Application of Xianglian intestine decoction in preparation of medicine for treating ulcerative colitis through targeted TLR4 / NLRP3 / GSDMD pathway

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of Xianglian intestine decoction in preparation of a medicine for treating ulcerative colitis through a targeted TLR4 / NLRP3 / GSDMD channel. According to the invention, the TLR4 is determined as a key target, and experiments show that the active ingredients in the Xianglian intestine soup and the TLR4 have very strong binding affinity. In-vivo experiments prove that the Xianglian intestine soup down regulates the protein levels of TLR4, NLRP3 and GSDMD-N and the mRNA expression (Plt; 0.05) of IL-1beta and IL-18, so that the pyroptosis is inhibited. Furthermore, the Xianglian intestine decoction inhibits NLRP3 inflammasome activation and GSDMD-mediated pyroptosis by targeting TLR4 so as to relieve UC inflammation and intestinal barrier injury. The invention provides a mechanism insight for the clinical curative effect of the Xianglian intestine decoction for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to the use of Xianglian Anchang Decoction in the preparation of a drug for treating ulcerative colitis by targeting TLR4, NLRP3 or GSDMD pathway. Background Art

[0002] Ulcerative colitis (UC) is a nonspecific inflammatory disease characterized by chronic inflammation or ulceration of the rectal and colonic mucosa. UC is recognized worldwide as one of the most challenging intestinal diseases and a significant risk factor for the development of colorectal cancer. Ongoing global industrialization has led to adverse shifts in lifestyle and nutritional behaviors, resulting in a significant epidemiological increase in the incidence and prevalence of UC. Current treatments for UC continue to face significant challenges, including suboptimal response rates, high recurrence rates, and significant adverse effects.

[0003] The pathogenesis of UC involves multiple interrelated factors: intestinal microbiota dysbiosis, intestinal immune dysbiosis, mucosal homeostasis disruption, and intestinal barrier dysfunction. Notably, intestinal barrier dysfunction plays a key role in the pathogenesis of UC, exhibiting a bidirectional "causal loop" characteristic. At the onset of intestinal inflammation, damaged intestinal epithelial cells promote the exposure of intestinal harmful substances to the immune system, thereby triggering and amplifying the inflammatory cascade. Therefore, elucidating the molecular mechanisms of intestinal epithelial cell damage may provide new therapeutic targets for the study of UC pathogenesis.

[0004] Increased epithelial cell death is one of the hallmarks of UC and is positively correlated with the severity of inflammation. Studies have found that inhibiting pyroptosis signals can alleviate UC. Pyroptosis is a type of programmed cell death triggered by inflammasomes, characterized by continuous cell swelling until the cell membrane ruptures, leading to the release of cellular contents, which in turn triggers a strong inflammatory response. Appropriate pyroptosis is an important innate immune response of the body and plays an important role in resisting infection, while excessive pyroptosis activity is highly proinflammatory and can lead to tissue damage. TLR4 is a key member of the Toll-like receptor family and plays a vital role in the innate immune response, particularly in recognizing bacterial infections and triggering inflammatory responses. TLR4 can lead to the activation of the NLRP3 inflammasome and promote pyroptosis. During the pyroptosis process, proinflammatory cytokines such as IL-1β and IL-18, as well as a large number of additional danger signals and intracellular antigens, are excessively released through pores formed by gasdermin proteins, thereby exacerbating the inflammatory response and intestinal mucosal damage. Summary of the Invention

[0005] This study employed network pharmacology and bioinformatics approaches to investigate the molecular targets of Xianglian Anchang Decoction (XLAC) in the treatment of UC. The findings revealed a key role for TLR4 in this process, which was preliminarily verified through molecular docking. Subsequent in vivo experiments further confirmed the crucial role of TLR4 in XLAC's treatment of UC. These findings are expected to provide a theoretical basis for the clinical efficacy of XLAC in this treatment.

[0006] The present invention provides the use of Xianglian Anchang Decoction in preparing a medicine for treating ulcerative colitis by targeting the TLR4 / NLRP3 / GSDMD pathway.

