Application of pseudoprevotella griseus in preparation of medicine for treating inflammatory bowel disease
By using Prevotella grey as a treatment for inflammatory bowel disease, the problems of low titer and major side effects of existing drugs have been solved, and safe and efficient treatment of inflammatory bowel disease has been achieved, which has significantly improved the patient's symptoms and Quality of life.
Patent Information
- Application Number
- CN202411565112.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-01-10
AI Technical Summary
The existing inflammatory bowel disease treatment drugs have problems such as low protective titer, short action time, and major toxic and side effects, and lack safe, efficient and side effects-free treatment methods.
Using Prevotella grey as a therapeutic drug, and through oral administration, it significantly improves symptoms of inflammatory bowel disease, such as shortening of the colon, shedding of intestinal villi, reduced goblet cells and inflammatory response in the colon, as a dominant strain Used for oral intestinal microbiota preparations.
It has achieved the prevention, relief or cure of inflammatory bowel disease, and has the advantages of small side effects, easy operation and low treatment cost, which has significantly improved the clinical manifestations of inflammatory bowel disease.
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Figure CN119280279A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedicine, and particularly relates to the application of Pseudomonas griseus in the preparation of a medicine for treating inflammatory bowel disease. Background Art
[0002] Inflammatory bowel disease, also known as inflammatory bowel disease (IBD), is an idiopathic, chronic but recurrent inflammatory disease of the gastrointestinal tract, including ulcerative colitis (UC) and Crohn's disease (CD). It is estimated that about 7 million people suffer from IBD worldwide, and the incidence of IBD is increasing significantly. IBD has become a common digestive disease and the main cause of chronic diarrhea, and most patients are young and middle-aged. The clinical manifestations of IBD are diarrhea, abdominal pain, and mucus, pus and blood in the stool, which seriously affect the quality of life of patients and impose a huge burden on public health. Therefore, it has received increasing research attention.
[0003] As research on IBD continues to deepen, recent studies have shown that the occurrence of inflammatory bowel disease may be related to genetics, environmental factors, and abnormal immune responses, but the etiology and pathogenesis of IBD have not yet been fully clarified. Studies and clinical data have shown that intestinal flora imbalance may play a key role in the pathogenesis of IBD. Therefore, regulating the intestinal flora and restoring the balance of the intestinal flora is a potential effective method for treating inflammatory bowel disease.
[0004] Over the years, the research and development of inflammatory bowel disease treatment has never stopped at home and abroad. Many drugs developed are difficult to promote and apply due to their low protective efficacy, short duration of action, and large toxic side effects. At present, there is still a lack of inflammatory bowel disease treatment drugs with good therapeutic effects, safety, low toxicity, and easy use. Therefore, there is an urgent need to develop effective IBD treatment drugs.
[0005] Probiotics are a type of active microorganisms that can improve the ecological balance of intestinal flora and are beneficial to the intestines. Due to their low price, convenience of use and good experience, they are increasingly attracting research attention.
[0006] Therefore, there is an urgent need in the art for a safe, highly effective, and side-effect-free therapy for inflammatory bowel disease. Summary of the invention
[0007] The purpose of the present invention is to provide a use of Pseudomonas griseus in the preparation of a drug for treating inflammatory bowel disease. The drug of the present invention can reduce DSS (dextran sulfate)-induced inflammatory bowel disease, has the effects of reducing colon shortening, intestinal villus shedding, goblet cell number reduction and colon inflammation, and has the advantages of small side effects and outstanding therapeutic effect.
[0008] The technical solution of the present invention is:
[0009] Use of Pseudomonas griseus in preparing medicines for treating inflammatory bowel disease.
[0010] The inflammatory bowel disease is colitis caused by DS.
[0011] The inflammatory bowel disease is caused by a shortening of the colon in mice.
[0012] The inflammatory bowel disease is caused by the loss of colon villi.
[0013] The inflammatory bowel disease is caused by a decrease in the number of crypts in the intestine.
[0014] The inflammatory bowel disease is caused by increased inflammation of the colon.
[0015] The drug is administered orally.
[0016] The oral bacterial amount is 1×10 9 CFU / day.
