Application of Zhongwinning in preparation of medicine for treating Crohn's disease

Abstract

The invention belongs to the technical field of new application of medicines, and provides application of Zhongwinning in preparation of a medicine for treating Crohn's disease. Experimental research proves that the Zhongwinning can obviously improve anorexia, hematochezia, body mass reduction and other conditions of CD model rats; the DAI, CMDI and TDI scores of CD model rats can be obviously reduced; the traditional Chinese medicine composition can obviously reduce the level of proinflammatory factors in colon tissues of rats, improve the level of anti-inflammatory factors, effectively improve intestinal inflammation symptoms and regulate and control secretion of inflammatory cytokines. The result shows that the Zhongwinning has a certain application prospect in CD treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of new drug applications, and in particular relates to the application of Zhonguning in preparing drugs for treating Crohn's disease. Background Art

[0002] Crohn's disease (CD) is a chronic inflammatory granulomatous disease of unknown cause that can affect the entire digestive tract. It is a nonspecific intestinal inflammatory disease and a type of inflammatory bowel disease (IBD). Its lesions can affect the entire digestive tract, from the mouth to the anus. The main lesions are peptic ulcers, the most common of which is intermittent transmural inflammation of the ileum or colon. As the disease progresses, half of the patients will develop complications (stricture, intestinal fistula or abscess).

[0003] The exact pathogenesis of CD is still unclear. It is generally believed that it is caused by the complex interaction between environment, genetics, immunity and microorganisms, and there are obvious regional and racial differences. The high-risk age group for CD is 15-40 years old, with no significant gender difference. The incidence of CD in developed Western countries has stabilized, while the incidence in my country is increasing year by year and is developing at a younger age.

[0004] There is no cure for CD. Symptoms can be relieved after treatment, but CD has a long treatment cycle, is prone to recurrence, and is difficult to cure. Surgery is usually required, but there is a high risk of recurrence. Therefore, how to promote intestinal mucosal healing and histological remission in CD patients is an urgent problem to be solved in current clinical work.

[0005] At present, the commonly used drugs for the treatment of CD include: aminosalicylic acid preparations, glucocorticoids, immunosuppressants, antibacterial drugs, etc. However, the above drugs only relieve the patient's symptoms and maintain treatment, and have the defects of easy relapse, excessive adverse reactions and high tolerance rate. For example, mesalazine is suitable for the mildest Crohn's disease, and its side effect is allergy; hormones are suitable for the acute phase, but can only relieve symptoms, cannot be used for long-term maintenance treatment, and may cause secondary infection, hypertension and peptic ulcers; immunosuppressants include azathioprine and methotrexate, which are used for maintenance treatment, and side effects include liver and kidney damage.

[0006] Mesaconine (CAS NO.: 6792-09-2; molecular formula: C 24 H 39NO9) is a new type of alkaloid with medicinal value. It has a clear chemical structure and can be obtained by raw material extraction or chemical synthesis. Zhongwu Ning has a significant effect on improving cardiac function and can effectively prevent and treat acute heart failure caused by myocardial ischemia-reperfusion injury. However, there is no report on the treatment of Crohn's disease with Zhongwu Ning. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the application of Zhongwu Ning in the preparation of drugs for treating Crohn's disease. Studies have shown that Zhongwu Ning can significantly improve the anorexia, bloody stools, and weight loss of CD model rats; can significantly reduce the DAI, CMDI, and TDI scores of CD model rats; can significantly reduce the level of pro-inflammatory factors in rat colon tissue, increase the level of anti-inflammatory factors, effectively improve the symptoms of intestinal inflammation, and regulate the secretion of inflammatory cytokines. This shows that Zhongwu Ning has a certain application prospect in the treatment of CD.

[0008] In order to achieve the above object, the solution adopted by the present invention is:

[0009] Application of Zhonguning or its pharmaceutically acceptable salt in preparing medicine for treating Crohn's disease.

[0010] Furthermore, in a preferred embodiment of the present invention, the Crohn's disease drug is a preparation prepared with Zhonguning or a pharmaceutically acceptable salt thereof as an active ingredient and adjuvants or auxiliary ingredients commonly used in pharmacy.

[0011] Furthermore, in a preferred embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt.

[0012] Furthermore, in a preferred embodiment of the present invention, the dosage form of the preparation includes a liquid preparation, a solid preparation or a semisolid preparation.