[0007] Preferably, the drug inhibits the expression of TLR4 protein, or the drug inhibits the expression of GSDMD protein, or the drug inhibits the expression of NLRP3 protein.

[0008] According to the application of the specific embodiment of the present invention, the dosage form of the drug for treating ulcerative colitis includes decoction, honey pills, granules or oral liquid. Xianglian Anchang Decoction can be further combined with other pharmaceutical excipients or active ingredients to prepare a drug for treating ulcerative colitis.

[0009] The above-mentioned drugs can be prepared into oral preparations or injections; the oral preparations include but are not limited to capsules, tablets, granules, and oral liquids; the injections include but are not limited to sterile powder for injection, aqueous injection, sodium chloride or glucose intravenous infusion.

[0010] Preferably, the ulcerative colitis drug comprises a pharmaceutically acceptable carrier.

[0011] In the above-mentioned medicine, the oral preparation includes additives, and the additives are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0012] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0013] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silicon oxide; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0014] The adhesive comprises distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

[0015] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or cross-linked carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or carboxymethyl starch sodium.

[0016] The surfactant includes Tween, sodium lauryl sulfate, sodium stearate sulfonate, etc.

[0017] The antioxidants include sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid, etc.

[0018] The preservatives or antibacterial agents include benzoic acid and sodium benzoate, sorbic acid, ethanol, parahydroxybenzoates (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0019] The flavoring agents include sweeteners such as saccharin sodium, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavorings such as citric acid, malic acid or tartaric acid; and aromatics such as fennel oil, mint oil, menthol, mint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

[0020] The method for using the above-mentioned drug comprises administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral, intravenous injection or transdermal permeation, and the drug is applied to the patient in need of treatment.

[0021] A pharmaceutically effective amount refers to a quantity sufficient to treat a disease at a reasonable benefit / risk ratio that can be obtained by applying a drug therapy. The level of an effective dose can be determined depending on factors including the patient's disease type, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration, rate of excretion, treatment cycle, concurrently used drugs, and other factors well known in the medical field. The drug of the present invention can be administered as an independent therapeutic agent or in combination with other therapeutic agents. Furthermore, the drug of the present invention can be added to a typical therapeutic agent continuously or simultaneously, and can be administered single or multiple times. It is important to consider all of the above factors and administer the minimum dose that produces the maximum effect without side effects, which can be determined by a physician based on the patient's condition, age, etc.

[0022] Beneficial effects of the present invention: Xianglian Anchang decoction (XLAC) significantly counteracted the adverse effects of UC, including bloody stools, abdominal pain, diarrhea, and weight loss, and improved mucosal inflammation and bleeding. Various results showed that the DAI scores, body weight, colon length, and spleen size of mice treated with XLAC approached those of the healthy control group.

[0023] The present invention further studies the molecular mechanism by which XLAC affects UC and uses network pharmacology experiments to confirm that XLAC intervenes in the relevant proteins and signaling pathways of UC.

[0024] The present invention replicated the therapeutic effect of XLAC on UC through animal experiments. Subsequently, the compound that actually exerted the therapeutic effect of XLAC was identified by LC-MS / MS. In addition, network pharmacology predicted the main targets of XLAC in the treatment of UC, including PIK3R1, PIK3CA, AKT, and MAPK3 - key regulators of inflammatory cytokine secretion, autophagy, and oxidative stress. Specifically, activation of the PI3K family can lead to the secretion of inflammatory factors TNF-α and IL-1, as well as cell apoptosis associated with inflammatory diseases. AKT is a downstream effector of PI3K, regulating cellular functions that are critical for the progression of inflammation. Similarly, MAPK, a member of the MAPK family, regulates the expression of inflammatory cytokines and cell proliferation after activation by cytokines or microbial stimuli. The above content shows that XLAC treatment of UC is closely related to the suppression of inflammation.

