Application of isopsoralen in preparation of medicine for treating ulcerative colitis

Abstract

The invention provides application of isopsoralen in preparation of a medicine for treating ulcerative colitis, and belongs to the technical field of medicines. The invention aims to provide a safer and more effective UC treatment medicine. The invention provides an application of isopsoralen in preparation of a medicine for treating ulcerative colitis. The invention provides a new medicine choice for preventing and treating ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to the application of isopsoralen in preparing a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease characterized by long-term inflammation that can cause symptoms such as diarrhea and bloody stools, and increase patients' risk of colorectal cancer. Current drug treatments often lack specificity and may cause adverse reactions. For example, aminosalicylic acid is used in clinical practice to treat mild to moderate UC, but over time, 30% of patients lose response to the drug or lose its efficacy. Steroids are used to relieve symptoms in moderate to severe patients, but due to their large side effects, they are not suitable for long-term use. Therefore, a new alternative treatment option for UC is urgently needed. Summary of the invention

[0003] The purpose of the present invention is to provide a safer and more effective drug for the treatment of UC. my country has abundant Chinese medicine resources, and Chinese herbal medicines are safe and effective. Therefore, it is urgent to find drugs for the treatment of ulcerative colitis from natural plants to meet the urgent clinical needs.

[0004] The present invention provides the use of isopsoralen in preparing a drug for treating ulcerative colitis, and the structure of the isopsoralen is as follows:

[0005]

[0006] The present invention provides the use of isopsoralen in preparing a drug for adjuvant treatment or relief of ulcerative colitis, and the structure of the isopsoralen is as follows:

[0007]

[0008] The present invention provides an application of isopsoralen in preparing a health product for assisting in the treatment or alleviation of ulcerative colitis. The structure of the isopsoralen is as follows:

[0009]

[0010] It is further defined that it contains at least one of isopsoralen and a pharmaceutically acceptable salt thereof.

[0011] It is further defined that the drug contains one or more pharmaceutically acceptable excipients.

[0012] It is further defined that the auxiliary material is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an antibacterial agent, a fragrance, an antioxidant, a pH adjuster, a protective agent, a diluent, a lubricant, and a solvent.

[0013] It is further defined that the dosage form of the drug is one of an injection, a capsule, a pill, a tablet, a granule, a tincture, an inhalation preparation, an oral solution, an oral suspension, and an oral emulsion.

[0014] It is further defined that the isopsoralen can improve the degree of intestinal mucosal barrier damage and increase the expression levels of MUC2, ZO-1, Occludin and Claudin.

[0015] It is further defined that the isopsoralen can reduce weight loss in mice, reduce diarrhea in mice, reduce blood in the stool in mice, reduce colon shortening in mice, inhibit excessive production of pro-inflammatory factors and increase the expression of anti-inflammatory factors.

[0016] It is further defined that the experimental ulcerative colitis is induced by dextran sulfate sodium or 2,4,6-trinitrobenzenesulfonic acid.

[0017] Beneficial effects: The present invention provides an application of Angelicin (Ang) derived from the Chinese herbal medicine Psoralea corylifolia in the preparation of a drug for relieving ulcerative colitis, providing a new drug option for the prevention and treatment of ulcerative colitis.

[0018] An Ang having the following structural formula:

[0019]

[0020] The present invention found that Ang described in the above structural formula can effectively treat and alleviate the symptoms of ulcerative colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS): including significantly relieving symptoms such as weight loss, colon shortening, and diarrhea and bloody stools; can significantly reduce inflammatory cell infiltration in colon tissue and protect colon tissue structure; can improve intestinal barrier function and regulate the expression level of inflammatory factors. Therefore, Ang can be used as an effective therapeutic drug for ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Body weight changes of ulcerative colitis mice in different treatment groups; A is the DSS group; B is the TNBS group; C is the DSS group; D is the TNBS group;

[0022] Figure 2 Disease activity index (DAI) scores of ulcerative colitis mice in different treatment groups;

[0023] Figure 3 Representative colonograms of ulcerative colitis mice in different treatment groups and statistical analysis results of colon length;

[0024] Figure 4 HE staining results of colon tissues of ulcerative colitis mice in different treatment groups;

[0025] Figure 5 Histological pathological injury scores of the colon of ulcerative colitis mice in different treatment groups;

[0026] Figure 6 Expression levels of inflammatory factors in the serum of ulcerative colitis mice in different treatment groups;

[0027] Figure 7 Expression levels of intestinal mucosal barrier proteins in colonic tissues of ulcerative colitis mice in different treatment groups;

[0028] Figure 8 Expression levels of MUC2 and ZO1 in colonic tissues of ulcerative colitis mice in different treatment groups;

