4,5,2'-trihydroxy-2,5'-dibromobenzophenone for use in a medicament for the treatment of inflammatory bowel disease

Abstract

The present application relates to a new use of halogenophenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone, in particular in the preparation of a drug for treating inflammatory bowel disease. The halogenophenol compound of the present application can significantly alleviate the weight loss and colon shortening of the colonitis model, reduce the disease activity index of the colonitis model, reduce the inflammatory cell infiltration of the colonitis model, reduce the expression of inflammatory factors, has an immune regulation function on the intestinal tract, and is superior to the clinical first-line drug mesalazine in most indicators, and has application prospects in the treatment of inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology and relates to new pharmaceutical uses of compounds, particularly new pharmaceutical uses of the halogenated phenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing, nonspecific inflammatory bowel disease characterized by diarrhea, rectal bleeding, abdominal pain, intestinal obstruction, and weight loss. It mainly includes two types: ulcerative colitis (UC) and Crohn's disease (CD).

[0003] The main differences between dysplasia cerebelli (CD) and ulcerative colitis (UC) lie in their pathophysiology and clinical manifestations. UC presents with recurrent abdominal pain, diarrhea, and bloody, mucous stools; the inflammatory changes in UC typically involve only the superficial mucosa and submucosa of the intestinal wall, starting in the rectum and eventually spreading to the rest of the colon. CD mainly presents with abdominal pain, fever, intestinal obstruction, diarrhea, and clinical signs such as abdominal masses and fistulas; CD usually occurs in the terminal ileum and adjacent colon, but can affect any area of ​​the intestine, and lesions can occur throughout the entire gastrointestinal tract. Idiopathic dysplasia (IBD) has become a global health concern. While more common in Europe and America, its incidence in my country is increasing year by year, making it a common digestive system disease in my country.

[0004] The exact cause and pathogenesis of IBD are still unclear, and its treatment still faces great challenges. However, some studies have reported that its pathogenesis may be related to genetic susceptibility, environmental factors, gut microbiota dysbiosis, and immune system disorders.

[0005] Medications used to treat IBD mainly include glucocorticoids, immunosuppressants, aminosalicylic acids, antibiotics, and biologics. While these drugs are effective in controlling the disease, they have significant side effects, are prone to relapse upon discontinuation, and are not suitable for long-term use. Therefore, finding effective and safe new drugs is of paramount importance for the treatment of inflammatory bowel disease.

[0006] CN 101602657A discloses a halogenated hydroxy aromatic ketone compound, its preparation method, and its uses. This class of compounds consists of benzophenone with one or more hydroxyl groups and halogen substituents attached to the two phenyl groups, respectively. Typical compounds include 4-chloro-3',4'-dihydroxybenzophenone, 2,3-dibromo-4,5-dihydroxybenzophenone, and 2,6-dibromo-3,3',4,4',5-pentahydroxybenzophenone. These halogenated hydroxy aromatic ketone compounds and their non-toxic salts, esters, and ethers possess antibacterial, antitumor, antioxidant, anti-vascular endothelial cell damage, and vascular smooth muscle cell proliferation inhibitory activities.

[0007] To date, there have been numerous reports on the medical applications of 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) in this class of compounds. For example, CN 102423308A discloses its application in the preparation of drugs to protect against myocardial ischemia-reperfusion injury, clarifying its protective function against myocardial ischemia-reperfusion injury; CN 103156829A discloses its application in the treatment and prevention of atherosclerosis, demonstrating that this class of compounds inhibits vascular endothelial damage and protects vascular endothelium by reducing the expression of serum TG and LDL-C, and inhibiting the expression of serum inflammatory factors IL-6, TNF-α, and adhesion molecule sICAM-1, thereby inhibiting the occurrence and development of atherosclerosis; CN 105193795A discloses its application in promoting angiogenesis, showing potential application prospects in the prevention and treatment of ischemic heart disease, stroke, muscle atrophy, and distal limb necrosis; CN 105816446A and CN 107007610A discloses its application in the preparation of drugs for type II diabetic nephropathy, as well as drugs for acute pyelonephritis and chronic nephritis, showing its important application prospects in the treatment of diabetic nephropathy, acute pyelonephritis and chronic nephritis; CN 115212192A provides its application in the preparation of drugs for renal fibrosis.