[0025] Furthermore, KEGG pathway analysis suggested that XLAC may improve UC by modulating the TLR signaling pathway. TLRs are a family of pattern recognition receptors primarily expressed on the plasma membrane of innate immune cells. As key components of innate immunity, TLRs serve as a first-line defense by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns. Studies have shown that TLRs expressed in the intestinal lamina propria regulate inflammatory responses. Due to the large number of TLR family members, we initially analyzed the GEO clinical database and identified differentially expressed genes, TLR2, TLR4, and TLR5, suggesting that these three genes may be involved in the pathogenesis of UC. To further elucidate the specific therapeutic target of XLAC, we subsequently performed qPCR validation and found that TLR4 mRNA expression was significantly upregulated in the model group and exhibited sensitivity to XLAC treatment. Finally, molecular docking experiments indicated that TLR4 may exhibit strong binding affinity to key components in drug-containing serum, such as trans-4-coumaric acid, methyl cinnamate, and 2-furylmethyl butyrate. These results indicate that TLR4 is a key target of XLAC in regulating UC.

[0026] TLR4, a membrane-bound pattern recognition receptor, is a key "starting signal" for inflammasome activation. NOD-like receptor pyrin domain-containing protein 3 (NLRP3) is an intracellular pattern recognition receptor that responds to pathogen- and damage-related stimuli, as well as other danger signals that disrupt cellular homeostasis, and participates in the innate immune response. The NLRP3 inflammasome is a supramolecular complex formed in the cytoplasm and composed of NLRP3 (an adaptor protein), ASC (an apoptosis-associated speck-like protein containing a CARD), and pro-aspartic protease 1.31. Under physiological conditions, NLRP3 and downstream pro-IL-1β and pro-IL-18 levels in cells are very low, maintaining a low-grade inflammatory state. However, activation of the NLRP3 inflammasome can lead to excessive inflammatory responses, thereby promoting pathological processes and is associated with a variety of human diseases, including type 2 diabetes, colitis, depression, and gout. Previous studies have demonstrated that the interaction between TLR4 and NLRP3 is prominent in a variety of diseases, including inflammatory bowel disease, Alzheimer's disease, and diabetic nephropathy. TLR4 activation can influence NLRP3 inflammasome activation through multiple pathways. Inflammasome activation leads to the proteolytic activation of caspase-1 and GSDMD. Upon cleavage by caspase-1, GSDMD, a direct downstream target of caspase-1, translocates its N-terminal domain to lipid membranes and oligomerizes to form pores approximately 10–14 nm in diameter, thereby forming pores that serve as pyroptotic effectors. These pores dissipate cellular ion gradients and allow water influx, leading to cell swelling, osmotic lysis, and the release of intracellular inflammatory cytokines such as IL-1β and IL-18.36.

[0027] Western blot and qRT-PCR were used to evaluate the inhibitory effect of XLAC on pyroptosis. We found that after TCM intervention, TLR4 and GSDMD-N protein levels in colon tissues were significantly reduced, as were the mRNA levels of TLR4, NLRP3, caspase-1, and GSDMD. We also used qRT-PCR to evaluate the anti-inflammatory effects of XLAC. The results suggest that abnormal activation of the LR4 / NLRP3 / GSDMD signaling pathway, leading to pyroptosis, is a key mechanism in UC. XLAC exerts its therapeutic effect by inhibiting this signaling pathway, thereby alleviating pyroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1showed the beneficial effects of XLAC on intestinal inflammation in UC mice, among which, (A) Timeline of DSS administration and different treatments of mice; (B) Body weight changes; (C) Representative images of colon length in each group; (D) Representative images of spleen in each group; (E) Comparison of colon length in each group; (F) Comparison of spleen weight in each group; (G) HE staining results (200×); ### Compared with the control group, P <0.001;***Compared with the model group, P <0.001.

[0030] Figure 2 showed the beneficial effects of XLAC on intestinal inflammation in UC mice, among which, (A) Representative images of PAS staining for each group; (B) Representative images of IF staining for ZO-1 from each group; (C) Representative images of IF staining for occludin from each group.