[0029] Fig. 9 Blood biochemical indices of liver and kidney function in ulcerative colitis mice in different treatment groups; A is the DSS-ALT group; B is the DSS-AST group; C is the DSS-BUN group; D is the DSS-CREA group; E is the TNBS-ALT group; F is the TNBS-AST group; G is the TNBS-BUN group; H is the TNBS-CREA group;

[0030] Fig.10 The organ / body weight ratios of liver, spleen and kidney of ulcerative colitis mice in different treatment groups; A is the DSS group; B is the DSS group; C is the DSS group; D is the TNBS group; E is the TNBS group; and F is the TNBS group. DETAILED DESCRIPTION

[0031] Isopsoralen (CAS No.: 523-50-2)

[0032] SPF-grade C57Bl / 6J male mice weighing 22-24 g were selected. The mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Animal Certificate No.: SCXK (Beijing) 2022-0006). The experimental animals were kept in an SPF-grade animal room with a temperature of 22±2℃ and a 12h day and night cycle. The animals were adaptively fed for one week before the experiment, and the animals were free to eat during the feeding period.

[0033] Example 1. Application of isopsoralen in the preparation of a drug for treating ulcerative colitis

[0034] 1. Observation of mouse physical signs and disease activity index (DAI) scoring

[0035] Methods: Aqueous solutions of Ang with concentrations of 3.15 mg / kg, 6.3 mg / kg and 12.6 mg / kg, aqueous solution of sulfasalazine (SASP) of 200 mg / kg, aqueous solution of 3% DSS and aqueous solution of 5% TNBS (0.2 mL / 100 g) were prepared.

[0036] The mice were randomly divided into 11 groups, with 10 mice in each group, namely, the control group (given distilled water), the DSS model group (given 3% DSS solution), the DSS-positive control group (given 3% DSS solution and gavage of 0.3 g / kg SASP every day), the DSS-Ang low-dose group (given 3% DSS solution and gavage of 3.15 mg / kg Ang every day), the DSS-Ang medium-dose group (given 3% DSS solution and gavage of 6.3 mg / kg Ang every day), and the DSS-Ang high-dose group (given 3% DSS solution and gavage of 12.6 mg / kg Ang every day). The mice were gavaged continuously for 9 days, and the mice were killed after 9 days. The mice were given TNBS model group (0.2mL / 100g 5% TNBS and 0.25mL 50% ethanol injected into the mouse anus about 4cm with a rubber tube on the 7th day, and no treatment was given for the first 7 days), TNBS-positive control group (control, 5% TNBS solution was given on the 7th day and then 0.3g / kg SASP was gavaged every day), TNBS-Ang low-dose group (Ang-L, 3.15mg / kg Ang was gavaged every day after 5% TNBS solution was given on the 7th day), TNBS-Ang medium-dose group (Ang-M, 6.3mg / kg Ang was gavaged every day after 5% TNBS solution was given on the 7th day), and TNBS-Ang high-dose group (Ang-H, 12.6mg / kg Ang was gavaged every day after 5% TNBS solution was given on the 7th day). The mice were gavaged for 9 consecutive days and then killed after 9 days.

[0037] The feces of the experimental animals were observed every day, and the body weight was recorded. The disease activity index (DAI) was scored for the weight change and fecal characteristics of the mice: 1) Weight change scoring criteria: 0 points - no change or increase in weight; 1 point - weight loss of 0-5% (including 5%); 2 points - weight loss of 5-10% (including 10%); 3 points - weight loss of 10-20% (including 20%); 4 points - weight loss greater than 20%; 2) Fecal characteristics scoring criteria: 0 points - normal; 1 point - soft feces; 2 points - wet and soft feces; 3 points - semi-loose feces; 4 points - loose feces; 3) Fecal occult blood scoring criteria: 0 points - no blood in the stool; 1 point - positive; 2 points - occult blood; 3 points - gross blood in the stool. On the last day of the experiment, the mice were killed by cervical dislocation, the colon was dissected, and the colon length was measured.

[0038] Results: See Figure 1The weight of mice in the control group (control) continued to grow steadily, while the weight of mice in the model group (DSS or TNBS) after modeling with DSS or TNBS was statistically significant compared with the control group (P<0.001). The weight of mice in the Ang treatment group (Ang-L, Ang-M, Ang-H) also decreased, but compared with the model group, the weight loss trend of mice in the Ang treatment group was improved, and the difference was statistically significant (P<0.01). At the same time, during the modeling period, it was found that the mice in the model group had severe blood in their stools, while the blood in their stools was significantly alleviated after Ang treatment and was better than the positive drug group (SASP). Figure 2 Compared with the control group, the DAI score of the model group was significantly increased (P<0.001), while the DAI score of the Ang group mice after administration was significantly reduced compared with the model group (P<0.001). Therefore, Ang treatment can significantly alleviate the symptoms of weight loss, diarrhea and bloody stool in DSS or TNBS mice, and the therapeutic effect is similar to that of positive drugs.

