an injection or an oral preparation.

[0040] In Example 2, mice were administered the drug via gavage at an effective dose of 12.5–50 mg / kg, using physiological saline with a final concentration of DMSO < 0.1%. In Example 5, cells were administered the drug via suspension drip at an effective dose of 25–100 μg / mL, using PBS solution with a final concentration of DMSO < 0.1%.

[0041] In a second aspect, the present invention provides the use of a combination of forsythoside A and at least one other drug for treating sepsis in the preparation of a drug for the prevention or treatment of sepsis-related encephalopathy.

[0042] In conjunction with a second aspect of the invention, in some embodiments, the other treatment drugs for sepsis include one or more of antibiotics, vasoactive drugs, or NLRP3 inhibitors.

[0043] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of forsythoside A and a pharmaceutically acceptable carrier, said pharmaceutical composition for the prevention or treatment of sepsis-associated encephalopathy.

[0044] As is well known to those skilled in the art, the cecal ligation and perforation (CLP) model is the internationally recognized gold standard animal model for simulating the pathophysiological process of human sepsis, exhibiting a high degree of similarity to human sepsis in terms of inflammatory cascade responses, organ dysfunction, and immune paralysis. Furthermore, the STAT3 / NLRP3 signaling pathway targeted in this application and its mediated microglial pyroptosis mechanism are key components of the mammalian innate immune system. Existing scientific research has shown that the key phosphorylation site of the STAT3 protein (Tyr705), the NLRP3 inflammasome assembly mechanism, and the pyroptosis executive protein GSDMD exhibit significant sequence homology and functional conservation of core mechanisms between mice and humans.

[0045] It should be noted that although there are certain differences between animal models and human clinical practice, the CLP model remains one of the important reference models for evaluating the efficacy of antiseptic drugs. Existing studies have shown that indicators such as improved survival rates, histopathological protection, and reduced levels of inflammatory factors observed in this model can, to some extent, reflect the potential clinical translational value of drugs.

[0046] The experimental data in this application show that, in the CLP model, forsythoside A can significantly improve the survival rate of test animals, reduce hippocampal tissue pathological damage, and reduce the levels of key inflammatory factors such as IL-6, TNF-α and IL-1β. These indicators are all biologically significant endpoints or surrogate indicators in the evaluation of sepsis and its complications.

[0047] In summary, considering the recognized effectiveness of the CLP model, the cross-species conservation of the STAT3 / NLRP3 pathway, and the molecular evidence of direct binding between forsythoside and STAT3 protein confirmed by surface plasmon resonance (SPR) experiments, those skilled in the art have reason to reasonably expect that forsythoside can exert a therapeutic effect in human subjects through a similar mechanism. Therefore, the animal experimental data of this application are sufficient to support the application of forsythoside in the preparation of drugs for the prevention or treatment of sepsis-related encephalopathy in humans.

[0048] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture and transfection, total mRNA and total protein extraction, protein sample preparation, molecular biological detection (such as ELISA, Western blot, immunofluorescence), cell function experiments (such as cell proliferation), animal model construction, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were purchased commercially and met the generally accepted quality standards in the art. Experimental animals were purchased commercially and then fed and modeled according to standard operating procedures in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental and control groups were set up according to the grouping methods commonly used in the art.

[0049] The mouse microglia BV2 and mouse hippocampal neuron cell lines HT22 used in the following examples were obtained from Seven Innovation (Beijing) Biotechnology Co., Ltd., and all cells were negative for mycoplasma. C57BL / 6 male mice were purchased from Hubei Provincial Experimental Animal Center and housed in an SPF-grade environment. All animal experiments were approved by the Animal Ethics Committee of Wuhan University (Approval No.: ZN2026085).

[0050] In this invention, all data collection and processing in the statistical analysis section were performed using a blinded method, and the quantitative data are expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. In the attached figures, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical difference (P ≥ 0.05).

[0051] Table 1. RT-qPCR primer sequence information

[0052] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0053] Example 1: Verification of the binding ability of PHI to STAT3 protein 1. Experimental Objective This embodiment aims to verify, through molecular docking simulation and surface plasmon resonance (SPR) biophysical technology, whether there is a direct physical binding interaction between forsythoside and signal transduction and transcription activator 3 (STAT3) protein, and to determine its binding affinity, so as to provide molecular-level evidence for the confirmation of the drug target of the present invention.

[0054] 2. Experimental Materials Test drug: Forsythoside A, purity ≥98.55%, purchased from MedChemExpress, catalog number HY-N0483.

[0055] Target protein: Recombinant human STAT3 protein (Proteintech, Ag0360).

[0056] Main instruments: Biacore, surface plasmon resonance spectrometer.