[0013] The beneficial effects of the application of Zhonguning provided by the present invention in preparing a drug for treating Crohn's disease are:

[0014] (1) This application first discovered the use of Zhongwu Ning in the preparation of drugs for the treatment of Crohn's disease. Studies have shown that Zhongwu Ning can significantly improve the anorexia, bloody stools, and weight loss of CD model rats, and improve the mental state, activity level, and eating conditions of CD model rats.

[0015] (2) Studies have shown that Zhongwuning can significantly reduce the DAI, CMDI, and TDI scores of CD model rats, indicating that Zhongwuning has significant therapeutic potential in reducing the disease activity of CD, improving the gross morphological damage of colon tissue, and improving histological damage;

[0016] (3) The present application confirms through research that Zhongwu Ning can reduce the levels of pro-inflammatory factors TNF-α, IL-1β, IL-6 in the serum of CD rats and increase the level of anti-inflammatory factor IL-10 in the serum of CD rats by regulating the expression of TNF-α, IL-1β, IL-6, IL-10, thereby alleviating the intestinal inflammatory response and mucosal barrier damage in CD rats, effectively reducing the level of inflammatory factors in the colon tissue of rats, improving the symptoms of intestinal inflammation, and regulating the secretion of inflammatory cytokines. It has certain application prospects in the treatment of CD. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0018] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0019] Example 1

[0020] 1. Experimental Materials

[0021] Experimental animals: SD rats, SPF grade, provided by Chengdu Dashuo Biotechnology Co., Ltd., male, body weight 170-210g. Production license number: SCXK (Chuan) 2015-030, fully nutritious, caged, in an environment with room temperature of 18-25°C, relative humidity of 50-60%, artificial 12h day / night cycle lighting, cages cleaned regularly every day, rats free to eat and drink water, and experiments were conducted after 1 week of adaptive feeding.

[0022] Main raw materials and reagents: Zhongwuning (provided by Good Doctor Pharmaceutical Group Co., Ltd., referred to as ZWN), mesalazine enteric-coated tablets (China National Pharmaceutical Group Co., Ltd., batch number: H20143164).

[0023] 2. Experimental methods

[0024] 2.1 Animal grouping: Forty newborn rats were randomly divided into 4 groups: Zhongwuning group, positive drug control group, model group and blank control group, with 10 rats in each group.

[0025] 2.2 Modeling method: Referring to the modeling method of MORRIS, 5% TNBS and 50% ethanol were mixed in a ratio of 1:1 to form TNBS enema solution. 3 mL kg -1 (Contains TNBS 80mg·kg -1 )CD model was prepared by enema.

[0026] The rats were fasted but not allowed to drink water for 24 h before modeling. The rats were weighed and the enema volume was calculated in advance. After intraperitoneal injection of 2% sodium pentobarbital, the rats were inverted and liquid paraffin was used to lubricate the anus and enema tube. Except for the blank group, all rats were enema with TNBS enema solution 3 mL kg -1 (Contains TNBS 80mg·kg -1 ) enema. After the enema, the rats were kept inverted for 1 minute to promote full absorption of the enema solution. The rats in the blank group were given enema with the same volume of sterile water. The enema was repeated once every 7 days, for a total of 2 times.

[0027] After modeling, the rats showed decreased appetite, weight loss, and bloody or loose stools. Two rats were randomly selected from each group and killed. The colon (15±2) cm was taken from the anus upward, cut open along the longitudinal axis, and rinsed with saline. The modeling was successful when the rats' colon wall thickened, mucosal congestion and edema or ulcers were observed.

[0028] 2.3 Administration method: All rats were allowed to eat and drink freely. Seven days after modeling, rats in each group were given drugs by gavage. The Zhongwuning group was given Zhongwuning 2 mg / kg by gavage; the positive drug group was given mesalazine enteric-coated tablets 0.2 g / kg by gavage; the model group and the blank control group were given an equal amount of 0.9% sodium chloride solution by gavage; each group was given drugs twice a day for 10 consecutive days.

[0029] 2.4 Measurement indicators and methods:

[0030] 2.4.1 General physical sign observation: Observe and record the rats’ daily mental state, activity level and diet during the experiment;

[0031] 2.4.2 Disease Activity Index (DAI) score, Colon Macroscopic Damage Index (CMDI) score, and Tissue Damage Index (TDI) score:

[0032] DAI score: Record the general information of rats every day, including stool status and weight changes. Evaluate the disease activity of enteritis in each group of rats according to the disease activity index.

[0033] Scoring criteria: The sum of the following three scores is the DAI score. The higher the score, the higher the disease activity.