[0039] See also Figure 3 , are the representative colonograms and statistical analysis results of the colon length of mice in each treatment group. Compared with the control group, the colon length of mice after DSS or TNBS modeling was significantly shortened (P<0.01 or P<0.001), while the colon length of mice after Ang or SASP treatment was significantly improved (P<0.05 or P<0.01).

[0040] 2. Colon pathological observation and histological pathological scoring

[0041] Methods: The terminal colon was sliced ​​and stained with hematoxylin-eosin (HE). The HE-stained colon specimens were observed and scored: 1) Scoring criteria for inflammation: 0 points - no inflammation; 1 point - inflammation limited to the mucosal layer; 2 points - inflammation gradually penetrates into the submucosal layer; 3 points - inflammation penetrates into the muscular layer; 4 points - transmural inflammation; 2) Scoring criteria for neutrophil infiltration: 0 points - no inflammatory infiltration; 1 point - mildly obvious; 2 points - moderately diffuse; 3 points - severely diffuse; 3) Crypt damage: 0 points - no damage; 1 point - basal 1 / 3 damage; 2 points - basal 2 / 3 damage; 3 points - all damage; 4 points - all damage and ulcers; 4) Crypt swelling: 0 points - no swelling; 1 point - mildly visible; 2 points - moderately diffuse; 3 points - severely diffuse; 5) Epithelial mucosal swelling: 0 points - no swelling; 1 point - mildly visible; 2 points - moderately diffuse; 3 points - severely diffuse; 6) Goblet cell loss: 0 points - no loss; 1 point - mild; 2 points - moderate; 3 points - severe; 7) Reactive epithelial hyperplasia: 0 points - no hyperplasia; 1 point - mildly visible; 2 points - moderately diffuse; 3 points - severely diffuse.

[0042] Results: See Figure 4 , Figure 4 The following are the results of HE staining of colon pathological sections of mice in each treatment group. The colon tissue structure of mice in the control group was intact, with no obvious neutrophil infiltration, neatly arranged goblet cells, complete morphology, and normal crypt structure. The integrity of the colon wall of mice in the model group was destroyed, neutrophil infiltration was severely diffuse, goblet cells were severely lost, the crypt structure was completely damaged, the mucosal layer and serosa layer were significantly thickened, and the arrangement of muscle layer cells was disordered. Compared with the model group, after Ang or SASP treatment, the morphology of the colon tissue structure of mice was significantly improved, the infiltration of inflammatory cells was significantly reduced, and the integrity of intestinal mucosal epithelial cells was improved. Figure 5 , Figure 5 is the colon tissue pathological score of mice in each treatment group. Compared with the control group, the pathological score of the model group was significantly increased (P<0.001). Compared with the model group, the pathological score of the mice in the Ang treatment group was significantly decreased (P<0.001).

[0043] 3. Elisa to detect the expression of related inflammatory factors

[0044] Methods: ELISA kits were obtained commercially, and biochemical analysis was performed according to the manufacturer's instructions. Blood samples were collected from mice and allowed to stand at room temperature until a serum supernatant was formed, then centrifuged, the supernatant was collected, and frozen at -80°C.

[0045] See also Figure 6 Compared with the blank group, the expression levels of IL-10 and TGF-β in the colon tissue of the model group mice were significantly reduced, and the expression levels of IL-1β, IL-6, and TNF-α were significantly increased. After Ang and SASP treatment, the expression levels of inflammatory factors were significantly reversed, indicating that Ang effectively inhibited the production of inflammatory factors, and the effect was similar to that of positive drugs.

[0046] 4. Western Blot Analysis of Intestinal Mucosal Barrier

[0047] Methods: Western blotting was performed under standard procedures. Colonic tissue protein samples were lysed on ice for 30 min using RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitors (RIPA lysis buffer: protease inhibitor: phosphatase inhibitor = 50:1:1). The protein supernatant was collected and centrifuged at 15,000 rpm for 10 min at 4°C. The protein concentration was determined using a BCA protein assay kit. After separation using 8%-12% sodium dodecyl sulfate polyacrylamide gel, the protein (40 μg) was transferred to a PVDF membrane. After blocking with 5% skim milk, the PVDF membrane was incubated with the primary antibody in a cold room overnight. After washing three times with phosphate-buffered saline Tween (PBST) for 15 min, the PVDF membrane was incubated with the primary antibody, and after washing three times with phosphate-buffered saline Tween (PBST) for 15 min, the PVDF membrane was incubated with the secondary antibody. Immunoreactive protein bands were observed using an ECL kit. Images were acquired using a biospectral gel imaging system.

