Application of angong niuhuang in preparation of medicine for treating ulcerative diseases

Abstract

The present invention relates to the technical field of medicine, in particular to a traditional Chinese medicine composition for treating ulcerative diseases, and especially to a new use of Angong Niuhuang in the treatment of ulcerative diseases. The Angong Niuhuang of the present invention is composed of Calculus Bovis, Cornu Bubali, Moschus, Margarita, Cinnabaris, Realgar, Rhizoma Coptidis, Radix Scutellariae, Fructus Gardeniae, Radix Curcumae, Borneolum Syntheticum, and Aurum Film. Clinical applications show that Angong Niuhuang Pills can effectively treat skin ulcers and peptic ulcers, including diseases such as diabetic ulcers, hypertensive ulcers, oral ulcers, and gastric ulcers. The results of animal studies indicate that Angong Niuhuang can significantly improve the clinical symptoms in a mouse model of DSS-induced ulcerative colitis, alleviate colon damage, and regulate intestinal barrier function through antioxidant and anti-inflammatory effects. Angong Niuhuang can significantly inhibit the expression of inflammatory factors (such as TNF-α and IL-6), and increase the level of the anti-inflammatory factor IL-10. In addition, Angong Niuhuang improves the expression of intestinal tight junction proteins, reduces intestinal permeability, so as to effectively repair the intestinal barrier function. No toxic reaction to the main organs of mice was found in the experiment, showing that it has relatively high safety.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to the application of a traditional Chinese medicine compound—Angong Niuhuang—in the treatment of ulcerative diseases. Background Technology

[0002] Ulcerative diseases include peptic ulcers and skin ulcers. Peptic ulcers, also known as digestive tract ulcers, include oral ulcers, gastric ulcers, and intestinal ulcers, among which ulcerative colitis is particularly difficult to treat.

[0003] Ulcerative colitis (UC) is a chronic intestinal disease characterized by persistent inflammation of the colonic mucosa, typically accompanied by symptoms such as abdominal pain, diarrhea, rectal bleeding, and weight loss. This disease is most common in young adults, with a peak incidence typically between 20 and 40 years of age. Its global incidence is increasing annually, particularly in certain regions of Europe, America, and Asia, where changes in lifestyle and environmental factors have led to a continuous rise in prevalence. Despite this, the pathogenesis of ulcerative colitis is not yet fully understood. Experts generally believe that the occurrence of ulcerative colitis is closely related to genetic susceptibility, immune system dysregulation, gut microbiota imbalance, and environmental factors. Genetic factors may make some individuals more sensitive to the immune response in the gut, thus making them more susceptible to the disease; simultaneously, adverse interactions between gut microbiota and the immune system are also an important pathogenic mechanism. Environmental factors, such as poor dietary habits, smoking, drug abuse, and infections, may further exacerbate the occurrence of ulcerative colitis.

[0004] Currently, treatment for ulcerative colitis mainly falls into two categories: drug therapy and surgical treatment. Drug therapy typically uses 5-aminosalicylic acid (5-ASA) drugs, suitable for relieving mild to moderate symptoms. For patients with acute flare-ups, steroids are often used to quickly control symptoms, although long-term use of steroids may lead to side effects such as immunosuppression and osteoporosis. Immunosuppressants and biologics are used to help patients who do not respond to traditional treatments, especially in moderate to severe cases. However, these treatments often come with significant side effects, and some patients respond poorly to these drugs, limiting their effectiveness. Furthermore, long-term use of immunosuppressants and biologics may lead to problems such as infection and drug resistance. Therefore, finding effective treatments with fewer side effects has become a key research focus in the field of ulcerative colitis treatment.

[0005] Traditional Chinese medicine (TCM) offers unique advantages as a traditional treatment for ulcerative colitis. TCM theory suggests that ulcerative colitis is often related to multiple factors, including spleen and stomach weakness, internal damp-heat, liver qi stagnation, and disharmony of qi and blood. TCM treatment not only focuses on symptomatic relief but also emphasizes regulating organ function, restoring qi and blood balance, and enhancing immune function. In treatment, TCM can effectively alleviate clinical symptoms by regulating the immune response, inhibiting excessive immune activation, and reducing chronic intestinal inflammation. Compared to traditional drugs, TCM has fewer side effects, can be used long-term, and is less likely to induce drug resistance. Furthermore, research shows that TCM can improve intestinal barrier function, enhance the integrity of intestinal epithelial cells, reduce intestinal permeability, and decrease the entry of harmful substances into the intestine, helping to prevent further intestinal damage. Due to these advantages, TCM has significant efficacy in the treatment of ulcerative colitis, particularly in improving patients' quality of life and alleviating long-term symptoms. Integrating the advantages of TCM theory with modern scientific research findings can provide more options and strategies for the clinical treatment of ulcerative colitis.