[0034] (1) Weight loss: no weight loss is 0 points; a weight loss of 1%-5% is 1 point; a weight loss of 6%-10% is 2 points; a weight loss of 11%-15% is 3 points; a weight loss of >15% is 4 points;

[0035] (2) Stool characteristics: 0 points for normal; 1 point for semi-loose stool; 2 points for loose stool;

[0036] (3) Occult blood in stool: negative, 0 points; positive, 1 point; gross blood in stool, 2 points.

[0037] CMDI score: After 10 days, rats were killed by cervical dislocation after fasting for 12 hours without deprivation of water. The colon tissues from 1 cm to 6 cm above the anus were obtained by laparotomy and rinsed with 0.9% sodium chloride solution. The gross morphological injury index of the colon tissue was evaluated by observing the gross injury condition.

[0038] Scoring criteria: 0 points for no damage; 1 point for mild congestion and edema without ulcers; 2 points for congestion, edema and thickening of the intestinal wall; 3 points for severe congestion and edema with ulcers less than 1 cm in diameter; 4 points for ulcers with a diameter greater than 1 cm on the basis of the 3 points; 5 points if there is adhesion between the intestinal tract and surrounding tissues.

[0039] TDI score: After CMDI scoring, colon tissue was collected, one portion was fixed with 10% formalin, dehydrated, embedded in paraffin, and sliced ​​for HE staining and Masson staining, and observed under an optical microscope. The remaining colon tissue was placed in liquid nitrogen for later use. The colon tissue was fixed and sliced ​​to evaluate the histological injury index.

[0040] Scoring criteria: 0 points for no damage; 1 point for mucosal and submucosal inflammatory infiltration / mild erosion; 2 points if the lesion range is >50% based on 1 point; 3 points if the ulcer extends to the submucosal layer and muscularis mucosa; 4 points if the lesion range is >50% based on 3 points; 5 points if cell necrosis reaches the basal layer of the mucosa; 6 points if the lesion range is >50% based on 5 points.

[0041] 2.4.3 Detection of inflammatory factor levels: Take about 100 mg of colon tissue preserved in liquid nitrogen, put it in a mortar, add liquid nitrogen three times, 10 mL each time, grind it quickly and thoroughly, add 1 mL of tissue lysis buffer, and continue grinding until there is no obvious block tissue; take 500 μL of liquid, transfer it to a 1.5 mL EP tube, ultrasonically treat it in an ice bath for 20-30 seconds, centrifuge it for 10 minutes (10000 r / min, r=8 cm), and take the supernatant;

[0042] The ELISA method was used to detect the levels of TNF-α, IL-1β, IL-6, and IL-10 in the serum of rats in each group: the double antibody sandwich method was used, and anti-rat inflammatory factor TNF-α, IL-1β, IL-6, and IL-10 antibodies were coated on the ELISA plate. The antibody was combined with the complex, and the surface impurities were washed away. Then, the antibody labeled with horseradish peroxidase was added to finally form an antibody-antigen-enzyme-labeled antibody complex. The free reagent components were washed away, and the color developing substrate was added. The optical density (OD) value was measured at a wavelength of 450nm using an ELISA instrument. The concentration of rat inflammatory factors was directly proportional to the value.

[0043] 2.5 Statistical methods: The experimental data were analyzed using SPSS 20.0 statistical software. The data results were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for analysis of variance, and the differences between the groups were compared using t-test. P < 0.05 was considered statistically significant.

[0044] 3. Experimental results

[0045] (1) General physical signs observation:

[0046] Model group: anorexia, decreased activity, loose stools, and bloody stools;

[0047] Blank control group: normal eating and normal stool characteristics;

[0048] Zhongwuning group: rats' appetite improved, stool became formed, and bloody stool decreased significantly;

[0049] Positive drug control group: The diarrhea and bloody stools of the rats were significantly improved, and their condition improved.

[0050] (2) Table 1 shows the disease activity index (DAI), colonic tissue gross morphological damage (CMDI) and histological damage (TDI) index scores:

[0051] Table 1 DAI, CMDI and TDI scores of rats in each group

[0052] Group No. DAI CMDI TDI Model Group <![CDATA[5.02±0.68 ## ]]> <![CDATA[5.88±0.48 ## ]]> <![CDATA[5.41±0.45 ## ]]> Blank control group 0 0 0 Zhongwuning Group <![CDATA[3.02±0.53 ** ]]> <![CDATA[3.13±0.51 ** ]]> <![CDATA[2.88±0.49 ** ]]> Positive drug control group <![CDATA[2.53±0.33 ** ]]> <![CDATA[2.79±0.44 ** ]]> <![CDATA[2.41±0.57 ** ]]>

[0053] Note: Compared with the model group, **P<0.01; compared with the blank control group, ## P<0.01.