[0008] However, there are no reports on the pharmacological effects of 4,5,2'-trihydroxy-2,5'-dibromobenzophenone in the treatment of inflammatory bowel disease, either domestically or internationally. Summary of the Invention

[0009] The object of this invention is to provide a novel pharmaceutical use of the halogenated phenolic compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) in the treatment of inflammatory bowel disease.

[0010] This invention provides the first in vivo experimental evidence that the halogenated phenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone has significant pharmacological activity in treating inflammatory bowel disease. The use of this benzophenone derivative and its pharmaceutically acceptable salts in the preparation of drugs for treating inflammatory bowel disease has great development and application value.

[0011] The structural formula of the halogenated phenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) is as follows:

[0012]

[0013] The compound in question is a halogenated phenol compound well known to those skilled in the art, and its preparation method is readily achievable. For example, CN 103156829A discloses a specific method for preparing 4,5,2'-trihydroxy-2,5'-dibromobenzophenone and its sodium salt.

[0014] Specifically, the inflammatory bowel disease described in this invention includes ulcerative colitis and Crohn's disease.

[0015] More specifically, Crohn's disease includes various perianal conditions such as perianal erythema, abscesses, ulcers, perianal fissures, or fistulas, and may also include symptoms of perianal hemorrhoid inflammation.

[0016] More specifically, Crohn's disease also includes intestinal fibrosis and intestinal stenosis caused by Crohn's disease.

[0017] Another object of the present invention is to provide a novel medicament for treating inflammatory bowel disease, wherein the medicament is prepared by adding a pharmaceutically acceptable common pharmaceutical carrier or excipient to a compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone or a pharmaceutically acceptable salt thereof as the active pharmaceutical ingredient.

[0018] Specifically, pharmaceutically acceptable salts include sodium, potassium, or ammonium salts.

[0019] Furthermore, the drug can be formulated into commonly used pharmaceutical preparations such as capsules, tablets, granules, injections, sustained-release preparations, oral liquids, or pills, depending on different requirements.

[0020] This invention uses a TNBS-induced rat inflammatory bowel disease model and a DSS-induced mouse acute ulcerative colitis model for in vivo experiments. By monitoring various indicators such as body weight, disease activity index, colonic histopathology, and inflammatory factors, the results confirm that the compound of this invention has a strong protective effect against inflammatory bowel disease, demonstrating its important application prospects in the treatment of inflammatory bowel disease.

[0021] The halogenated phenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone has strong anti-inflammatory and anti-immune effects. Its diverse activities are highly consistent with the pathogenesis of inflammatory bowel disease and can effectively improve intestinal damage in mice with inflammatory bowel disease. It is superior to mesalazine, a current first-line clinical drug, in terms of disease activity index (DAI) and colon and rectal length. Attached Figure Description

[0022] Figure 1 These are the fecal bleeding conditions of rats in different experimental groups.

[0023] Figure 2 The changes in colon and rectal length and gross morphological damage of colon tissue in rats from different experimental groups are shown.

[0024] Figure 3 These are HE staining images of intestinal tissue from rats in different experimental groups.

[0025] Figure 4 These are the results of Western blotting analysis of intestinal mucosal barrier proteins in rats from different experimental groups.

[0026] Figure 5 The changes in colon and rectum length in mice from different experimental groups.

[0027] Figure 6 These are HE staining images of intestinal tissue from mice in different experimental groups.

[0028] Figure 7 These are the results of Western blotting analysis of intestinal mucosal barrier proteins in mice from different experimental groups. Implementation

[0029] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can better understand and utilize the present invention, and are not intended to limit the scope of protection of the present invention.