[0006] In the pathological mechanisms of ulcerative colitis (UC), abnormal immune responses and impaired intestinal barrier function are core factors. Damage to the intestinal barrier function allows harmful substances, pathogens, and cytokines to penetrate the intestinal epithelium, activating local immune responses and leading to chronic inflammation. In recent years, increasing research has shown that regulating signaling pathways related to intestinal barrier function can effectively alleviate ulcerative colitis symptoms and promote intestinal repair and regeneration.

[0007] In ulcerative colitis research, the TGF-β and Wnt / β-catenin pathways have become key pathways for intestinal barrier function repair. The TGF-β pathway participates in regulating intestinal immune responses and epithelial cell proliferation and repair. Studies have shown that TGF-β can promote the growth and regeneration of intestinal epithelial cells, repair damaged intestinal barriers, and thus alleviate intestinal inflammation. The Wnt / β-catenin pathway plays an important role in the homeostasis and regeneration of intestinal epithelial cells, regulating their proliferation, differentiation, and migration.

[0008] Currently, the TGF-β and Wnt / β-catenin pathways have received widespread attention in the treatment of ulcerative colitis. By modulating these signaling pathways, researchers have found that they can effectively improve intestinal barrier function and reduce intestinal inflammation. For example, enhanced TGF-β signaling can promote the repair of intestinal epithelial cells, improve the integrity of the intestinal barrier, reduce intestinal permeability, and reduce the overactivation of the intestinal immune system, thereby improving the pathological symptoms of ulcerative colitis.

[0009] Furthermore, the Wnt / β-catenin pathway is also considered to play a key role in the restoration of intestinal barrier function. By promoting the self-repair of the intestinal epithelium, Wnt / β-catenin signaling not only helps enhance the integrity of the intestinal barrier but also improves clinical symptoms by reducing intestinal inflammation.

[0010] With in-depth research into the TGF-β and Wnt / β-catenin pathways, the treatment strategy for ulcerative colitis has gradually shifted from single-target immunosuppression to multi-target therapy, especially by regulating the repair of intestinal barrier function to achieve therapeutic effects. By using specific drugs or natural Chinese herbal medicines, these key signaling pathways can be effectively regulated, promoting the repair and reconstruction of the intestinal barrier, thereby alleviating the symptoms of ulcerative colitis.

[0011] In summary, by regulating the TGF-β and Wnt / β-catenin signaling pathways and combining them with the advantages of traditional Chinese medicine, a safer and more effective treatment plan can be provided for ulcerative colitis.

[0012] Angong Niuhuang Wan originated from the "Treatise on Warm Diseases - Upper Jiao Chapter," written by Wang Mengying, a renowned physician of the Qing Dynasty and one of the founders of the Warm Disease School. The main ingredients include bezoar, concentrated buffalo horn powder, musk, pearl, cinnabar, realgar, Coptis chinensis, Scutellaria baicalensis, gardenia, turmeric, and borneol, all carefully selected under the guidance of traditional Chinese medicine theory. Traditionally, Angong Niuhuang Wan is believed to have the effects of "clearing heat and detoxifying, calming the nerves and relieving convulsions," and is mainly used to treat brain diseases caused by the invasion of heat toxins, such as high fever with delirium, epilepsy, meningitis, and other acute conditions.

[0013] Whether Angong Niuhuang can treat ulcerative diseases has not yet been reported, and further research is needed to provide a new drug for clinical use. Summary of the Invention

[0014] The purpose of this invention is to provide a novel use of Angong Niuhuang in the preparation of drugs for treating ulcerative diseases.