[0054] As shown in Table 1, compared with the blank control group, the DAI, CMDI, and TDI scores of the model group rats were significantly increased (P < 0.01). Compared with the model group, the positive drug control group and Zhongwuning group had a very obvious effect after intervention, and the scores were significantly reduced; the effect of the Zhongwuning group after intervention was close to that of the positive drug control group.

[0055] The above results show that Zhonguning can significantly reduce DAI, CMDI, and TDI scores, and the effect is close to that of the positive drug mesalazine enteric-coated tablets, indicating that Zhonguning has obvious therapeutic potential in reducing the disease activity of CD, improving the gross morphological damage of colon tissue, and improving histological damage.

[0056] (3) Table 2 shows the test results of inflammatory factor levels in rats in each group:

[0057] Table 2 Levels of inflammatory factors in rats in each group

[0058] Group No. IL-1β (ng / mg) IL-6 (ng / mg) TNF-α(ng / mg) IL-10 (ng / mg) Model Group <![CDATA[45.32±2.53 ## ]]> <![CDATA[155.12±4.11 ## ]]> <![CDATA[134.33±3.32 ## ]]> <![CDATA[20.12±4.51 ## ]]> Blank control group 14.34±2.18 58.25±3.64 45.28±3.05 35.56±5.22 Zhongwuning Group <![CDATA[23.25±2.45 ** ]]> <![CDATA[85.28±3.15 ** ]]> <![CDATA[58.71±3.18 ** ]]> <![CDATA[40.68±4.35 ** ]]> Positive drug control group <![CDATA[21.41±2.33 ** ]]> <![CDATA[80.34±4.08 ** ]]> <![CDATA[55.24±3.24 ** ]]> <![CDATA[42.44+3.86 ** ]]>

[0059] Note: Compared with the model group, **P<0.01; compared with the blank control group, ## P<0.01.

[0060] The data in Table 2 show that compared with the blank control group, the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of rats in the model group were significantly increased, and the level of anti-inflammatory factor IL-10 was significantly decreased, and the difference was statistically significant (P < 0.05).

[0061] Compared with the model group, the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of rats in the positive drug control group and Zhongwuning group were significantly lower than those in the model group, and the level of anti-inflammatory factor IL-10 was significantly increased, with statistically significant differences (P < 0.05). The levels of inflammatory factors in the colon tissue of rats in the Zhongwuning group were close to those in the positive drug control group.

[0062] The above results show that Zhongwuning can alleviate the intestinal inflammatory response and mucosal barrier damage in CD rats by regulating the expression of TNF-α, IL-1β, IL-6, and IL-10, effectively reduce the level of inflammatory factors in the colon tissue of rats, improve the symptoms of intestinal inflammation, and regulate the secretion of inflammatory cytokines. It has certain application prospects in the treatment of CD.

[0063] In summary, the present invention provides the use of Zhongwu Ning in the preparation of drugs for treating Crohn's disease. Experimental studies have confirmed that Zhongwu Ning can significantly improve anorexia, bloody stools, weight loss and the like in CD model rats, and improve the mental state, activity level and eating conditions of CD model rats; Zhongwu Ning can significantly reduce the DAI, CMDI and TDI scores of CD model rats, indicating that Zhongwu Ning has obvious therapeutic potential in reducing the disease activity of CD, improving the gross morphological damage of colon tissue, and improving histological damage; Zhongwu Ning can reduce the intestinal inflammatory response and mucosal barrier damage of CD rats by regulating the expression of TNF-α, IL-1β, IL-6 and IL-10, effectively reduce the level of inflammatory factors in the colon tissue of rats, improve the symptoms of intestinal inflammation, and regulate the secretion of inflammatory cytokines, and has certain application prospects for the treatment of CD.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Zhonguning or its pharmaceutically acceptable salt in preparing a drug for treating Crohn's disease.

2. The use according to claim 1, characterized in that: The Crohn's disease drug is a preparation prepared by taking Zhonguning or its pharmaceutically acceptable salt as an active ingredient and adding auxiliary materials or auxiliary ingredients commonly used in medicine.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable salt is an inorganic salt or an organic salt.

4. The use according to claim 2, characterized in that: The dosage form of the preparation includes a liquid preparation, a solid preparation or a semisolid preparation.