[0030] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or conditions recommended by the manufacturer.

[0031] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0032] The following examples verified the application of the halophenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) in the preparation of drugs for treating inflammatory bowel disease. The drugs for treating inflammatory bowel disease include the active ingredient 4,5,2'-trihydroxy-2,5'-dibromobenzophenone, and common pharmaceutical carriers or excipients used in combination with the active ingredient, and can be made into common pharmaceutical preparations such as capsules, tablets, granules, injections, sustained-release agents, oral liquids or dripping pills according to specific requirements.

[0033] Example 1: Effect of LM49 on TNBS-induced inflammatory bowel disease in rats

[0034] Experimental animals: SPF-grade healthy male SD rats, weighing 210-250 g.

[0035] Animal source: Beijing Vital River Laboratory Animal Technology Co., Ltd.; License number: SCXK (Beijing) 2021-0011; Certificate number: 110011220105069712.

[0036] The animals used in this experiment and related disposals complied with the requirements of animal welfare, and were ethically reviewed by the Animal Welfare Committee of this institution before the experiment was carried out.

[0037] Drugs and reagents: LM49 (prepared in the laboratory, purity ≥99.5%, batch number 20210420); Mesalazine (Sunflower Pharmaceutical Group Jiamusi Luling Pharmaceutical Co., Ltd., 210608); TNBS (Sigma-Aldrich Corporation, USA, SLCK4178); Rat TNF-α enzyme-linked immunosorbent assay kit (Shanghai Fanke Industrial Co., Ltd., F40565-A); Rat IL-17A enzyme-linked immunosorbent assay kit (Shanghai Fanke Industrial Co., Ltd., F40260); BCA protein quantification kit (Wuhan Boster Biological Engineering Co., Ltd., 16I18B97); TRIzol reagent (9108); PrimerScript RT reagent Kit (RR047A) and SYBR Premix Ex Taq™ II Kit (RR820A), TaKaRa Corporation.

[0038] Healthy male SD rats were fasted but allowed to drink water for 24 h. After anesthesia, a 1 mL syringe connected to a gastric gavage needle was used to aspirate the TNBS modeling agent with a modeling dose of 60 mg / kg (a 5% TNBS solution mixed with 0.25 mL of 50% ethanol). The gastric gavage needle was carefully inserted into the anus of the rat to a depth of 7-8 cm. During the process, the intestinal wall was avoided from being injured, and the drug solution was slowly pushed in. After the enema was completed, the gastric gavage needle was slowly withdrawn. The rat was inverted at a 30° angle with its head down for 3 min and then put back into the cage.

[0039] If rats exhibit symptoms such as reduced appetite, decreased activity, weight loss, diarrhea, bloody stools, and decreased coat luster, it indicates that the rat inflammatory bowel disease model has been successfully established.

[0040] The normal group received the same volume of physiological saline.

[0041] After successful modeling, except for 12 normal rats, the other rats were randomly divided into 5 groups: model group, low-dose LM49 group, medium-dose LM49 group, high-dose LM49 group, and mesalazine positive control group, with 12 rats in each group.

[0042] The normal group and the model group were given 1.0 ml / 100 g of 0.5% CMC-Na solution. The low, medium and high dose groups of LM49 were given 0.5% CMC-Na suspensions of different concentrations of drug, namely 22.5 mg / kg, 45 mg / kg and 90 mg / kg, respectively. The mesalazine positive control group was given 0.5% CMC-Na suspension at a concentration of 225 mg / kg. The administration volume was 1.0 ml / 100 g.

[0043] The medication was administered via gavage once a day for one week after the modeling was completed.

[0044] Rats were weighed daily, and the characteristics of their feces and the presence of occult blood were observed in each group. Finally, the Disease Activity Index (DAI) was calculated according to the DAI scoring criteria: DAI = (weight loss score + fecal characteristics score + fecal blood score) / 3.