[0015] The Angong Niuhuang formula of this invention is made from multiple raw medicinal materials, including bezoar, concentrated buffalo horn powder, musk or artificial musk, pearl, cinnabar, realgar, Coptis chinensis, Scutellaria baicalensis, gardenia, turmeric, and borneol. The principal ingredients, bezoar and concentrated buffalo horn powder, have the effects of clearing heat and detoxifying, cooling blood and eliminating toxins. The assistant ingredients, musk and borneol, open the orifices and refresh the mind, improving central nervous system function; pearl and cinnabar calm the nerves and relieve convulsions; gardenia and Scutellaria baicalensis clear heat and cool blood; Coptis chinensis has anti-inflammatory and detoxifying effects; turmeric regulates qi and eliminates dampness; and realgar kills parasites and detoxifies. All these are adjuvant ingredients, working synergistically to enhance anti-inflammatory, intestinal barrier repair, and immune regulation functions. The combined effects of these herbs clear heat and detoxify, cool blood and reduce inflammation, showing significant efficacy in the treatment of ulcerative diseases. Attached Figure Description

[0016] Figure 1 Effects of Angong Niuhuang on activity scores in mice with ulcerative colitis. Compared to the control group, **** P < 0.0001, *** P<0.001, ** P<0.01, * P < 0.05 ns No significant difference.

[0017] Figure 2 Effects of Angong Niuhuang on white blood cells, red blood cells, and platelets in mice with ulcerative colitis. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference.

[0018] Figure 3 Effects of Angong Niuhuang on ALT and AST in mice with ulcerative colitis. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference.

[0019] Figure 4 Effects of Angong Niuhuang on inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in mice with ulcerative colitis. Compared with the control group, **** P<0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference.

[0020] Figure 5 Effects of Angong Niuhuang on the levels of MDA (malondialdehyde), SOD (superoxide dismutase), and GSH (glutathione) in mice with ulcerative colitis. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference.

[0021] Figure 6 Effects of Angong Niuhuang on the levels of tight junction proteins ZO-1, Oecludin, and Claudin in mice with ulcerative colitis. Compared with the control group, ****P < 0.0001, *** P < 0.001, *** P < 0.01, * P < 0.05 ns No significant difference.

[0022] Figure 7 Effects of Angong Niuhuang on intestinal permeability markers (FITC-dextran penetration and LPS levels) in mice with ulcerative colitis. Compared with the control group, **** P < 0.0001, *** P<0.001, ** P < 0.01, * P < 0.05 ns No significant difference. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Specific Example 1: A successful case of using Angong Niuhuang Wan to prevent and treat ulcerative diseases

[0025] Case 1

[0026] Mr. Huang, 45 years old, developed an oral ulcer after biting his lip while eating. The ulcer was irregularly white with red edges and was painful. He broke off about 1 / 3 of a pill of Angong Niuhuang Wan (3g / pill), dissolved it in a small amount of boiling water, and applied it to the ulcer with chopsticks. There was no irritation, and the pain was significantly reduced after about half an hour. He applied it three times a day, and the pain of the ulcer completely disappeared after the second day. After 5 days of continuous use, the ulcer healed.

[0027] Case 2

[0028] Ms. Kong, 48 years old, had suffered from premenstrual oral ulcers for more than 20 years, mostly on the tongue, lips, and inner walls of the mouth. In the early stages, it would heal in about 7 days, but in recent years it has taken about 10 days to heal. This time, she just felt pain on the tip of her tongue and saw small round ulcers. She applied Angong Niuhuang Wan suspension to the ulcers as described above, and the pain quickly subsided. On the second day, only small red spots were visible on the ulcers, and they healed completely on the third day. The ulcers were cured before they could develop.

[0029] Case 3

[0030] Mr. Zhao, 38 years old, had suffered from gastric ulcers for many years with recurrent flare-ups. This time, the flare-up was caused by alcohol consumption. He experienced upper abdominal pain with a burning sensation. He broke Angong Niuhuang Wan (3g / pill) in half, chewed and swallowed it, and took one pill daily. The pain disappeared after one day.

[0031] To consolidate the therapeutic effect, take it continuously for 7 days, and all symptoms will disappear.

[0032] Case 4

[0033] Mr. Zhang, 53 years old, had been suffering from diabetes for more than 20 years. He developed a fistula on his back, with symptoms including pus, blood and other fluid oozing out. There was an ulcer at the opening of the fistula, which was red, swollen and painful. After dissolving a small amount of Angong Niuhuang Wan in a small amount of boiling water, he used a clean gauze soaked in the medicine to insert into the fistula and applied the medicine to the outside as well. After one day, the redness and swelling subsided and the pain at the ulcer disappeared. He continued to use the medicine for two weeks, and the fistula closed and healed, and the ulcer healed completely.