[0031] Figure 3 This is a network pharmacology analysis of XLAC in the treatment of UC, (A) Total ion chromatography of XLAC; (B) Venn diagram of XLAC-UC related targets; (C) Construction of public gene PPI network; (D) Results of GO analysis; (E) Results of KEGG enrichment analysis.

[0032] Figure 4 Bioinformatics analysis of UC targets, including: (A) Comparison of TLRs based on bioinformatics analysis; (B) In vivo validation of TLRs; ### Compared with the control group, P < 0.001; Compared with the model group, * P <0.05,** P <0.01,*** P <0.001.

[0033] Figure 5 The docking results of three compounds with the central target are shown in Figure 2. (A) trans-4-coumaric acid with TLR4; (B) methyl cinnamate with TLR4; (C) methyl 2-furanylbutyrate with TLR4; (D) Molecular docking result scoring table.

[0034] Figure 6 This was verified by in vivo animal experiments, in which (A) ELISA was used to detect the expression level of TLR4 in each group; (B, C, and D) Western blot analysis was used to evaluate the protein bands and relative protein expression of TLR4 and GSDMD-N in each group; (E) qPCR was used to detect the expression level of Caspase-1 in each group; (F) qPCR was used to detect the expression level of NLRP3 in each group. (G) qPCR was used to detect the expression level of IL-18 in each group; (H) qPCR was used to detect the expression level of IL-1β in each group; ### Compared with the control group, P <0.001; compared with the model group, * P <0.05,** P <0.01,*** P <0.001. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0036] experimental animals Fifty 8-week-old male C57BL / 6J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The experimental animals were housed in a specific pathogen-free environment, maintained at a constant temperature (25 ± 0.5°C) and humidity (50 ± 5%), with a 12-h light / dark cycle, and provided with standard food.

[0037] Preparation of XLAC XLAC is composed of 15 medicinal ingredients (10g of Scutellaria baicalensis, 9g of Coptis chinensis, 30g of Pueraria lobata, 15g of Agrimoniae herb, 2g of Panax notoginseng powder, 9g of Saposhnikovia divaricata, 15g of Poria cocos, 6g of Aucklandia lappa, 15g of Cynanchum wilfordii, 10g of Scorched Shenqu, 15g of Corydalis yanhusuo, 10g of Angelica dahurica, 15g of White Peony Root, 15g of Plantago seed, and 15g of Honeysuckle). Xianglian Anchang Decoction is obtained by decocting.

[0038] After dose conversion (9.1× clinical equivalent), two therapeutic concentrations, 1.24 g / mL (XLAC-L) and 2.48 g / mL (XLAC H), were prepared by standardized extraction, which involved a 1:10 (w / v) water infusion, a two-cycle atmospheric pressure extraction (1 hour after boiling), vacuum concentration at 70°C, and storage at 4°C. In subsequent experiments, the prepared extracts were administered by gavage for 7 consecutive days. Quality parameters included Latin nomenclature verification, extraction yield monitoring (68 ± 3%), and HPLC fingerprint verification to ensure batch consistency.

[0039] Example 1 Fifty C57BL / 6J male mice were randomly divided into a control group (n=10) and a model group (n=40). After one week of adaptive feeding, the model group was given 2% (w / v) DSS (molecular weight, 36-50 kDa; MP Biomedicals, Santa Ana, CA, United States) in drinking water to induce acute UC.

[0040] Simultaneously with modeling, drug intervention was initiated, and the model group was randomly divided into four groups: model group, low-dose XLAC group (XLAC-L, 1.24 g / mL), high-dose XLAC group (XLAC-H, 2.48 g / mL), and sulfasalazine group (SASP, 0.5 g / 10 mL). The control and model groups were gavaged with equal volumes of distilled water.

[0041] During the experiment, the general condition and body weight of the mice were observed every day. After the last administration, the mice were fasted for 24 hours with no restriction on drinking water. The mice were then anesthetized with 3% pentobarbital (45 mg / kg) by intraperitoneal injection, followed by eyeball bleeding. After centrifugation, the serum was separated. The colon and spleen were collected, and the samples were frozen in liquid nitrogen and stored at -80°C or fixed in 10% (w / v) formalin solution. In this study, the colon length and spleen weight were measured. The entire experimental process was as follows. Figure 1 As shown in A.