[0045] After the last administration, the rats were fasted but allowed free access to water for 12 hours. Blood was then collected from the abdominal aorta of the anesthetized rats, centrifuged at 3000 r / min for 20 min, and the supernatant was collected for ELISA to detect changes in serum inflammatory factors TNF-α and IL-17A.

[0046] After blood was drawn from the abdominal aorta, the rats were euthanized. The abdominal cavity was fully dissected to expose the colon. The length from the cecum to the anal margin was recorded as the colon length. The colonic tissue was harvested from the rats, and the presence of edema, adhesions, ulcers, necrosis, etc., was observed. Photos were taken and measurements were recorded. Approximately 1 cm of colon was taken, fixed with tissue fixative, and subjected to HE staining and histopathological scoring (HS).

[0047] The colon tissue was longitudinally cut open, washed, and laid flat. The tissue damage index (CMDI) was observed and scored. The remaining colon tissue was aliquoted, quickly frozen in liquid nitrogen, and stored at -80°C for ELISA, RT-PCR, and Western Blot analysis.

[0048] Spleen and mesenteric lymph node tissues from rats were collected for flow cytometry analysis.

[0049] Statistical methods were employed using IBM SPSS Statistics 26.0 software for data processing. The data were presented in... This indicates that one-way ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0050] Results and Analysis

[0051] 1. Blood in rat stool

[0052] Figure 1 The study presented the fecal hemorrhage observed in each group of rats after the last administration. The results showed that the model group rats experienced more severe fecal hemorrhage compared to the normal group, indicating successful model establishment. Treatment with different doses of LM49 resulted in varying degrees of relief from fecal hemorrhage in each treatment group.

[0053] 2. Changes in rat body weight and Disease Activity Index (DAI) score

[0054]

[0055] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0056] Table 1 shows the changes in rat body weight and DAI results, indicating that compared with the normal group, the model group showed significant differences in both body weight decrease and DAI score increase. P <0.01), indicating successful model establishment. Compared with the model group, the body weight of the high-dose LM49 group and the positive control group was significantly increased ( P <0.05); the DAI scores of the medium- and high-dose LM49 groups and the positive control group were significantly reduced ( P <0.05 or P <0.01).

[0057] 3. Changes in colorectal length and gross morphological damage (CMDI) of colonic tissue in rats.

[0058]

[0059] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0060] Combination Figure 2 According to the data in Table 2, compared with the normal group, the colorectal length in the model group was significantly shorter. P <0.01); compared with the model group, the colorectal length of the high-dose LM49 group and the positive control group was significantly increased ( P <0.05 or P <0.01).

[0061] Figure 2 Gross morphological observation of the colonic tissue in the model group showed significant thickening, hardening, congestion, and adhesion of the colonic wall. After drug administration, all drug-treated groups showed significant recovery. Table 2 shows the results of the gross morphological damage score of the colonic tissue, indicating that the gross morphological score of the colonic tissue in the model group was significantly higher than that in the normal group. P <0.01); compared with the model group, the gross score of colon tissue in the positive control group was significantly lower in the medium and high dose groups of LM49 ( P <0.05 or P <0.01).

[0062] 4. HE staining and histopathological scoring of rat intestinal tissue (HS)

[0063] Figure 3 HE staining of intestinal tissue showed that in the normal group, the intestinal mucosa was intact, the lamina propria crypts were neatly arranged, goblet cells were abundant, and the submucosa and muscularis propria were clearly defined, with no obvious inflammatory changes. In the model group, focal necrosis was observed in the intestinal tissue, with damage extending to the submucosa, crypt disappearance, and amyloid deposition; inflammatory edema was present in the submucosa; connective tissue proliferated and repaired; the inflammatory response was severe, extending to the muscularis propria, with abundant infiltration of neutrophils and lymphocytes. Compared with the model group, the inflammation was improved in each of the treatment groups, and the crypts and epithelium recovered to varying degrees.