[0034] Case 5

[0035] Ms. Li, 72 years old, had suffered from hypertension for more than ten years. She experienced unbearable itching in her calves, which worsened at night. Each time it flared up, she could only relieve the pain by soaking her legs in hot water. When she scratched, ulcers formed, causing pain, burning, and itching. She dissolved Angong Niuhuang Wan (a traditional Chinese medicine) in a small amount of boiling water and then applied the medicine to the ulcers with a cotton swab. The pain disappeared after one day. After using the medicine for a week, the ulcers healed completely.

[0036] Example 2: The intervention effect of Angong Niuhuang on a mouse model of ulcerative colitis

[0037] 1. Experimental Objective

[0038] This study aimed to investigate the therapeutic effect of Angong Niuhuang on a DSS-induced ulcerative colitis (UC) model mouse, exploring its pharmacological mechanisms through multiple pathways, including improving the intestinal barrier, alleviating intestinal inflammation, and regulating the immune response. The study analyzed its protective effect against colonic injury and its efficacy in alleviating clinical symptoms through oral administration of a suspension.

[0039] 2. Experimental materials:

[0040] Laboratory animals:

[0041] Eight-week-old male C57BL / 6 mice, weighing approximately 20-25g, were purchased from the laboratory animal center and acclimatized for one week.

[0042] The experimental mice were randomly divided into 5 groups (n=8 per group): normal group, model group, low-dose Angong Niuhuang group (50 mg / kg), medium-dose Angong Niuhuang group (100 mg / kg), and high-dose Angong Niuhuang group (200 mg / kg).

[0043] drug:

[0044] Angong Niuhuang Suspension: Angong Niuhuang powder was prepared according to the prescription ratio of Angong Niuhuang Pill in the Chinese Pharmacopoeia, and then dissolved in 0.9% sodium carboxymethyl cellulose to obtain Angong Niuhuang suspension.

[0045] DSS (2,4,6-trinitrobenzenesulfonic acid) solution was used to induce an ulcerative colitis model.

[0046] Reagents and instruments:

[0047] ELISA kit (detection of cytokines such as TNF-α, IL-1β, IL-6, and IL-10).

[0048] HE staining reagent.

[0049] Blood routine analysis reagents.

[0050] Blood biochemistry analysis reagents.

[0051] Microscope (used for observing histological sections).

[0052] 3. Experimental Methods

[0053] 3.1 Establishment of an ulcerative colitis model

[0054] After one week of acclimatization, an ulcerative colitis model was established by oral administration of DSS solution (2% concentration) for 7 consecutive days. Mice in both the model group and the treatment group received this treatment.

[0055] Starting on day 1 of DSS induction, patients were treated with oral administration of Angong Niuhuang Suspension according to their respective groups: low-dose group (50 mg / kg), medium-dose group (100 mg / kg), and high-dose group (200 mg / kg), for 14 consecutive days.

[0056] 3.2 Activity Scoring (Truelove-Witts Scoring System)

[0057] During the experiment, the mice were observed for clinical symptoms daily, and their activity levels were assessed. The activity scoring system was based on behavioral changes such as diarrhea and bloody stools, and was evaluated according to the following criteria:

[0058] Truelove-Witts rating criteria:

[0059] Diarrhea (score range 0-3):

[0060] 0: Normal, stool remains unchanged.

[0061] 1: Mild diarrhea (1-2 loose stools).

[0062] 2: Moderate diarrhea (2-3 loose stools).

[0063] 3: Severe diarrhea (more than 3 episodes of diarrhea).

[0064] Blood in stool (score range 0-3):

[0065] 0: No blood in stool.

[0066] 1. Occasional bloody stools.

[0067] 2: Persistent bloody stools.

[0068] 3: Severe bloody stools or obvious fresh blood in the stool.

[0069] A total score is calculated by combining the scores from the two aspects mentioned above, ranging from 0 to 6. The higher the total score, the more severe the disease activity.

[0070] 3.3 Post-Experiment Processing and Sample Collection

[0071] After the experiment, the mice were euthanized and their colon tissue was removed.

[0072] HE staining was used to prepare colon tissue sections to observe pathological changes in the colon, such as ulceration and degree of inflammation.