[0042] The mice in the control group were in good spirits, active, with shiny and bright fur, quick reactions, steady weight gain, and black-brown granular feces. The mice in the model group were listless, unresponsive, with rough and brittle fur, weight loss, foul odor around the anus, and sticky and bloody stools. The mice in the XLAC-H and SASP groups recovered well, their activities gradually recovered, their fur gradually became smooth and shiny, their feces gradually formed, and there was occasional perianal contamination. The mice in the XLAC-L group were listless, with rough and brittle fur, low gloss, reduced activity, loose stools, and no significant improvement overall. Compared with the control group, the weight of the mice in the other four groups decreased to varying degrees, among which the weight loss in the model group was the most significant ( P <0.001). There was no significant difference between the XLAC-L and SASP groups. The weight of the XLAC-H group was closest to the control group, but there was still a statistically significant difference ( Figure 1 B).

[0043] Compared with the control group, the colon length of the mice in the model group was significantly shortened ( P <0.001), the colon length of the XLAC group, especially the XLAC-H group, was longer than that of the model group ( P <0.001) ( Figure 1 C and 1E).

[0044] The spleen weight of mice in the model group was significantly higher than that in the other four groups. After XLAC treatment, the splenomegaly of mice was reduced and the spleen weight approached that of the control group, indicating that XLAC can alleviate the inflammatory response of UC ( Figure 1 D and 1F).

[0045] Hematoxylin and eosin staining (H&E) H&E staining was performed to observe histological changes in colonic tissue. Colonic tissue was immersed in 4% paraformaldehyde for 24 hours and then embedded in paraffin blocks. Subsequently, the paraffin-embedded tissue was cut into 4-μm-thick sections, dewaxed with xylene, dehydrated with a graded alcohol series, and stained with hematoxylin and eosin. Histopathology of the gastric mucosa was observed under a light microscope.

[0046] HE staining results showed that the intestinal tissue structure of mice in the control group was intact, the mucosal epithelial cells were tightly arranged, the crypt structure was normal, and there was little or no inflammatory cell infiltration; the intestinal tissue of mice in the model group was ulcerated, the crypts and villi were atrophied, and the inflammatory cell infiltration was obvious; the intestinal mucosa of mice in the XLAC-H group was free of ulcers, the crypt structure was basically restored, and the inflammatory cell infiltration was significantly alleviated; the intestinal mucosa of mice in the XLAC-L group and the SASP group was free of ulcers, the crypt structure was partially restored, and the inflammatory cell infiltration was reduced ( Figure 1 G).

[0047] Periodic acid-Schiff (PAS) staining The colonic mucosal barrier was assessed using a PAS staining kit. Paraffin-embedded sections were dewaxed and rehydrated using graded alcohols. Sections were then oxidized with periodic acid solution, stained with Schiff's reagent, and counterstained with hematoxylin. After standard dehydration and procedure, mucin-producing goblet cells were examined under a light microscope.

[0048] Neutral mucin, normally secreted by colonic goblet cells, can be stained red by AB-PAS. In this study, colonic tissue from the control group demonstrated adequate mucus secretion and orderly arrangement of goblet cells, while the model group exhibited significant depletion of goblet cells and a complete absence of the mucus layer. Notably, XLAC-H treatment effectively restored mucus production and normalized goblet cell structure to near-physiological levels. Both the XLAC-L and SASP groups showed partial but significant improvement in these pathological features ( Figure 2 A).