[0017] The beneficial effects of the present invention are:
[0018] The gray pseudoprevotella prepared by the present invention can significantly improve the symptoms associated with inflammatory bowel disease, such as shortened colon, loss of intestinal villi, reduced number of goblet cells and colonic inflammatory response, thereby preventing, alleviating or curing inflammatory bowel disease. The present invention provides a superior strain for preventing, alleviating or curing inflammatory bowel disease, which can be used as an oral intestinal flora preparation. At present, the drugs for the clinical treatment of inflammatory bowel disease mainly include: traditional anti-inflammatory drugs, immunosuppressants and antibiotics. However, these therapeutic drugs have certain side effects, cannot be used for a long time or have high costs. Gray pseudoprevotella can overcome the above-mentioned shortcomings and achieve the advantages of small side effects, easy implementation and low treatment cost for the treatment of inflammatory bowel disease (gray pseudoprevotella can be obtained by large-scale culture, with low preparation cost; gray pseudoprevotella can be directly orally administered, which is easy to operate).
[0019] The gray Pseudo-Prevotella of the present invention is Alloprevotella rava strain. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 P. griseus ameliorates DSS-induced colon shortening.
[0021] Figure 2 Pathological section showing that Prevotella griseus reduces intestinal villus loss in colitis.
[0022] Figure 3 Statistical results for the increase in the number of colitis goblet cells by Pseudomonas griseus.
[0023] Figure 4 Pseudoprevotella griseus reduces inflammation in colitis. DETAILED DESCRIPTION
[0024] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.
[0025] Reagent Source
[0026] 1. Male C57BL / 6J mice aged 6-8 weeks and weighing 20-22 g (Wei Tong Li Hua Laboratory Animal Technology Co., Ltd., Beijing)
[0027] 2. Carnoy's fixative (G1120-500mL, Seville, Hubei)
[0028] 3. Ethanol (Sinopharm, Chongqing)
[0029] 4. Xylene (X112051, Aladdin, Shanghai)
[0030] 5. Hematoxylin and eosin (HE) staining kit (C0105S, Bio-Tech, Shanghai)
[0031] 6. Neutral gum (ZLI-9555, Zhongshan Jinqiao, Beijing)
[0032] 7. Scanning microscope (M8, PreciPoint, France)
[0033] 8. PBS phosphate buffer (pH 7.2-7.4) (ZLI-9062, Zhongshan Jinqiao, Beijing)
[0034] 9. Automatic biological tissue pathology dehydration machine (ZT-12M2, Yaguang Pathology, Hubei)
[0035] 10. Biological tissue paraffin embedding machine (YB-7LF, Yaguang Pathology, Hubei)
[0036] 11. Paraffin slicer (Leica RM2245, Leica, Germany)
[0037] 12. Alcian blue-nuclear fast red staining kit (C0155M, Bio-Tech, Shanghai)
[0038] 13. Pseudomonas griseus (358370, Beina Biotech, Beijing)
[0039] 14.DSS (9011-18-1, MP Biomedicals, USA)
[0040] Other reagents were commercially available.
[0041] Example 1: Improvement effect of Pseudomonas griseus on colon shortening in colitis
[0042] 1. Experimental groups:
[0043] Male C57BL / 6J mice were randomly divided into four groups (n=10 in each group): (1) control group (Sham); (2) P. griseus group; (3) DSS group; and (4) P. griseus + DSS group.
[0044] 2. Experimental treatment:
[0045] The DSS group and P. griseus + DSS group were given 3% DSS drinking water; the control group and P. griseus group were given normal drinking water.
[0046] 3. Administration and dosage:
[0047] The administration method was oral administration. The mice in the control group and the DSS group were gavaged with 200 μl of normal saline every other day for 14 consecutive days. The mice in the P. griseus group and the P. griseus + DSS group were gavaged with 1×10 9 CFU / day of P. griseus for 14 consecutive days. After the drug treatment, the mice in the DSS group and P. griseus + DSS group were given 3% DSS drinking water, and the weight changes of the mice in each group were observed and recorded every day.
[0048] 4. Experimental observation indicators:
[0049] On the 7th day of the experiment, the mice in each group were killed and the colons were collected for photography and length measurement.
[0050] 5. It has been tested that oral administration of Pseudomonas griseus before irradiation can significantly increase the colon length of mice with DSS-induced colitis (see Figure 1 ).
[0051] Example 2: Effect of Pseudomonas griseus on intestinal villus shedding caused by colitis 1. Animal model construction and drug administration:
[0052] Referring to steps 1-4 in Example 1, a colitis mouse model was established and the mice were administered Pseudomonas griseus.