[0057] Molecular docking software: AutoDock Vina 1.1.2.

[0058] Chip: CM5 sensor chip.

[0059] Main reagents: HBS-EP+ analysis buffer (pH 7.4), regeneration buffer (10 mM glycine-hydrochloric acid buffer, pH 2.0).

[0060] 3. Experimental Methods 3.1 Molecular docking simulation Protein pretreatment (removing water molecules and redundant ligands, adding hydrogen atoms) was performed using PyMol 2.4. AutoDock Tools 1.5.6 was used to generate the PDBQT file for docking simulations. The docking box was set to its maximum size to enclose the protein, while other parameters remained at their default values. The docking results were set to output the 10 optimal docking sites. The docking conformation with the lowest binding energy and highest clustering frequency was considered the most potential binding mode between the ligand and protein. Molecular docking results were expressed as binding energies, and the average value was calculated after three repeated runs, with the standard deviation also calculated. Finally, the docking results were visualized using the PLIP online tool and PyMol 2.4 software.

[0061] 3.2 Surface Plasmon Resonance (SPR) Combined Experiment The binding kinetics of PHI and STAT3 protein were determined using SPR technology.

[0062] 3.2.1 Chip Activation and Protein Coupling 400 mM EDC and 100 mM NHS were mixed instantaneously and injected onto the chip surface at a flow rate of 10 μL / min, followed by activation for 420 s. STAT3 protein was diluted to 20 μg / mL with 10 mM sodium acetate buffer (pH 5.0) and coupled to the detection channel Fc2 at a flow rate of 10 μL / min, with a fixation volume of 12700 RU. The reference channel Fc1 was similarly activated, but only blocking buffer was injected without protein coupling. Subsequently, 1 M ethanolamine hydrochloride was bubbled in at a flow rate of 10 μL / min for 420 s for blocking.

[0063] 3.2.2 Combined with kinetic measurements PHI was serially diluted twofold with analytical buffer to prepare seven concentrations: 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM. The samples were injected sequentially at a flow rate of 20 μL / min, with a binding time of 60 s and a dissociation time of 90 s. After each injection cycle, the chip surface was regenerated with regeneration buffer at a flow rate of 30 μL / min for 30 s to restore its integrity. Each concentration was measured once.

[0064] 4. Experimental Results 4.1 Molecular docking results Molecular docking simulation results ( Figure 1 A) shows that PHI can stably embed in the binding pocket of the STAT3 protein. Its molecular backbone is highly matched with the hydrophobic region within the pocket, and it forms non-covalent bonds such as hydrogen bonds and hydrophobic interactions with key amino acid residues such as TRP-501, SER-540, ALA-505 and LEU-525. From a structural biology perspective, this suggests that there is a potential stable physical binding between PHI and STAT3.

[0065] 4.2 SPR combined with validation results SPR sensor map ( Figure 1 (B) shows that within the concentration range of 1.56–100 μM, PHI exhibits a concentration-dependent binding response to immobilized STAT3 protein. The binding signal increases with increasing PHI concentration and gradually saturates, suggesting that PHI can directly bind to STAT3 protein. The dissociation phase kinetics curve shows slow dissociation of the complex, indicating that the PHI-STAT3 interaction has a certain degree of stability, with an affinity on the order of μM (K). D = 0.42 μM).

[0066] 5. Conclusion This embodiment, through a combination of computer simulation prediction and biophysical experimental verification, confirms that forsythoside can directly bind to STAT3 protein with an affinity at the μM level. This result reveals the molecular initiation mechanism of forsythoside regulating the STAT3 signaling pathway, providing a structural biology basis for its subsequent inhibition of STAT3 phosphorylation and the downstream NLRP3 pyroptosis pathway, and confirming the scientific validity of the target selection in this invention.

[0067] Example 2: In vivo pharmacodynamic evaluation of forsythoside A in mice with sepsis-associated encephalopathy 1. Experimental Objective This embodiment aims to evaluate the effect of forsythoside A on the survival rate, disease severity, and cognitive impairment of septic mice using a cecal ligation and perforation (CLP) induced SAE mouse model, and to verify the therapeutic effect of the drug of the present invention in vivo.

[0068] 2. Experimental Materials Laboratory animals: 90 male C57BL / 6 mice, 8 weeks old, SPF grade, weighing 20-25 g, purchased from Hubei Provincial Laboratory Animal Center.

[0069] Test drug: Forsythoside A (PHI, MedChemExpress, HY-N0483, purity ≥ 98.55%). Dissolved in DMSO before administration and diluted with PBS (final concentration DMSO < 0.1%).

[0070] Main instruments: Morris water maze system, Y maze, surgical microscopy instruments.

[0071] Main reagents: sodium pentobarbital, physiological saline, etc.