[0073] 3.4 Complete blood count and blood biochemistry analysis

[0074] Blood samples were collected for routine blood tests and blood biochemistry analysis, mainly to detect indicators such as white blood cell count, red blood cell count, platelet count, liver function, and kidney function, in order to assess the effects of Angong Niuhuang on the blood system and liver and kidney function.

[0075] 3.5 ELISA Detection

[0076] The levels of serum cytokines such as TNF-α, IL-1β, IL-6, and IL-10 were detected by ELISA to evaluate the regulatory effect of Angong Niuhuang on inflammatory factors.

[0077] 4 Experimental Results

[0078] 4.1 Activity score and clinical symptoms

[0079] According to the Truelove-Witts scoring system, the clinical symptoms of mice differed significantly among different groups. Normal group: No obvious diarrhea, bloody stools, or other clinical symptoms were observed; the activity score was 0, indicating the lowest disease severity. Control group (DSS treatment group): Mice exhibited obvious diarrhea, bloody stools, and decreased intestinal motility; the activity score was significantly increased, demonstrating severe colitis. Angong Niuhuang intervention groups (high-dose, medium-dose, and low-dose groups): The activity scores of mice in all intervention groups were significantly lower than those in the control group, especially the high-dose group, where diarrhea and bloody stool symptoms were significantly alleviated, and the activity score was significantly lower than that in the control group (P<0.01). These results indicate that Angong Niuhuang can significantly alleviate the symptoms of ulcerative colitis induced by DSS.

[0080] 4.2 Histological observation

[0081] Normal group: The colonic tissue was intact, the intestinal wall structure was clear, and there was no obvious inflammatory infiltration. Control group: The colonic wall showed obvious ulceration, infiltration, and bleeding, and the mucosal layer was severely damaged, indicating severe damage to the colonic tissue. Angong Niuhuang intervention group: - The degree of colonic tissue damage gradually decreased in each dose group, especially in the high-dose group, where the intestinal wall structure was relatively intact, and the ulcer area and...

[0082] Inflammatory infiltration was significantly reduced, with a statistically significant difference compared to the control group (P<0.05).

[0083] 4.3 Complete blood count and blood biochemistry analysis:

[0084] Blood routine

[0085] Normal group: White blood cell, red blood cell, and platelet counts remained at normal levels. Control group: White blood cell count was significantly elevated, while red blood cell and platelet counts decreased, reflecting a severe inflammatory response. Angong Niuhuang intervention group: The white blood cell count in mice in the high-dose group was significantly lower than that in the control group, while red blood cell and platelet counts were close to normal levels, with the most significant changes observed in the high-dose group.

[0086] Blood biochemistry

[0087] Normal group: All blood biochemical indicators were normal. Control group: Hypoalbuminemia, and significantly elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating impaired liver function. Angong Niuhuang intervention group: Serum ALT and AST levels in the high-dose group were significantly lower than those in the control group, while serum albumin levels were close to normal, indicating that Angong Niuhuang has a protective effect on liver function.

[0088] 4.4 ELISA detection of cytokine levels:

[0089] Normal group: Levels of all cytokines were within the normal range. Control group: Levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 were significantly increased, while IL-10 levels were decreased, indicating a significant inflammatory response. Angong Niuhuang intervention group: The serum concentrations of TNF-α, IL-1β, and IL-6 in the high-dose group were significantly lower than those in the control group (P<0.01), while IL-10 levels were significantly increased, suggesting that Angong Niuhuang can effectively inhibit the excessive secretion of inflammatory factors.

[0090] 5. Conclusion

[0091] This study demonstrates that Angong Niuhuang has a significant interventional effect on DSS-induced ulcerative colitis in mice. By improving activity scores, reducing colonic tissue damage, and inhibiting excessive secretion of inflammatory factors, Angong Niuhuang significantly alleviated the symptoms of ulcerative colitis. The high-dose group exhibited the best efficacy, indicating that Angong Niuhuang has a strong anti-inflammatory effect and can effectively protect the intestinal barrier and improve intestinal function. Furthermore, complete blood count and blood biochemistry analyses further support the protective effect of Angong Niuhuang on the liver and overall health. These results provide experimental evidence for the use of Angong Niuhuang in the treatment of ulcerative colitis.

