Application of polydatin in preparation of medicine for treating ulcerative colitis

Abstract

The invention provides application of polydatin in preparation of a medicine for treating ulcerative colitis. The application proves that polydatin can improve pathological injury of colon tissues, inhibit Th1 / Th17 immunoreaction abnormally activated in intestinal tracts, remarkably reduce expression of proinflammatory factors and relieve oxidative stress, so that symptoms of ulcerative colitis are effectively relieved, and the problem of how to safely and effectively treat ulcerative colitis is solved. The invention also provides a medicine for treating ulcerative colitis, the medicine comprises polydatin and a pharmaceutically acceptable carrier, the polydatin is prepared into the medicine for treating ulcerative colitis, and the medicine has a wide clinical application prospect.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and mainly to the application of polydatin in the preparation of drugs for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease caused by a variety of endogenous and external factors. Because patients need to go to the toilet frequently, their social activities are limited, and rectal bleeding can cause fear and anxiety, seriously affecting their quality of life. In addition, chronic and recurrent inflammation increases the risk of cancer.

[0003] In clinical practice, 5-aminosalicylic acid drugs (5-ASA, trade name: mesalazine) are one of the first choice drugs for the treatment of UC, which can relieve symptoms by reducing intestinal inflammatory response. However, some patients are resistant to 5-ASA, resulting in poor treatment effect. In addition, long-term use of 5-ASA may cause some adverse reactions, such as headache, nausea, liver and kidney damage, and acute pancreatitis. Azathioprine is an immunosuppressant that reduces inflammatory response by inhibiting the activity of the immune system. However, long-term use of azathioprine may cause serious side effects such as bone marrow suppression, infection, and liver damage. Glucocorticoids can quickly relieve the symptoms of UC, but long-term use can lead to osteoporosis, hypertension, and diabetes.

[0004] Based on this, finding a substance that can safely and effectively treat ulcerative colitis is a problem worth studying. Summary of the invention

[0005] In order to solve the problem of how to safely and effectively treat ulcerative colitis, the present application provides the use of polydatin in the preparation of a drug for treating ulcerative colitis.

[0006] Polydatin is a natural polyphenol compound extracted from plants such as Polygonum cuspidatum. Polydatin can improve pathological damage to colon tissue, inhibit abnormally activated Th1 / Th17 immune response in the intestine, significantly reduce the expression of pro-inflammatory factors, and alleviate oxidative stress, thereby effectively alleviating the symptoms of ulcerative colitis and solving the problem of how to safely and effectively treat ulcerative colitis.

[0007] Optionally, the polydatin treats ulcerative colitis by inhibiting Th1 / Th17 cell immune response.

[0008] Optionally, the polydatin treats ulcerative colitis by reducing the level of pro-inflammatory cytokines.

[0009] Optionally, the pro-inflammatory factors include IFN-γ, IL-1β, IL-17 and TNF-α.

[0010] Optionally, the polydatin treats ulcerative colitis by reducing Th1 / Th17 cell infiltration in intestinal mucosal tissue.

[0011] Optionally, the polydatin treats ulcerative colitis by activating Nfe2l2 and downstream signaling pathways to reduce oxidative stress.

[0012] One of the most prominent immunopathological hallmarks of intestinal inflammation in UC is CD4 + Dysregulation of T cell-mediated immune response. There is an overactive Th1 / Th17 cell response in the intestinal mucosa and peripheral blood of UC patients. Th1 cells and Th17 cells are two major T cell subsets, which play an important role in immune regulation and inflammatory response. Th1 cells mainly produce IFN-γ, which plays a key role in anti-bacterial and cell-mediated immune responses. IL-17A produced by Th17 cells has important functions in anti-fungal and autoimmune responses. Studies have found that in the inflamed intestinal mucosal tissue of UC patients, the infiltration of Th1 and Th17 cells is significantly increased, and the secretion of inflammatory factors such as IFN-γ, IL-17A and TNF-α is increased. This over-activated Th1 / Th17 cell response leads to the continuous aggravation of the inflammatory response of the intestinal mucosa and continuous damage to the intestinal tissue.

[0013] Optionally, the dosage form of the drug includes oral preparations and enemas.

[0014] Optionally, the oral preparation includes tablets, granules, capsules and oral liquids.

[0015] Optionally, the enema includes enema solution and suppository.