[0072] 3. Experimental Methods 3.1 SAE Model Establishment A sham-operated endoscopic seizure (SAE) model was induced using cecal ligation and puncture (CLP). Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. Under aseptic conditions, a midline laparotomy was performed to expose and ligate the mid-cecum. The cecum was punctured with an 18G needle, and a small amount of feces was gently squeezed out. After repositioning the cecum, the abdomen was closed layer by layer. Postoperatively, pre-warmed sterile saline was injected subcutaneously for fluid resuscitation. The sham-operated group underwent only laparotomy and cecal manipulation, without ligation and puncture.

[0073] 3.2 Grouping and Dosing Regimen Mice were randomly divided into the following groups (n=10 for behavioral experiments, n=5 for biochemical and histological experiments, and n=15 for survival analysis experiments): Sham group: sham surgery group, mice underwent only abdominal and cecal incision without ligation or perforation, and were administered 0.2 mL PBS (final concentration DMSO < 0.1%) solution by gavage once 1 hour before surgery; CLP group: Mice underwent cecal ligation and perforation to establish an SAE model. One hour before the operation, mice were administered 0.2 mL of PBS (final concentration DMSO < 0.1%) solution by gavage. PHI monotherapy group: Mice underwent only abdominal and cecal surgery without ligation or perforation. One hour before surgery, mice were given a single oral gavage of PHI solution, with a volume of 0.2 mL and a dose of 50 mg / kg. CLP + PHI-L group: SAE model was prepared by CLP procedure. PHI solution was administered by gavage once 1 hour before the procedure, with a volume of 0.2 mL and a dose of 12.5 mg / kg. CLP + PHI-M group: SAE model was prepared by CLP procedure. PHI solution was administered by gavage once 1 hour before the procedure, with a volume of 0.2 mL and a dose of 25 mg / kg. CLP + PHI-H group: SAE model was prepared by CLP procedure. PHI solution was administered by gavage once 1 hour before the procedure, with a volume of 0.2 mL and a dose of 50 mg / kg.

[0074] 3.3 Pharmacodynamic evaluation indicators 3.3.1 Survival rate The survival of mice was recorded within 7 days after CLP surgery, Kaplan-Meier survival curves were plotted, and Log-rank test analysis was performed.

[0075] 3.3.2 Sepsis Severity Score Twenty-four hours post-surgery, the mice were assessed for appearance, consciousness, and activity according to the Murine Sepsis Score. The scoring criteria included five items: coat cleanliness, eye discharge, respiratory rhythm, spontaneous activity, and alertness. Each item was graded and scored, with a higher total score indicating more severe sepsis and more aggressive disease progression.

[0076] 3.3.3 Morris Water Maze Assess spatial learning and reference memory. After several days of continuous training, record the escape latency of mice when they find the hidden platform; after removing the platform, record the time the mice spend in the target quadrant and the number of times they cross the original platform location.

[0077] 3.3.4 Y-maze Experiment The experiment was conducted after the water maze was completed, and the alternation response rate of the mice was recorded.

[0078] 4. Experimental Results 4.1 Survival rate Kaplan-Meier survival curve results ( Figure 2 A) shows that the 7-day survival rate of mice in the CLP model group was significantly reduced to 40%. PHI pretreatment significantly improved the survival rate, with the CLP + PHI-H group increasing the survival rate to 80%, which was statistically different from the CLP group (P<0.05).

[0079] 4.2 Severity of sepsis The sepsis score was significantly elevated in CLP-induced mice. Intervention with medium-dose (25 mg / kg) and high-dose (50 mg / kg) PHI significantly reduced this score, indicating that PHI can alleviate the severity of CLP-induced systemic disease. Figure 2 B).

[0080] 4.3 Spatial Learning and Memory 4.3.1 Avoiding the incubation period: In the navigation training, the time required for mice in the CLP group to find the hidden platform (escape latency) was significantly longer than that in the Sham group, suggesting that the spatial learning ability of septic mice is impaired. Compared with the CLP group, PHI-M and PHI-H treatments significantly shortened the escape latency of mice, suggesting that PHI can alleviate CLP-induced spatial learning impairment (P<0.05). Figure 2 CD).

[0081] 4.3.2 Space Exploration: In the platform removal exploration experiment, the CLP group mice spent significantly less time in the target quadrant and crossed the original platform location less often than the Sham group, suggesting impaired spatial reference memory in septic mice. Compared with the CLP group, PHI-M and PHI-H treatments significantly increased the time spent in the target quadrant and the number of platform crossings (P<0.05), indicating that PHI can improve CLP-induced spatial reference memory retention impairment. Figure 2 EF).