[0048] For immunohistochemical staining (IHC), colon tissue sections were de-affinity and rehydrated. After antigen extraction, they were blocked with bovine serum albumin (5%, 1.5h), immersed in primary antibodies MUC2 (1:2000) and ZO-1 (1:100) and incubated overnight at 4°C. Then, the slides were incubated with secondary antibodies for 1 hour and stained with hematoxylin for 10 minutes to mark cell nuclei. The samples were observed with an optical microscope.

[0049] Results: See Figure 7 , the expression of tight junction proteins MUC2, ZO-1, Occludin and Claudin1 decreased significantly after DSS or TNBS induction, indicating that the intestinal barrier was severely damaged. After Ang treatment, the expression of MUC2, ZO-1, Occludin and Claudin increased significantly, indicating that Ang has a good protective effect on the intestinal barrier of UC mice. The present invention also used IHC experiments to detect changes in key proteins MUC2 and ZO1. The results showed that Ang treatment reversed the phenomenon of decreased MUC2 secretion and decreased ZO-1 expression in UC mice ( Figure 8 ). This shows that Ang can repair the intestinal barrier and maintain the integrity of the intestinal mucosa.

[0050] 5. Ang toxicological safety evaluation

[0051] Methods: After the administration, blood was collected from the eye sockets to examine blood biochemical indicators and evaluate liver function (ALT, AST) and renal function (BUN, CREA). The liver, kidney and spleen were weighed after dissection, and the organ / body weight ratio was calculated to evaluate whether it was different from the blank group.

[0052] Results: See Fig. 9Compared with the blank group, the hematological indices (ALT, AST, BUN, CREA) of the mice in the Ang group did not increase significantly; Fig.10 The results of the organ / body weight ratio of the Ang group showed no significant difference compared with the blank group. Therefore, the 12.6 mg / kg dose of Ang had no obvious toxicity to the liver, kidney function and organ index of mice.

[0053] In summary, the present invention establishes an acute UC mouse model induced by DSS or TNBS, and detects the general clinical symptoms, DAI scores, colon length, colon histopathological scores, inflammatory factors and intestinal mucosal barrier index expression of UC mice treated with Ang. It is found that Ang can effectively alleviate the inflammatory symptoms of UC, including reducing weight loss, diarrhea, blood in the stool and shortened colon, as well as inhibiting the excessive production of proinflammatory factors, increasing the expression of anti-inflammatory factors, and significantly improving the damaged intestinal mucosal barrier. And the results of body weight, DAI score, colon length, colon histopathological score and inflammatory factors show that Ang has similar improvement effects as the positive drug SASP. This suggests that Ang may alleviate UC by protecting the colon tissue structure, reducing colon inflammation and improving colon damage, which can provide a new choice for UC treatment drugs.

Claims

1. Use of isopsoralen in the preparation of a medicament for treating ulcerative colitis, characterized in that: The structure of the isopsoralen is as follows:

2. Use of isopsoralen in the preparation of a drug for adjuvant treatment or relief of ulcerative colitis, characterized in that: The structure of the isopsoralen is as follows:

3. The use of isopsoralen in the preparation of a health product for assisting the treatment or relieving ulcerative colitis, characterized in that: The structure of the isopsoralen is as follows:

4. The use according to any one of claims 1 to 3, characterized in that: Contains at least one of isopsoralen and its pharmaceutically acceptable salt.

5. The use according to any one of claims 1 to 3, characterized in that: The drug contains one or more pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The auxiliary material is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an antibacterial agent, a fragrance, an antioxidant, a pH regulator, a protective agent, a diluent, a lubricant, and a solvent.

7. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is one of injection, capsule, pill, tablet, granule, tincture, inhalation preparation, oral solution, oral suspension and oral emulsion.

8. The use according to any one of claims 1 to 3, characterized in that: The isopsoralen can improve the degree of intestinal mucosal barrier damage and increase the expression levels of MUC2, ZO-1, Occludin and Claudin.

9. The use according to any one of claims 1 to 3, characterized in that: The isopsoralen can reduce weight loss in mice, reduce diarrhea in mice, reduce blood in stool in mice, reduce shortening of the colon in mice, inhibit excessive production of pro-inflammatory factors, and increase the expression of anti-inflammatory factors.

10. The use according to any one of claims 1 to 3, characterized in that: The experimental ulcerative colitis was induced by dextran sulfate sodium or 2,4,6-trinitrobenzenesulfonic acid.

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