[0049] Immunofluorescence staining Colonic tissue sections were microwaved for antigen retrieval using 0.01 M citrate-EDTA antigen retrieval buffer (Biyuntian, P0086, dilution 1:50) and then cooled naturally. After incubation with hydrogen peroxide solution at 37°C for 10 minutes, sections were blocked with 5% BSA (Biyuntian, ST023) for 30 minutes. Sections were then incubated with ZO-1 (Servicebio, GB111402, dilution 1:500, China) and Occludin (Servicebio, GB11141, dilution 1:50, China) overnight at 4°C. After washing with PBS, sections were incubated with Alexa Fluor 488 (Servicebio, GB25303, dilution 1:500, China) and Coralite 594 (Proteintech, SA00013-3-100, dilution 1:300, USA). Images were captured using an inverted fluorescence microscope. Immunofluorescence analysis further confirmed the significant upregulation of tight junction proteins ZO-1 and Occludin in ileal tissue after XLAC administration ( Figure 2 B and 2C).

[0050] Collectively, the AB-PAS staining and immunofluorescence results provided convincing evidence that XLAC treatment promoted intestinal barrier repair and maintained mucosal homeostasis by restoring the mucus layer and epithelial tight junction complexes.

[0051] Quantitative real-time PCR (qRT-PCR) Total RNA was isolated from colonic tissue using the TransZol Up Plus RNA Kit. cDNA was synthesized from 1 μg of total RNA using the GoScript™ Reverse Transcription System. qRT-PCR amplification was performed using the MonAmp™ ChemoHS qPCR Mix (Monad, MQ00401S, China). All experiments were performed according to the manufacturer's instructions.

[0052] Table 1 Primer list

[0053] Western blot analysis Colonic tissue was weighed and homogenized in protein lysis buffer (1:6, w / v) using a tissue homogenizer. The homogenate was incubated on ice for 30 minutes, vortexed every 10 minutes, and then centrifuged at 12,000 × g for 30 minutes at 4°C. The supernatant was collected, and protein concentration was determined using the BCA assay. Aliquots were stored at −80°C for subsequent analysis. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk in TBST, the membranes were incubated overnight at 4°C with primary antibodies against TLR4 (Proteintech, 19811-1-AP, 1:2000) and GSDMD-N (Affinity, DF13758, 1:1000). After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at 37°C for 2 hours with gentle shaking. Protein bands were visualized using WesternLightning™ chemiluminescent reagent and captured on X-ray film. Quantitative analysis was performed using ImageJ software, and β-actin was used as an internal control for normalization.

[0054] Enzyme-linked immunosorbent assay (ELISA) Blood was collected from mice via ocular bleeding. Serum was obtained by centrifugation. TLR4 concentration in serum was quantified according to the instructions of a specific ELISA kit (Elabscience, E-EL-M2417).

[0055] Medicinal Chemistry Analysis Sample preparation The preconcentration decoction was allowed to stand overnight at 4°C, and the supernatant was collected. Metabolites were extracted with 50% methanol buffer. Briefly, 20 μL of sample was extracted with 120 μL of pre-cooled 50% methanol, vortexed for 1 minute, and incubated at room temperature for 10 minutes; the extraction mixture was then stored at −20°C overnight. After centrifugation at 4000 g for 20 minutes, the supernatant was transferred to a new 96-well plate. Samples were stored at −80°C prior to LC-MS analysis. A pooled QC sample was also prepared by combining 10 μL of each extraction mixture.