[0082] 4.4 Working Memory Results of the Y-maze experiment ( Figure 2 The results showed that the spontaneous alternation rate (reflecting working memory) of mice in the CLP group was significantly reduced, suggesting that the spatial working memory of septic mice was impaired and their ability to explore independently was reduced. Compared with the CLP group, PHI-M and PHI-H treatments significantly increased the spontaneous alternation rate of mice (P<0.05), suggesting that PHI can improve CLP-induced short-term spatial working memory impairment.

[0083] 5. Conclusion This embodiment demonstrates that forsythoside A intervention significantly improves the survival rate of septic mice, reduces disease severity scores, and effectively reverses sepsis-induced spatial learning and memory impairment and working memory deficits. These results prove that forsythoside A has a clear anti-SAE efficacy, exhibiting a cognitive-improving effect and supporting its use as a candidate drug for treating SAE.

[0084] Example 3: Protective effect of forsythoside on hippocampal pathological morphology and synaptic ultrastructure in SAE mice 1. Experimental Objective To evaluate the protective effect of forsythoside A on CLP-induced pathological damage and synaptic ultrastructure of hippocampal neurons in SAE mice.

[0085] 2. Experimental Materials Experimental animals and grouping: Same as in Example 2.

[0086] Main reagents: Hematoxylin-eosin (HE) staining solution, Nissl staining reagent.

[0087] Main instruments: optical microscope, transmission electron microscope.

[0088] 3. Experimental Methods 3.1 Tissue sampling and fixation After the behavioral tests, mice were anesthetized and perfused with physiological saline and 4% paraformaldehyde via the heart. Brain tissue was harvested, the hippocampus was separated, a portion was fixed in 4% paraformaldehyde for paraffin sectioning, and the other portion was cut into 1 mm pieces. 3 Small pieces were fixed in 2.5% glutaraldehyde for use as electron microscopy samples.

[0089] 3.2 H&E staining Hippocampal tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. The morphology of neurons in the CA1, CA3, and DG regions of the hippocampus was observed under an optical microscope, and the number of surviving neurons was counted.

[0090] 3.3 Nissl staining Paraffin sections were stained with Nissl to observe the distribution and density of Nissl bodies in neurons and to assess neuronal functional status.

[0091] 3.4 Transmission Electron Microscopy Fresh hippocampal tissue was collected, double-fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated, embedded in epoxy resin, and then ultrathin sections were prepared. After staining with uranium acetate and lead citrate, the ultrastructure of neurons, such as mitochondria, presynaptic vesicles, postsynaptic dense area (PSD) thickness, and synaptic cleft width, was observed under a transmission electron microscope, and damage scores were performed.

[0092] 3.5 Statistical Analysis Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0093] 4. Experimental Results 4.1 Pathological damage to hippocampal neurons: H&E staining ( Figure 3 A) shows that, compared with the Sham group, the CLP group exhibited significant pathological changes in neurons of the CA3 region of the hippocampus, including disordered cell arrangement, cell body shrinkage, and nuclear pyknosis. After intervention with medium and high doses of PHI, the aforementioned pathological damage was significantly reduced.

[0094] Quantitative analysis ( Figure 3 C) indicates that the number of surviving neurons in the CA3 region was significantly reduced in the CLP group, while PHI treatment dose-dependently increased the number of surviving neurons in the CA3 region (P<0.05).

[0095] 4.2 Neuronal functional state: Nissl staining ( Figure 3 B and 3D showed that, compared with the Sham group, the number of Nissl bodies in the CA3 region of the hippocampus of mice in the CLP group was significantly reduced, suggesting that sepsis induces neuronal structural damage and protein synthesis dysfunction. Compared with the CLP group, medium and high doses of PHI intervention significantly increased the number of Nissl bodies in the CA3 region (P<0.05), while low doses of PHI had no significant protective effect, indicating that medium and high doses of PHI can alleviate neuronal structural and functional damage caused by sepsis.

[0096] 4.3 Synaptic ultrastructure: Transmission electron microscopy observation ( Figure 3 E) It was found that the hippocampal synaptic structure in the CLP group was severely damaged, manifested as mitochondrial swelling, cristae breakage, reduction of presynaptic vesicles, thinning of postsynaptic dense area (PSD), and widening or disorder of synaptic cleft. The synaptic ultrastructural damage in the PHI treatment group was reduced in a dose-dependent manner. Among them, after high-dose PHI intervention, the mitochondrial morphology was more complete and the synaptic structure was clearer, which was close to the level of the sham-operated group.

[0097] Quantitative analysis ( Figure 3 FG confirmed that, compared with the sham surgery group, the CLP group had significantly reduced PSD thickness (P<0.01) and abnormally widened synaptic cleft width (P<0.05). After high-dose PHI intervention, PSD thickness increased significantly and synaptic cleft width tended to normal (P<0.05), suggesting that high-dose PHI can effectively repair sepsis-induced synaptic ultrastructural damage.