[0064]

[0065] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0066] The intestinal histopathological scoring results in Table 3 show that, compared with the normal group, the histopathological score of the model group was significantly higher ( P <0.01); compared with the model group, the histopathological scores of the positive control group in the medium and high dose LM49 groups were significantly lower ( P <0.05 or P<0.01).

[0067] 5. Changes in MPO, MDA, and SOD in rat intestinal tissue

[0068]

[0069] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0070] The results in Table 4 show that, compared with the normal group, the MPO activity and MDA content in the intestinal tissue of rats in the model group were significantly increased. P <0.01), SOD activity was significantly reduced ( P <0.01), which proves that the model was successfully created.

[0071] After administration, compared with the model group, the MPO activity in the intestinal tissue of rats in the LM49 medium- and high-dose groups and the positive control group was significantly reduced. P <0.05 or P <0.01), MDA content decreased significantly ( P <0.05 or P <0.01); In the high-dose LM49 group, SOD activity in the intestinal tissue of rats in the positive control group was significantly reduced ( P <0.05 or P <0.01).

[0072] 6. Changes in serum inflammatory factors TNF-α and IL-17A in rats

[0073]

[0074] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0075] The results of ELISA detection of inflammatory factors TNF-α and IL-17A in rats are listed in Table 5. Compared with the normal group, the levels of TNF-α and IL-17A in the model group rats were significantly increased. P <0.01).

[0076] After administration, compared with the model group, the levels of TNF-α and IL-17A in rats in the low, medium, and high dose LM49 groups and the positive control group were significantly reduced. P <0.05 or P <0.01).

[0077] 7. Flow cytometry detection of the proportion of T cells in rat spleen

[0078]

[0079] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0080] Table 6 shows the flow cytometry results of Th17 cells, indicating that the proportion of Th17 cells in the model group rats was significantly increased compared with the normal group. P <0.01). Compared with the model group, the proportion of Th17 in rats in the low, medium, and high dose LM49 groups and the positive control group was significantly reduced ( P <0.05 or P <0.01).

[0081] Table 6 shows that the flow cytometry results of Treg cells indicate that the proportion of Treg cells in the model group was significantly lower than that in the normal group. P <0.01). Compared with the model group, the proportion of Treg rats in the positive control group was significantly reduced in the medium and high dose LM49 groups ( P <0.01).

[0082] 8. RT-PCR detection of mRNA levels of related factors in rat intestinal tissue

[0083]

[0084] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0085] RT-PCR was used to detect the mRNA levels of IL-1β, IL-10, and ROR-γt in rat intestinal tissue. The results in Table 7 show that, compared with the normal group, the levels of the inflammatory factors IL-1β and ROR-γt in the model group rats were significantly increased. P <0.01), IL-10 levels were significantly reduced ( P <0.01)

[0086] Compared with the model group, the levels of IL-1β and ROR-γt in rats in the medium- and high-dose LM49 groups and the positive control group were significantly reduced. P <0.01), IL-10 levels were significantly elevated ( P <0.01).

[0087] 9. Effects of Western Blot on the levels of Claudin-1, Occludin, and ZO-1 proteins in the intestinal tissue of CD rats

[0088] Disruption of the intestinal mucosal barrier is an important pathophysiological basis of Crohn's disease (CD). Intestinal tight junction proteins ZO-1, Occludin, and Claudin-1 are markers of intestinal epithelial integrity. Therefore, Western blotting was used to detect Claudin-1, Occludin, and ZO-1 proteins in the rat intestinal mucosal barrier. The results are as follows: Figure 4 As shown.

[0089] Compared with the normal group, the relative content of Claudin-1 in the model group rats was significantly reduced ( P <0.01). Compared with the model group, the relative content of Claudin-1 in rats in the high-dose LM49 group and the positive control group was significantly increased ( P <0.05 or P <0.01).

[0090] Compared with the normal group, the relative content of Occludin in the model group rats was significantly reduced ( P <0.01). Compared with the model group, the relative content of Occludin in rats in the medium and high dose groups of LM49 was significantly increased in the positive control group ( P <0.05 or P <0.01).