[0053] 2. Identification of intestinal tissue damage:
[0054] After 7 days of DSS treatment, mice were killed by cervical dislocation, and the colon segments were removed and rinsed with pre-cooled PBS. The small intestine was then rolled up with a small wooden stick, with the villi facing inward, and fixed with a pin. After fixation with Carnoy's fixative overnight, the tissue was dehydrated with gradient alcohol concentrations and the alcohol in the tissue was replaced with xylene. The transparent tissue block was placed in the dissolved paraffin and kept warm in a wax dissolving box. After the embedded wax block was fixed on a microtome, it was cut into 4 μm slices and stained with hematoxylin and eosin (HE) for observation.
[0055] 3. HE staining:
[0056] Use xylene to remove paraffin from mouse intestinal tissue sections, then transfer to distilled water through high-concentration to low-concentration alcohol. Take out the sections, put them in hematoxylin for 2 minutes, tap water for 1 minute, 0.6% hydrochloric acid alcohol for 15 seconds, rinse with tap water for 15 minutes, and place the sections in distilled water for a while. Move to 1% alcohol solution eosin for 30 seconds, dehydrate the sections with alcohol, make them transparent with xylene, drop neutral gum on them, and cover them with coverslips for sealing.
[0057] 4. The pathological changes of small intestinal tissues observed by Precipoint M8 scanning microscope showed that the colon mucosal barrier of DSS-treated mice was severely damaged, intestinal epithelial cells were significantly shed, and intestinal villi were atrophied; administration of Pseudomonas griseus before DSS treatment significantly alleviated intestinal epithelial shed and intestinal villi maintained a good morphology (see Figure 2 ). The scale bar indicates 200 μm.
[0058] Example 3: Effect of Pseudomonas griseus on the number of colitis goblet cells
[0059] According to steps 3-4 in Example 2, the mouse colon tissue was stained with Alcian blue-nuclear fast red and observed. The number of goblet cells in the colon tissue was detected by Precipoint M8 scanning microscope. Figure 3 As shown: The number of goblet cells in 6 different fields of view was detected in each slide. The number of goblet cells in normal mice and mice treated with Pseudomonas griseus alone was 107 and 113 / mm, respectively; after DSS treatment, the number of goblet cells decreased to 36 / mm. Administration of Pseudomonas griseus maintained the number of goblet cells at 69 / mm, which was significantly different from that of mice treated with DSS.
[0060] Example 4: Effect of Pseudomonas griseus on inflammatory factors of colitis
[0061] 1. Referring to steps 1-4 in Example 1, a colitis mouse model was established and mice were administered Pseudomonas griseus.
[0062] 2. On the 7th day after DSS treatment, mice were killed by cervical dislocation, and the colon segments were removed and rinsed with pre-cooled PBS.
[0063] 3. The intestinal tissues of each group were homogenized and protein was quantified using BCA.
[0064] 4. Use IL-1β and IL-6 kits to detect and quantify the content of IL-1β and IL-6 in the colon tissue of each group, and calculate them.
[0065] 5. Tested as Figure 4 As shown: the IL-6 of normal mice and mice treated with Pseudomonas griseus alone was 131pg / mg and 135pg / mg, and the IL-1β was 125pg / mg and 122pg / mg; after DSS treatment, IL-6 and IL-1β were 187pg / mg and 249pg / mg, respectively. The IL-6 and IL-1β levels of Pseudomonas griseus were 137pg / mg and 175pg / mg, which were significantly different from those of mice treated with DSS.
[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.
Claims
1. Application of Pseudomonas griseus in the preparation of medicines for treating inflammatory bowel disease.
2. The use according to claim 1, characterized in that: The inflammatory bowel disease is colitis caused by DSS.
3. The use according to claim 1, characterized in that: The inflammatory bowel disease is caused by a shortening of the colon in mice.
4. The use according to claim 1, characterized in that: The inflammatory bowel disease is caused by the loss of colon villi.
5. The use according to claim 1, characterized in that: The inflammatory bowel disease is caused by a decrease in the number of crypts in the intestine.
6. The use according to claim 1, characterized in that: The inflammatory bowel disease is caused by increased inflammation of the colon.
7. The use according to claim 1, characterized in that: The drug is administered orally.
8. The use according to claim 1, characterized in that: The oral bacterial amount is 1×10 9 CFU / day.