[0098] 5. Conclusion This embodiment demonstrates that sepsis can cause severe pathological morphological changes and synaptic ultrastructural damage in mouse hippocampal neurons. After intervention with forsythoside A, neuronal shrinkage and loss in the CA3 region of the hippocampus were significantly reduced, Nissl body density was restored, and the thickness of the postsynaptic compact area (PSD) and the width of the synaptic cleft were repaired in a dose-dependent manner. These results demonstrate the neuroprotective effect of forsythoside A at the histopathological and ultrastructural levels.

[0099] Example 4: In vivo verification of the mechanism by which forsythoside A reduces hippocampal neuroinflammation and microglia pyroptosis by inhibiting the STAT3 / NLRP3 / GSDMD pathway. 1. Experimental Objective This embodiment aims to verify the mechanism of action of forsythoside A in SAE mice from a molecular biology perspective, focusing on the regulatory effect of PHI on the phosphorylation level of STAT3 in hippocampus, activation of NLRP3 inflammasomes, and GSDMD-mediated microglia pyroptosis pathway, and to elucidate its neuroprotective molecular targets.

[0100] 2. Experimental Materials Experimental animals and grouping: Same as in Example 2.

[0101] Sample source: Hippocampal tissue from mice in Example 2.

[0102] Main reagents: anti-p-STAT3 antibody (Tyr705, Abmart, T56566), anti-STAT3 antibody (Abmart, T55292), anti-NLRP3 antibody (Cell Signaling Technology, 15101), anti-Caspase-1 antibody (Adipogen, AG-20B-0042), anti-GSDMD antibody (Cell Signaling Technology, 39754), anti-β-actin antibody (Proteintech, 66009-1-Ig); qPCR related reagents; mouse IL-6, TNF-α, IL-1β, IL-18 ELISA kits were all purchased from Beijing Sizhengbai Biotechnology Co., Ltd.

[0103] RT-qPCR primers: Sequence information is shown in Table 1, synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0104] Main instruments: chemiluminescence imaging system, laser confocal microscope, enzyme-linked immunosorbent assay (ELISA) reader.

[0105] 3. Experimental Methods 3.1 ELISA detection of inflammatory factors: After homogenizing hippocampal tissue, the concentrations of pro-inflammatory factors IL-6, TNF-α, IL-1β, and pyroptosis-related factor IL-18 were detected using an ELISA kit.

[0106] 3.2 Western Blot detection of protein expression: Total protein was extracted from hippocampal tissue, and the protein levels of p-STAT3, STAT3, NLRP3, pro-Caspase-1, CleavedCaspase-1, GSDMD-FL, and GSDMD-NT were detected. Normalization was performed using β-actin as an internal control.

[0107] 3.3 RT-qPCR detection of mRNA expression: Total RNA was extracted from hippocampal tissue, reverse transcribed into cDNA, and the relative expression levels of Stat3 and Nlrp3 mRNA were detected by qPCR, with β-actin as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0108] 3.4 Immunofluorescence Double immunofluorescence staining of paraffin sections of hippocampal tissue: p-STAT3 / IBA1: to locate whether phosphorylated STAT3 occurs in activated microglia.

[0109] GSDMD-NT / IBA1: To determine whether the active fragment of the pyroptosis executive protein GSDMD accumulates in activated microglia.

[0110] 3.5 Image Analysis and Statistics: Quantitative analysis of Western blot bands and fluorescence images was performed using software such as ImageJ. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0111] 4. Experimental Results 4.1 Inhibition of hippocampal neuroinflammation: ELISA results ( Figure 4 The results showed that, compared with the sham-operated group, the levels of pro-inflammatory factors IL-6, TNF-α, IL-1β and IL-18 in the hippocampus of mice in the CLP model group were significantly increased; medium and high doses of PHI intervention could significantly reduce the levels of these inflammatory factors (P<0.05), indicating that PHI can effectively inhibit the inflammatory response in the brain related to SAE.

[0112] 4.2 Inhibiting STAT3 signaling pathway activation: RT-qPCR results ( Figure 4I) showed that, compared with the Sham group, the expression of Stat3 mRNA in the hippocampus was significantly upregulated in the CLP group, and PHI treatment could downregulate it.

[0113] Western Blot ( Figure 4 EF showed that, compared with the Sham group, the CLP group had a significantly increased level of p-STAT3 (Tyr705) protein in the hippocampus, while the total STAT3 level remained relatively unchanged. PHI treatment significantly inhibited the increase in p-STAT3 level (P<0.05).