[0091] Compared with the normal group, the relative content of ZO-1 in the model group rats was significantly reduced ( P <0.01). Compared with the model group, the relative content of ZO-1 in the high-dose LM49 group and the positive control group was significantly increased ( P <0.05 or P <0.01).

[0092] Example 2: Effect of LM49 on DSS-induced acute ulcerative colitis in mice

[0093] Experimental animals: Healthy C57BL / 6 mice aged 6 - 8 weeks, with a body weight of 20 g ± 1 g.

[0094] Source of animals: Beijing Vital River Laboratory Animal Technology Co., Ltd.; License number: SCXK (Beijing) 2021 - 0006, Certificate number: 110011220114500912.

[0095] The animals used in this experiment and related disposals comply with animal welfare requirements, and the experiment has passed the ethical review of the animal welfare committee of this institution before it is carried out.

[0096] Drugs and reagents: LM49 (prepared in the laboratory, purity ≥ 99.5%, batch number 20150320); Mesalazine (Sunflower Pharmaceutical Group Jiamusi Luling Pharmaceutical Co., Ltd., 210608); DSS (Dalian Meilun Biotechnology Co., Ltd., MO510B); Mouse IL-10 enzyme-linked immunosorbent assay kit (Shanghai Fanke Industrial Co., Ltd., F40565 - A); Mouse IL-17 enzyme-linked immunosorbent assay kit (Shanghai Fanke Industrial Co., Ltd., F2176 - A); BCA protein quantification kit (Wuhan Boster Biological Engineering Co., Ltd., 16I18B97); TRIzol reagent (9108), PrimerScript RT reagent Kit (RR047A) and SYBR Premix Ex Taq™ II Kit (RR820A), TaKaRa Co., Ltd.

[0097] After 7 days of adaptive feeding of healthy C57BL / 6 mice aged 6 - 8 weeks, they were randomly divided into 6 groups with 12 mice in each group, namely normal group, model group, low-dose LM49 group, medium-dose LM49 group, high-dose LM49 group, and mesalazine positive control group.

[0098] During the experiment, except for the normal group which was given tap water, the other groups were given 2.5% dextran sulfate sodium (DSS) solution for free drinking for 7 days, and the fresh DSS solution was replaced every morning to induce an acute ulcerative colitis model in mice. When there was weight loss, softening, thinning, and bloody stools, decreased activity, and positive occult blood test, it was considered that the acute ulcerative colitis (UC) model was successfully established.

[0099] After successful modeling, the normal group and the model group were given 1.0 ml / 100 g of 0.5% CMC-Na solution. The low, medium and high dose groups of LM49 were given 0.5% CMC-Na suspensions of different concentrations of drug, namely 32.5 mg / kg, 65 mg / kg and 130 mg / kg, respectively. The mesalazine positive control group was given 0.5% CMC-Na suspension at a concentration of 290 mg / kg. The administration volume was 1.0 ml / 100 g.

[0100] The medication was administered via gavage once a day for one week after the modeling was completed.

[0101] Rats were weighed daily, and the characteristics of their feces and the presence of occult blood were observed in each group. Finally, the DAI score was calculated as follows: DAI = (weight loss score + fecal characteristics score + fecal blood score) / 3.

[0102] After the last administration, the mice were fasted for 12 hours but allowed free access to water. Blood was then collected from the abdominal aorta of the mice after anesthesia. The blood was centrifuged at 3000 r / min for 20 min, and the supernatant was collected for ELISA to detect changes in serum inflammatory factors IL-10 and IL-17 in rats.

[0103] Mice were euthanized after blood was drawn from the abdominal aorta. The abdominal cavity was fully dissected to expose the colon. The length from the cecum to the anal margin was recorded as the colon length. Colorectal tissue was harvested from the mice to observe for edema, adhesions, ulcers, necrosis, etc., and photographs and measurements were taken. Approximately 1 cm of colon was taken, fixed with tissue fixative, and subjected to HE staining and histopathological scoring (HS).