[0056] Liquid phase parameters All samples were acquired by the LC-MS system according to machine orders. All chromatographic separations were performed using an ultra-performance liquid chromatography (UPLC) system (SCIEX, UK). An ACQUITY UPLC T3 column (100 mm x 2.1 mm, 1.8 µm, Waters, UK) was used for reversed-phase separation. The column oven was maintained at 35°C. The flow rate was 0.4 ml / min, and the mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid). The gradient elution conditions were as follows: 0–0.5 min, 5% B; 0.5–7 min, 5% to 100% B; 7–8 min, 100% B; 8–8.1 min, 100% to 5% B; 8.1–10 min, 5% B. The injection volume for each sample was 4 µl. Mass spectrometry parameters Metabolites eluted from the column were detected using a high-resolution tandem mass spectrometer, a TripleTOF 5600plus (SCIEX, UK). The Q-TOF was operated in both positive and negative ion modes. The curtain gas was set to 30 PSI, ion source gas 1 to 60 PSI, and ion source gas 2 to 60 PSI. The interface heater temperature was 650°C. For positive ion mode, the ion spray voltage was set to 5000 V. For negative ion mode, the ion spray voltage was set to 4500 V. Mass spectral data were acquired in IDA mode. The TOF mass range was 60 to 1200 Da. Survey scans were acquired in 150 ms, and up to 12 integrated ion scans were collected if the threshold of 100 counts per second (counts / s) was exceeded and the sample was in a 1+ charge state. The total cycle time was fixed at 0.56 seconds. Four time segments for each scan were summed at an 11 kHz pulse frequency using a 40 GHz multichannel TDC detector with four anodes / channel. Dynamic exclusion was set to 4 seconds. During acquisition, mass accuracy was calibrated every 20 samples. In addition, to assess the stability of the LC-MS throughout the acquisition, a quality control sample was collected after every 10 samples.

[0057] Network Pharmacology XLAC's target To identify bioactive compounds from XLAC, a UPLC-Q-TOF-MS method was employed, successfully detecting 460 compounds. Subsequently, a comprehensive list of XLAC-associated targets was obtained by collecting, filtering, and excluding targets from the PubChem, Swiss Target Prediction, and UniProt databases for subsequent analysis.

[0058] Obtain disease-related targets Using "UC" as the disease term, disease-related targets were obtained from the GeneCards database, OMIM database, DisGenNET database, and Drugbank database. The Venny 2.1 platform was used to draw a Venn map to screen for common targets between herbal medicine targets and disease targets.

[0059] Constructing protein-protein interaction (PPI) networks The potential active targets were imported into the String platform, “Homo sapiens” was set as the species, the minimum interaction score was set to 0.4, nodes without interaction links were hidden, and the downloaded TSV files were imported into Cytoscape 3.8.2 software to obtain the protein interaction (PPI) network diagram between XLAC and UC.

[0060] GO function and KEGG pathway enrichment analysis In order to clarify the role of XLAC's potential targets in gene function and signaling pathways, the cross-targets of XLAC in treating UC were input into the DAVID 6.8 platform for gene ontology (GO) functional analysis of cross-target protein genes. P <0.05, the top 10 enriched terms in biological processes, cellular components, and molecular functions were selected. KEGG pathway enrichment analysis was performed using the Metascape platform, and the relevant signaling pathways enriched from cross-targets were filtered by P value to obtain the top 30 pathways.

[0061] A total of 460 XLAC compounds were identified by UPLC-Q-TOF-MS ( Figure 3 A). There are 239 cross-targets between XLAC drug targets and UC disease targets ( Figure 3 B). 239 potential targets were imported into the STRING platform to construct the PPI network diagram of XLAC and UC ( Figure 3 C). According to the degree value, the top three are PIK3R1, PIK3CA, and MAPK3. Among them, inflammatory cytokine-related indicators account for the majority. To further clarify the potential targets and biological functions of XLAC, GO and KEGG pathway analysis were performed. Figure 3As shown in Figures D and E, GO enrichment analysis indicated that multiple genes may synergistically exert biological effects. The molecular functions of XLAC in UC intervention mainly include kinase activity and protein kinase activity, while the biological processes involve hormone responses, cellular responses to nitrogen compounds, and active regulation of responses to external stimuli. KEGG pathway analysis showed that targets were mainly enriched in inflammation-related pathways and signal transduction. Notably, key inflammatory pathways include: chemokine signaling pathway, Toll-like receptor signaling pathway, T cell receptor signaling pathway, C-type lectin receptor signaling pathway, and FcεRI signaling pathway. These findings suggest that the therapeutic effects of the XLAC formula are mainly mediated by inhibiting the inflammatory response.