[0092] Example 3: The intervention effect of Ben'an Gong Niuhuang in an ulcerative colitis model

[0093] 1. Experimental Objective

[0094] This study aimed to investigate the effect of Angong Niuhuang on intestinal barrier repair in mice with ulcerative colitis. The activity of antioxidant enzymes such as MDA, SOD, and GSH was analyzed to assess the role of Angong Niuhuang in improving oxidative stress. Simultaneously, the expression of intestinal tight junction proteins (ZO-1, Occludin, and Claudin) was analyzed using ELISA and RT-qPCR to detect the intestinal barrier repair effect. Furthermore, the degree of intestinal barrier damage was assessed by measuring intestinal permeability markers (such as FITC-dextran and LPS), thereby revealing the pharmacological mechanism by which Angong Niuhuang treats ulcerative colitis by repairing the intestinal barrier.

[0095] 2 Experimental Materials

[0096] 2.1 Animals

[0097] The animals used in the experiment were 6-8 week old female C57BL / 6 mice purchased from the laboratory animal center. Mice were acclimatized for one week under environmental conditions controlled at 22±2℃, humidity 50±5%, and a 12-hour light / 12-hour dark cycle. They were fasted for one week prior to the experiment to ensure an empty gut.

[0098] 2.2 Antioxidant enzyme detection reagents

[0099] MDA assay kit, SOD assay kit, and GSH assay kit. All reagents were used according to the manufacturer's instructions. Each indicator was measured using a microplate reader (550nm).

[0100] 2.3 Cytokine Detection

[0101] TNF-α, IL-1β, IL-6, IL-10 ELISA kit

[0102] 2.4 Intestinal barrier-related factors:

[0103] ZO-1, Occludin, Claudin, and other ELISA kits

[0104] 2.5 RT-qPCR reagent

[0105] SYBR Green PCR Master Mix

[0106] cDNA Synthesis Kit

[0107] PCR primers: Specific primers for tight linker protein genes such as ZO-1, OcCludin, and Claudin.

[0108] FITC-dextran (4kDa) was administered orally via gavage, and intestinal permeability was assessed by serum FITC concentration.

[0109] The LPS ELISA kit is used to detect LPS levels in mouse serum and assess intestinal barrier damage.

[0110] 3 Experimental Methods

[0111] 3.1 Oxidative Stress Analysis

[0112] The extracted colon tissue was frozen at -80°C, homogenized using a tissue homogenizer, and then the MDA, SOD, and GSH biochemical reagent kits were tested according to their instructions. The MDA content, SOD activity, and GSH activity were measured respectively.

[0113] MDA is the final product of lipid peroxidation and can reflect the level of oxidative stress. SOD is superoxide dismutase, which mainly catalyzes the conversion of superoxide anions in cells and is a key enzyme in antioxidant reactions. GSH, as an important antioxidant molecule, can scavenge free radicals and maintain the antioxidant function of cells.

[0114] During the assay, the procedure for each sample was followed according to the kit instructions. The absorbance (OD value) of the reaction solution was measured using an ELISA reader (550nm), and then the MDA content and the activities of SOD and GSH were calculated.

[0115] 3.2 Molecular analysis related to the intestinal barrier

[0116] The effects of Angong Niuhuang on ulcerative colitis-related cytokines were analyzed by detecting inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-10 in mouse serum using an ELISA kit.

[0117] At the end of the experiment, the serum of the mice was collected, and the samples were added to the pre-coated ELISA plate according to the kit instructions. The corresponding secondary antibody and substrate were added, and the absorbance value was measured using an ELISA reader (450nm). The concentration of each factor was calculated according to the standard curve.

[0118] 3.3 RT-qPCR detection of tight junction protein expression

[0119] Total RNA was extracted from colon tissue, cDNA was synthesized using a reverse transcription kit, and qPCR was performed using SYBR Green.

[0120] Specific primers were designed for intestinal barrier-related genes such as ZO-1, Occludin, and Claudin, and their expression levels were finally analyzed by real-time quantitative PCR.

[0121] By calculating the relative expression levels of CT values ​​and GAPDH internal reference genes, we evaluated whether Angong Niuhuang could improve the expression of tight junction proteins (such as ZO-1, Occludin, and Claudin) and thus repair the intestinal barrier.

[0122] 3.4 Intestinal permeability analysis

[0123] Using FITC-dextran (4kDa) as a marker molecule, the drug was administered orally, and changes in intestinal permeability were assessed by measuring the concentration of FITC-dextran in serum.

[0124] After the mouse experiment, FITC-dextran (40 mg / kg) was administered by gavage. Blood was collected from the mice 30 minutes later, and the fluorescence intensity of FITC in the serum (485 / 530 nm) was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The intestinal permeability was quantitatively analyzed by comparing the results with a standard curve.