[0114] Immunofluorescence results ( Figure 4 G) showed that the sepsis model induced p STAT3 expression is upregulated, and p is elevated. STAT3 is primarily co-localized in the nuclei of IBA1-labeled microglia in the CA3 region of the hippocampus. Quantitative fluorescence statistics (QFS) Figure 4 H) indicates that medium- and high-dose PHI intervention can significantly downregulate intranuclear p in microglia. The fluorescence expression intensity of STAT3 (P<0.05) suggests that high-dose PHI can exert an anti-inflammatory regulatory effect by inhibiting the phosphorylation level of STAT3 in microglia.

[0115] 4.3 Inhibition of NLRP3 inflammasome activation and pyroptosis: qPCR results ( Figure 4 The results showed that, compared with the Sham group, the expression of Nlrp3 mRNA in the hippocampus of the CLP group was significantly increased; high-dose PHI intervention could significantly reverse the abnormal upregulation of Nlrp3 mRNA.

[0116] Western Blot results ( Figure 4 JM indicated that the expression levels of NLRP3, Cleaved Caspase-1 (p20), and the pyroptosis functional fragment GSDMD-NT in the hippocampus of the CLP group were significantly upregulated compared with those of the Sham group; high-dose PHI intervention downregulated the protein expression of NLRP3, Cleaved Caspase-1, and GSDMD-NT (P<0.05), indicating that high-dose PHI can inhibit the assembly of the NLRP3 inflammasome, the activation of caspase-1, and the cleavage of the pyroptosis executive protein GSDMD.

[0117] Immunofluorescence images ( Figure 4 O) showed that GSDMD-NT signals and IBA1-positive microglia were co-localized in the CA3 region of the hippocampus. Quantitative analysis ( Figure 4The results showed that, compared with the Sham group, the GSDMD-NT fluorescence signal in microglia of the CLP group was significantly enhanced (P<0.001); high-dose PHI (50 mg / kg) treatment significantly reduced this signal intensity (P<0.05), which directly demonstrated that PHI can inhibit the pyroptosis process of sepsis-induced microglia.

[0118] 5. Conclusion The core mechanism of action of PHI in vivo is to target and inhibit the STAT3 / NLRP3 / GSDMD signaling axis in microglia, thereby suppressing microglial pyroptosis and related neuroinflammation, ultimately protecting neurons. This provides mechanistic support for its use in the treatment of sepsis-related encephalopathy.

[0119] Example 5: In vitro cell safety evaluation of forsythoside A and its inhibitory effect on inflammation-induced secretion of inflammatory factors in microglia. 1. Experimental Objective This embodiment aims to evaluate the safety of forsythoside A (PHI) at the cellular level on microglia (BV2) and neurons (HT22), and to verify the inhibitory effect of PHI on the secretion of inflammatory factors in microglia under inflammatory stimulation, providing in vitro cellular evidence for in vivo efficacy and safety data.

[0120] 2. Experimental Materials Cell lines: Mouse microglia BV2 and mouse hippocampal neuronal cell line HT22 were both purchased from Seven Innovation (Beijing) Biotechnology Co., Ltd.

[0121] Main reagents: Nigerian styracin, CCK-8 kit (TargetMol, C0005), ELISA kit (same as in Example 4), anti-PSD95 antibody (Cell Signaling Technology, 3409), anti-Synaptophysin (SYN, Affinity, AF0257).

[0122] Main instruments: ELISA reader, CO2 cell incubator, inverted microscope.

[0123] 3. Experimental Methods 3.1 Cell Culture BV2 was cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2.

[0124] 3.2 Safety evaluation of PHI cells BV2 microglia were treated with different concentrations (0~100 μg / mL) of PHI for 6 hours, then incubated with CCK-8 reagent for 2 hours. The absorbance at 450 nm was measured using an ELISA reader, and the cell viability was calculated.

[0125] 3.3 Establishment of a microglial cell inflammatory activation model and PHI intervention BV2 cells were pretreated with different concentrations of PHI for 30 minutes, followed by stimulation with LPS (500 ng / mL) for 4 hours. Finally, they were stimulated with nigericin (10 μM) for 45 minutes to strongly activate the NLRP3 inflammasome. HT22 cells were used only for subsequent co-culture in conditioned medium to detect neuronal damage.