[0062] Bioinformatics analysis To explore gene expression patterns associated with UC, we searched the Gene Expression Omnibus (GEO) database using the query "UC." Our research enabled us to access the GSE11223 dataset, which includes 63 samples from patients with active UC and 69 samples from healthy subjects. We then shifted our focus to identifying genes associated with Toll-like receptors (TLRs). Using the search queries "TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10," we retrieved relevant gene information from two different disease gene databases. The expression levels of TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 in UC were analyzed using the GSE11223 dataset. Figure 4 As shown in Figure 2, the expression of TLR2, TLR4, and TLR5 in the intestinal epithelium of UC patients was significantly upregulated compared with the normal control group ( P <0.05), suggesting their potential involvement in the pathogenesis of UC. Subsequently, qRT-PCR validation in colonic tissue of UC mice confirmed elevated TLR4 expression compared with normal controls and that TLR4 expression was effectively downregulated after XLAC treatment. In summary, integrated bioinformatics and experimental approaches identified TLR4 as a key mediator of XLAC treatment in UC.

[0063] Molecular docking Based on the results of network pharmacology, the top five core active ingredients ranked by degree value were searched in the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and their 3D structure files were downloaded. The protein structure of TLR4 was searched in the PDB protein structure database (https: / / www.rcsb.org / ), and its protein structure file was downloaded. Molecular docking was performed using the CB-Dock2 platform (https: / / cadd.labshare.cn / cb-dock2 / php / index.php), and the binding between the active ingredient and the target protein was understood based on the minimum Vina score, with lower values indicating tighter binding. A docking score lower than -5 was considered significant and represented the docking score of the compound and protein. The ggplot package was used to complete the information display, depicting the optimal docking image of the receptor and ligand after visualization. Finally, the screened targets were verified by animal experiments.

[0064] The binding interaction between the active ingredient and the target protein was evaluated based on the minimum Vina score, where lower values indicate stronger binding affinity. The docking score threshold was set to <-5 to define significant binding activity, representing the interaction strength between the compound and the protein ( Figure 5 D). Visualization of the optimal receptor-ligand docking conformation using the ggplot package ( Figure 5 A, 5B and 5C).

[0065] The results showed that TLR4 has good binding affinity to key components of XLAC, including trans-4-coumaric acid, methyl cinnamate, and 2-furylmethyl butyrate. These findings suggest that XLAC may exert its therapeutic effects through TLR4-related signaling pathways.

[0066] ELISA assay showed that the expression of TLR4 in the serum of mice in the model group was significantly increased compared with that in the control group ( P <0.001), XLAC treatment effectively reduced the expression of TLR4 ( Figure 6 A). WB analysis showed that the protein expressions of TLR4 and GSDMD-N in the colon tissue of the model group were significantly increased compared with the control group ( P <0.001); compared with the model group, the XLAC treatment group significantly reduced the protein levels of TLR4 and GSDMD-N in the colon of UC mice in a dose-dependent manner ( P <0.05) ( Figure 6 B and 6D).

[0067] qRT-PCR analysis revealed that the expression of IL-1β and IL-18 mRNA, which are associated with pyroptosis, increased significantly in the model group, while the expression levels decreased after XLAC treatment ( P <0.001), indicating that XLAC can play a therapeutic role by inhibiting TLR4-mediated pyroptosis ( Figure 6 G and 6H).

[0068] Further detection of the TLR4-mediated pyroptosis pathway revealed that the expression of TLR4, NLRP3, and Caspase-1 mRNA in the colon tissue of the model group mice was significantly increased compared with the control group ( P <0.001) ( Figure 6 E and 6F).

[0069] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Application of Xianglian Anchang Decoction in the preparation of drugs for the treatment of ulcerative colitis by targeting the TLR4 / NLRP3 / GSDMD pathway.

2. The use according to claim 1, characterized in that The drug inhibits the expression of TLR4 protein.

3. The use according to claim 1, characterized in that The drug inhibits the expression of GSDMD protein.

4. The use according to claim 1, characterized in that The drug inhibits the expression of NLRP3 protein.

5. The use according to claim 1, characterized in that The dosage form of the medicine includes decoction, honey pills, granules or oral liquid.

6. The use according to claim 1, characterized in that The drug includes a pharmaceutically acceptable carrier.

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