[0104] The remaining colon tissue was aliquoted, quickly placed in liquid nitrogen for cryopreservation, and then transferred to -80°C for storage for ELISA, RT-PCR, and Western Blot detection.

[0105] Spleen and mesenteric lymph node tissues from mice were collected for flow cytometry analysis.

[0106] Statistical methods were employed using IBM SPSS Statistics 26.0 software for data processing. The data were presented in... This indicates that one-way ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0107] Results and Analysis

[0108] 1. Effects on body weight and DAI in mice with acute ulcerative colitis

[0109]

[0110] Compared with the normal group, * P <0.05,** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0111] Table 8 shows the changes in mouse body weight and DAI results, indicating that compared with the normal group, the model group had a decrease in body weight and a significant increase in DAI score. P <0.01). Compared with the model group, the body weight of the high-dose LM49 group and the positive control group was significantly increased ( P <0.01); In the medium and high dose groups of LM49, the DAI score of the positive control group was significantly reduced ( P <0.01).

[0112] 2. Effect on colon length in mice with acute ulcerative colitis

[0113]

[0114] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0115] Results of changes in colon length in mice are as follows Figure 5 Table 9 shows that, compared with the normal group, the colon length of mice in the model group was significantly reduced ( P <0.01). Compared with the model group, the body weight of the high-dose LM49 group and the positive control group was significantly increased ( P <0.05 or P <0.01).

[0116] 3. Effect on histopathological scores in mice with acute ulcerative colitis

[0117]

[0118] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0119] Figure 6HE staining results showed that in the normal group, the colonic folds were clear, the intestinal epithelium of the mucosa was intact, consisting of a single layer of columnar epithelium, the epithelial cells were normal in morphology and structure, the lamina propria had abundant intestinal glands and a large number of goblet cells, the muscle layer was uniformly stained, the muscle fibers were normal in morphology and structure and arranged regularly, and no obvious inflammation was observed. In the model group, the colonic mucosa showed partial erosion, accompanied by a large number of lymphocyte infiltrations, and the number of goblet cells in the surrounding intestinal epithelium was reduced, with obvious inflammatory symptoms. Compared with the model group, the inflammation in each treatment group was improved, and the number of goblet cells recovered to varying degrees.

[0120] Table 10 shows the histopathological scores of mice in the model group, which were significantly higher than those in the normal group. P <0.01). Compared with the model group, the histopathological scores of the high-dose LM49 group and the positive control group were significantly reduced ( P <0.01).

[0121] 4. Effects on intestinal permeability in mice with acute ulcerative colitis

[0122] The concentration of FITC-dextran in serum can reflect changes in colonic epithelial permeability, thereby evaluating the integrity of the intestinal mucosal barrier. Increased intestinal permeability indicates impaired intestinal mucosal barrier function. The results are shown in Table 11.

[0123]

[0124] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0125] The results showed that, compared with the normal group, the intestinal permeability of mice in the model group was significantly increased ( P <0.01). Compared with the model group, the intestinal permeability of mice in the medium and high dose LM49 groups and the positive control group was significantly reduced ( P <0.01).

[0126] 5. Effects on antioxidant activity in mice with acute ulcerative colitis

[0127]

[0128] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group,# P <0.05, ## P <0.01.

[0129] The results of antioxidant activity in mice (see Table 12) showed that, compared with the normal group, the levels of MPO and MDA in the model group were significantly increased. P <0.01), SOD content decreased significantly ( P <0.01).

[0130] Compared with the model group, the MPO content in the high-dose LM49 group and the positive control group was significantly increased. P <0.05 or P <0.01); MDA levels were significantly increased in the medium- and high-dose LM49 groups and the positive control group ( P <0.01); SOD levels were significantly increased in the high-dose LM49 group and the positive control group ( P <0.01).