[0126] The cell processing and grouping are as follows: PBS group: control group, cells were not subjected to inflammatory stimulation, and were pretreated with an equal volume of physiological saline (final concentration DMSO < 0.1%) for 30 minutes; LPS model group: An inflammation activation model was established using the above method, and an equal volume of physiological saline solution (final concentration DMSO < 0.1%) was added before treatment for 30 minutes; PHI monotherapy group: cells were not subjected to inflammatory stimulation and were pretreated with 2 mL of 100 μg / mL PHI solution for 30 minutes. LPS + PHI-L group: An inflammation activation model was established as described above, and 2 mL of 25 μg / mLPHI solution was added beforehand and the patient was treated for 30 minutes. LPS + PHI-M group: An inflammation activation model was established as described above, and 2 mL of 50 μg / mLPHI solution was added beforehand and treated for 30 minutes. LPS + PHI-H group: An inflammation activation model was established as described above, and the patient was pre-treated with 2 mL of 100 μg / mL PHI solution for 30 minutes. 3.4 Detection Indicators 3.4.1 Cell viability The direct toxicity of PHI to BV2 cells and the effect of combined treatment with inflammatory stimuli on BV2 cell viability were detected by the CCK-8 assay.

[0127] 3.4.2 Release of inflammatory factors After BV2 cells were processed into groups, cell supernatants were collected, and the secretion levels of TNF-α, IL-6, IL-1β, and IL-18 were detected using an ELISA kit.

[0128] 3.4.3 Detection of neuronal synaptic proteins After HT22 cells were treated in groups, total cellular protein was extracted, and the expression levels of postsynaptic dense protein PSD95 and synaptophysin SYN were detected.

[0129] 4. Experimental Results 4.1 Cell safety evaluation like Figure 5 As shown in Figure A, after treating BV2 cells with PHI alone (maximum concentration 100 μg / mL) for 6 hours, the cell viability was not significantly different from the control group (P>0.05), indicating that PHI had no significant cytotoxicity to microglia within the experimental concentration range; Figure 5 As shown in Figure B, under LPS inflammatory stimulation, the PHI concentration groups did not aggravate cell damage, and the cell survival rate of the high-concentration PHI group remained above 85%, further confirming the safety of the drug.

[0130] 4.2 Protective effect on neuronal synaptic proteins like Figure 5 As shown in CE, LPS damage significantly downregulated the expression of synaptic proteins PSD95 and Synaptophysin in HT22 neurons; medium and high doses of PHI pretreatment significantly restored the protein expression levels of PSD95 and SYN in HT22 cells (P<0.05), suggesting that PHI can directly or indirectly protect the integrity of neuronal synaptic structure.

[0131] 4.3 Inhibition of microglial cell inflammatory factor secretion like Figure 5 As shown in FI, compared with the control group, the secretion of TNF-α, IL-6, IL-1β and IL-18 in the cell supernatant of the LPS model group was significantly increased (P<0.01); the level of inflammatory factors in the PHI-only group was not different from that in the control group; compared with the LPS model group, the secretion of inflammatory factors in the PHI intervention group decreased in a dose-dependent manner, and medium and high doses of PHI pretreatment significantly inhibited the release of these inflammatory factors (P<0.05), indicating that PHI can effectively inhibit the inflammatory activation of microglia.

[0132] 5. Conclusion In vitro experiments have confirmed that PHI can safely and effectively inhibit the neurotoxic activation of microglia and indirectly protect neurons, providing direct cellular evidence for its in vivo treatment of SAE by regulating microglia function.

[0133] Example 6: Validation of in vitro cell mechanisms 1. Experimental Objective In an in vitro BV2 microglia model, this study thoroughly examines whether forsythoside A exerts its anti-inflammatory and pyroptosis-inhibiting effects by inhibiting the STAT3 / NLRP3 / GSDMD signaling axis at the gene transcription, protein expression, and cellular localization levels, thereby elucidating its molecular mechanism at the cellular level.

[0134] 2. Experimental Materials Cell line: Mouse microglia BV2.

[0135] Cell culture and grouping: Same as in Example 5.

[0136] Main reagents and instruments: Same as in Example 4.

[0137] 3. Experimental Methods 3.1 RT-qPCR detection of mRNA expression Total RNA was extracted from cells in each group and reverse transcribed into cDNA. Using specific primers, qPCR was performed using the SYBR Green method to detect the relative mRNA expression levels of Stat3 and Nlrp3, with β-actin as an internal reference gene. -ΔΔCt Calculation by method.

[0138] 3.2 Western Blot detection of protein expression: Total cellular protein was extracted, and the expression levels of p-STAT3, STAT3, NLRP3, Cleaved Caspase-1, and GSDMD-NT were detected, with β-actin as an internal control.

[0139] 3.3 Immunofluorescence staining Cells were cultured on slides, treated according to groups, fixed, and permeabilized. Primary antibodies were used for incubation with anti-p-STAT3 or anti-GSDMD-NT antibodies and anti-IBA1 antibodies. Secondary antibody incubation was followed by DAPI staining of the nuclei. Fluorescence intensity and distribution were observed under a laser confocal microscope.