[0125] The degree of intestinal barrier disruption was further assessed by measuring serum LPS (lipopolysaccharide) levels. LPS is a marker of intestinal barrier damage caused by gut microbiota dysbiosis, and serum LPS levels reflect changes in intestinal permeability. ELISA detection was performed according to the LPS kit instructions.

[0126] 4. Experimental Results

[0127] 4.1 Antioxidant enzyme activity and oxidative stress

[0128] Normal group: MDA content was (3.2±0.5) nmol / mg, indicating that intestinal oxidative stress was at a normal level. Control group (DSS-induced group): MDA content was significantly increased, reaching (7.1±1.0) nmol / mg, indicating that the colonic inflammatory response induced by DSS led to a significant increase in oxidative damage. Angong Niuhuang intervention group: MDA content in the high-dose group was (4.0±0.8) nmol / mg, the medium-dose group was (5.2±0.7) nmol / mg, and the low-dose group was (6.3±0.9) nmol / mg. Compared with the control group, the high-dose group showed the most significant reduction in MDA, and the difference was statistically significant (P<0.01).

[0129] Normal group: SOD activity was (120.3±12.4) U / mg. Control group: SOD activity was significantly reduced, measured at (56.8±9.2) U / mg, indicating that DSS-induced colitis aggravated oxidative stress. Angong Niuhuang intervention group: SOD activity in the high-dose group was (96.5±14.1) U / mg, in the medium-dose group it was (78.4±11.8) U / mg, and in the low-dose group it was (63.7±9.9) U / mg. The SOD activity in the high-dose group was significantly higher than that in the control group (P<0.01), indicating that Angong Niuhuang can significantly improve oxidative stress.

[0130] Normal group: GSH content was (110.2±15.7) μmol / g. Control group: GSH content decreased to (52.3±8.6) μmol / g, reflecting a decline in the function of the antioxidant system during ulcerative colitis. Angong Niuhuang intervention group: GSH content in the high-dose group was (94.5±13.0) μmol / g, in the medium-dose group it was (75.1±10.8) μmol / g, and in the low-dose group it was (58.2±8.3) μmol / g. The GSH level in the high-dose group was significantly higher than that in the control group (P<0.01), indicating that Angong Niuhuang can effectively enhance the antioxidant capacity of the intestine.

[0131] 4.2 Expression of tight junction proteins:

[0132] Normal group: ZO-1 expression level was (1.0±0.2) ng / mg protein. Control group: ZO-1 expression significantly decreased to (0.2±0.1) ng / mg protein, indicating that DSS-induced intestinal damage significantly reduced the expression of tight junction proteins, leading to impaired intestinal barrier function. Angong Niuhuang intervention group: ZO-1 expression was (0.7±0.1) ng / mg protein in the high-dose group, (0.5±0.1) ng / mg protein in the medium-dose group, and (0.4±0.1) ng / mg protein in the low-dose group. The ZO-1 expression level in the high-dose group was significantly higher than that in the control group (P<0.01), indicating that Angong Niuhuang can effectively repair the intestinal barrier.

[0133] Occludin (tight junction protein):

[0134] Normal group: Occludin expression was (0.9±0.1) ng / mg protein. Control group: Occludin expression was significantly reduced to (0.3±0.1) ng / mg protein. Angong Niuhuang intervention group: Occludin expression in the high-dose group was (0.8±0.1) ng / mg protein, in the medium-dose group it was (0.6±0.1) ng / mg protein, and in the low-dose group it was (0.5±0.1) ng / mg protein. The Occludin level in the high-dose group was significantly higher than that in the control group (P<0.01), demonstrating the repair effect of Angong Niuhuang on the intestinal barrier.

[0135] Claudin (Claudin protein):

[0136] Normal group: Claudin expression was (1.1±0.1) ng / mg protein. Control group: Claudin expression was significantly decreased to (0.4±0.1) ng / mg protein. Angong Niuhuang intervention group: Claudin expression in the high-dose group was (1.0±0.1) ng / mg protein, in the medium-dose group it was (0.8±0.1) ng / mg protein, and in the low-dose group it was (0.6±0.1) ng / mg protein. The Claudin level in the high-dose group was significantly higher than that in the control group (P<0.01).