[0131] 6. Effects on serum IL-10 and IL-17 levels in mice with acute ulcerative colitis

[0132]

[0133] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0134] The levels of IL-10 and IL-17 in mouse serum were measured. Table 13 shows that, compared to the normal group, the IL-10 level in the model group was significantly lower. P <0.01); IL-17 levels were significantly increased ( P <0.01).

[0135] Compared with the model group, the IL-10 levels in the high-dose LM49 group and the positive control group were significantly increased. P <0.01); IL-17 levels were significantly decreased in the high-dose LM49 group and the positive control group ( P <0.05 or P <0.01).

[0136] 7. Effects on the levels of CD4+IL-17+;CD4+CD25+Foxp3 in the spleen of mice with acute ulcerative colitis

[0137]

[0138] Compared with the normal group, * P <0.05, ** P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0139] Flow cytometry was used to detect Th17 and Treg cells in the spleen of mice. The statistical results in Table 14 show that the proportion of Th17 cells in the model group was significantly increased compared with the normal group. P <0.01); the proportion of Treg cells was significantly reduced ( P <0.01).

[0140] Compared with the model group, the proportion of Th17 cells in the medium- and high-dose LM49 groups and the positive control group was significantly decreased. P <0.05 or P <0.01); the proportion of Treg cells was significantly increased in the high-dose LM49 group and the positive control group ( P <0.01).

[0141] 8. Effects on intestinal tight junction proteins in mice with acute ulcerative colitis

[0142] During the development of ulcerative colitis (UC), the intestinal mucosal barrier is damaged, and the intestinal epithelium is an important component of this barrier. Intestinal tight junction proteins ZO-1, Occludin, and Claudin-1 are markers of intestinal epithelial integrity. Therefore, Western blotting was used to detect the protein levels of these proteins. Figure 7 The results showed that, compared with the normal group, the relative levels of Claudin-1, Occludin, and ZO-1 in the model group were significantly decreased ( P <0.01).

[0143] Compared with the model group, the relative levels of Claudin-1 were significantly increased in the high-dose LM49 group and the positive control group. P <0.01); the proportion of Occludin cells in the medium- and high-dose LM49 groups and the positive control group increased significantly ( P <0.05 or P <0.01); the proportion of ZO-1 cells was significantly increased in the high-dose LM49 group and the positive control group ( P <0.01).

[0144] The above series of experimental results indicate that LM49 has a protective effect against acute ulcerative colitis in mice. The effects of LM49 on UC were observed through changes in body weight, colon length, colonic histological score, and DAI (diuretic acid). Histological scores showed a significant decrease in the treatment groups compared to the model group. FITC-glucan assays of mouse intestinal permeability showed a decrease in intestinal permeability in all treatment groups compared to the model group. Antioxidant results showed that the levels of MPO and MDA in the colon of mice in all treatment groups were decreased to varying degrees, while SOD was increased to varying degrees compared to the model group. ELISA assays of serum IL-10 and IL-17 showed that serum IL-10 in all treatment groups was significantly increased compared to the model group, while IL-17 was significantly decreased. Flow cytometry analysis of the effects of LM49 on Th17 and Treg cells showed that Th17 (CD4+IL-17+) in the spleen of mice in all treatment groups was lower than that in the model group, while the levels of Treg cells (CD4+CD25+Foxp3), which have anti-inflammatory effects, were higher than those in the model group. Western cytometry analysis of LM49 also showed a significant increase in IL-10 and IL-17 levels in the spleen of mice in all treatment groups. Blot analysis revealed that the expression of tight junction proteins was increased to varying degrees compared to the model group.

[0145] The above results demonstrate that LM49 has a protective effect against acute ulcerative colitis in mice and can effectively reduce enteritis inflammation in mice, with the high-dose group showing the most significant effect.

[0146] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

The application of 1,4,5,2'-trihydroxy-2,5'-dibromobenzophenone in the preparation of drugs for treating inflammatory bowel disease.

2. The application according to claim 1, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

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