[0140] 4. Experimental Results 4.1 PHI inhibits STAT3 signaling pathway activation mRNA levels: such as Figure 6 As shown in Figure E, compared with the control group, LPS stimulation significantly increased Stat3 mRNA expression in BV2 cells. There was no difference between the PHI monotherapy group and the control group; however, the Stat3 mRNA level in the PHI intervention group decreased in a dose-dependent manner (P<0.01).

[0141] Protein phosphorylation: such as Figure 6 As shown in AB, compared with the control group, the p-STAT3 / STAT3 ratio in the LPS model group was significantly increased; after PHI intervention, the ratio decreased significantly, and the high-dose group recovered to a level close to that of the PBS group.

[0142] Cell localization: Immunofluorescence images ( Figure 6C) showed that p-STAT3 signaling was co-localized with IBA1-positive BV2 microglia; compared with the PBS control group, the p-STAT3 fluorescence signal in the nucleus of the LPS model group was significantly enhanced (P<0.001), suggesting that LPS can induce STAT3 phosphorylation and nuclear translocation in microglia; while after PHI pretreatment, the p-STAT3 fluorescence intensity decreased in a concentration-dependent manner, and the differences between the medium and high dose PHI intervention groups and the LPS model group were statistically significant (P<0.05), confirming that PHI can effectively inhibit LPS-induced activation of the STAT3 pathway in microglia. Figure 6 D).

[0143] 4.2 PHI inhibits NLRP3 inflammasome activation and pyroptosis mRNA levels: such as Figure 6 As shown in Figure J, compared with the control group, Nlrp3 mRNA expression was significantly upregulated in the LPS model group, while no significant change was observed in the PHI-only group. PHI intervention significantly reduced LPS-induced high expression of Nlrp3 mRNA (P<0.001), suggesting that PHI can effectively inhibit the transcriptional activation of the NLRP3 inflammasome.

[0144] Protein levels: such as Figure 6 As shown in FI, compared with the control group, the expression levels of NLRP3, Cleaved Caspase-1 (p20) and GSDMD-NT proteins were significantly upregulated in the LPS model group; after PHI intervention, the expression levels of the above proteins were significantly downregulated.

[0145] Cell localization and quantification: Immunofluorescence images ( Figure 6 K) showed that GSDMD-NT pyroptosis signaling was co-localized with IBA-1 positive microglia; compared with the PBS control group, the GSDMD-NT fluorescence signal in the LPS model group was significantly enhanced (P<0.001), suggesting that LPS can induce microglia pyroptosis activation; while after PHI pretreatment, the GSDMD-NT fluorescence intensity was significantly reduced, and the difference between the LPS + PHI-H intervention group and the LPS model group was statistically significant (P<0.05), confirming that PHI can effectively inhibit LPS-induced microglia pyroptosis pathway activation. Figure 6 L).

[0146] 5. Conclusion In vitro experiments fully demonstrated at the cellular and molecular levels that PHI inhibits the inflammatory activation and pyroptosis processes of microglia by targeting and suppressing the STAT3 / NLRP3 / GSDMD signaling axis. This provides direct mechanistic evidence for the neuroprotective effect of PHI in vivo and corroborates the results of in vivo experiments.

[0147] The above results demonstrate that forsythoside A not only treats sepsis-related encephalopathy, but also achieves this through a clear mechanism of directly binding to STAT3, inhibiting its phosphorylation, and blocking the NLRP3 / GSDMD pyroptosis pathway, with dose-dependent effects and good safety profile.

[0148] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0149] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0150] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. The use of forsythoside A in the preparation of drugs for the prevention or treatment of sepsis-associated encephalopathy with cognitive impairment.

2. The application according to claim 1, characterized in that: The drug is used to improve cognitive deficits caused by sepsis-associated encephalopathy and / or improve the survival rate of subjects.

3. The application according to claim 1, characterized in that: The drug is available in either injectable or oral formulation.

4. The application according to claim 1, characterized in that: The dosage of the forsythoside A is 12.5~50 mg / kg.

5. The use of a combination of forsythoside and at least one other drug for treating sepsis in the preparation of a drug for the prevention or treatment of sepsis-associated encephalopathy with cognitive impairment.

6. The application according to claim 5, characterized in that: Other medications for treating sepsis include one or more of antibiotics, vasoactive drugs, or NLRP3 inhibitors.

7. The use of a pharmaceutical composition comprising a therapeutically effective amount of forsythoside and a pharmaceutically acceptable carrier in the preparation of a medicament for the prevention or treatment of sepsis-associated encephalopathy with cognitive impairment.

8. The application according to claim 7, characterized in that: The forsythoside is the only active ingredient, and the drug is an injectable or oral preparation.

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