[0137] 4.3 Detection of intestinal permeability markers:

[0138] FITC-dextran penetration:

[0139] Normal group: FITC-dextran penetration was (6.5±0.8) μg / mL. Control group: FITC-dextran penetration significantly increased to (15.3±1.2) μg / mL, indicating that DSS treatment led to increased intestinal permeability. Angong Niuhuang intervention group: FITC-dextran penetration in the high-dose group was (8.2±1.0) μg / mL, in the medium-dose group it was (10.1±1.0) μg / mL, and in the low-dose group it was (12.5±1.1) μg / mL. The intestinal permeability of the high-dose group was significantly lower than that of the control group (P<0.01), indicating that Angong Niuhuang can alleviate intestinal barrier damage.

[0140] LPS (lipopolysaccharide) levels:

[0141] Normal group: LPS content was (0.2±0.05) ng / mL. Control group: LPS content increased to (1.2±0.1) ng / mL. Angong Niuhuang intervention group: LPS content in the high-dose group was (0.5±0.1) ng / mL, the medium-dose group was (0.8±0.1) ng / mL, and the low-dose group was (1.0±0.1) ng / mL. The LPS level in the high-dose group was significantly lower than that in the control group (P<0.01), further demonstrating that Angong Niuhuang can effectively repair the intestinal barrier.

[0142] 5. Conclusion

[0143] The results of this experiment indicate that Angong Niuhuang can significantly improve oxidative stress, repair the intestinal barrier, and alleviate colonic damage in mice with DSS-induced ulcerative colitis. By increasing the activity of antioxidant enzymes (such as SOD and GSH), decreasing MDA levels, and enhancing the expression of tight junction proteins (such as ZO-1, Occludin, and Claudin), Angong Niuhuang effectively repairs the intestinal barrier. Furthermore, the inhibitory effect of Angong Niuhuang on intestinal permeability markers such as FITC-dextran and LPS further demonstrates its protective effect on the intestinal barrier. These results provide experimental evidence for the treatment and prevention of ulcerative colitis and reveal its possible pharmacological mechanism.

Claims

1. The use of Angong Niuhuang in the preparation of drugs for treating ulcerative diseases, characterized in that, Angong Niuhuang is made from raw materials including bezoar, buffalo horn, musk, pearl, cinnabar, realgar, coptis, scutellaria, gardenia, turmeric, borneol, and gold leaf (or without gold leaf).

2. The use according to claim 1, characterized in that, The treatment of ulcerative diseases includes peptic ulcers and skin ulcers.

3. The use according to claim 1, characterized in that, The aforementioned peptic ulcers include oral ulcers, pharyngeal ulcers, gastric ulcers, duodenal ulcers, intestinal ulcers, etc.

4. The use according to claims 1-3, characterized in that, Angong Niuhuang Wan can significantly improve pain and edema caused by ulcers; Angong Niuhuang Wan can significantly improve clinical symptoms in a mouse model of ulcerative colitis induced by DSS. Experiments show that it achieves its therapeutic effect by reducing diarrhea, bloody stools, abnormal intestinal motility, and colon damage.

5. The use according to claims 1-3, characterized in that, Angong Niuhuang can significantly reduce the expression levels of inflammatory factors (such as TNF-α and IL-6) in a mouse model of ulcerative colitis and significantly increase the level of the anti-inflammatory factor IL-10, thus exhibiting significant anti-inflammatory effects.

6. The use according to claims 1-3, characterized in that, Angong Niuhuang can improve intestinal barrier function in a mouse model of ulcerative colitis, significantly increase the expression of tight junction proteins (such as ZO-1, Occludin, and Claudin), reduce intestinal permeability, and repair colonic damage.

7. The use according to claims 1-6, characterized in that, The aforementioned Angong Niuhuang raw material and pharmaceutically acceptable excipients are used to prepare a clinical preparation; the dosage form of the drug includes, but is not limited to, liquid preparations, solid preparations and semi-solid preparations.

8. The use as described in any one of claims 1-7, characterized in that, The human preventive dose of the Angong Niuhuang raw material is 0.01-1.0g / 60kg / d.

9. The use as described in any one of claims 1-7, characterized in that, The human therapeutic dose of the Angong Niuhuang raw material is 0.1-5g / 60kg / day.

10. The use as described in claim 9, characterized in that, The preferred human therapeutic dose of the Angong Niuhuang raw material is 0.2-3g / 60kg / d.

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