[0016] The present application also provides a drug for treating ulcerative colitis, which comprises polydatin and a pharmaceutically acceptable carrier.

[0017] The present application provides the use of polydatin in the preparation of a drug for treating ulcerative colitis. The present application confirms that polydatin can improve pathological damage to colon tissue, inhibit abnormally activated Th1 / Th17 immune responses in the intestine, significantly reduce the expression of proinflammatory factors, and reduce oxidative stress, thereby effectively alleviating the symptoms of ulcerative colitis and solving the problem of how to safely and effectively treat ulcerative colitis. The present application also provides a drug for treating ulcerative colitis, the drug comprising polydatin and a pharmaceutically acceptable carrier, and polydatin is prepared into a drug for treating ulcerative colitis, which has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0019] Figure 1 Schematic diagram of model construction and results of Example 1 (Figure A is a schematic diagram of model construction; Figure B is a schematic diagram of mouse weight change results; Figure C is a schematic diagram of colon tissue HE staining and pathological scoring);

[0020] Figure 2 This is a schematic diagram showing the effect of polydatin on the body weight changes of mice with chronic colitis induced by DSS;

[0021] Figure 3 Schematic diagram of model construction and results of Example 2 (Figure A is a schematic diagram of model construction; Figure B is the change in mouse weight; Figure C is a schematic diagram of DAI results; Figure D is a representative photo of the colon of each group, colon length statistics and histopathological scores; Figure E is HE staining and pathological scores of colon tissue);

[0022] Figure 4 The differentiation and activation of Th1 / Th17 cells in intestinal mucosal tissue in Example 2;

[0023] Figure 5 To analyze the transcriptional levels of Th1 / Th17 cell-related transcription factors and cytokines in colon tissue;

[0024] Figure 6 Analysis of pro-inflammatory cytokine protein levels in colon tissue culture supernatants;

[0025] Figure 7 To analyze the transcriptional levels of Nfe2l2 and downstream anti-oxidative stress molecules in colon tissue;

[0026] Figure 8 Schematic diagram of the experimental results in Example 3 (Figure A is a flow cytometry comparison of the effect of polydatin on the differentiation of Th1 / Th17 cells in peripheral blood of UC patients and healthy subjects, and Figure B is a flow cytometry comparison of the effect of polydatin on the differentiation of CD4+ / -17 cells in peripheral blood of UC patients and healthy subjects) + (Histogram showing the effect of each T cell subtype ratio). DETAILED DESCRIPTION

[0027] The following embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following embodiments do not represent all implementations consistent with the present application. They are only examples of systems and methods consistent with some aspects of the present application as detailed in the claims.

[0028] In order to solve the problem of how to enable antioxidants to achieve efficient antioxidant effects at lower dosages, the present application provides the use of polydatin in the preparation of a drug for treating ulcerative colitis.

[0029] Polydatin is a natural polyphenol compound extracted from plants such as Polygonum cuspidatum. Polydatin can improve pathological damage to colon tissue, inhibit abnormally activated Th1 / Th17 immune response in the intestine, significantly reduce the expression of pro-inflammatory factors, and alleviate oxidative stress, thereby effectively alleviating the symptoms of ulcerative colitis and solving the problem of how to safely and effectively treat ulcerative colitis.

[0030] In some embodiments, the polydatin treats ulcerative colitis by inhibiting Th1 / Th17 cell immune response.

[0031] In some embodiments, the polydatin treats ulcerative colitis by reducing the levels of pro-inflammatory cytokines.

[0032] In some embodiments, the pro-inflammatory factors include IFN-γ, IL-1β, IL-17 and TNF-α.

[0033] In some embodiments, the polydatin treats ulcerative colitis by reducing Th1 / Th17 cell infiltration in intestinal mucosal tissue.

[0034] In some embodiments, the polydatin treats ulcerative colitis by activating Nfe2l2 and downstream signaling pathways to reduce oxidative stress.

[0035] One of the most prominent immunopathological hallmarks of intestinal inflammation in UC is CD4 + Dysregulation of T cell-mediated immune response. There is an overactive Th1 / Th17 cell response in the intestinal mucosa and peripheral blood of UC patients. Th1 cells and Th17 cells are two major T cell subsets, which play an important role in immune regulation and inflammatory response. Th1 cells mainly produce IFN-γ, which plays a key role in anti-bacterial and cell-mediated immune responses. IL-17A produced by Th17 cells has important functions in anti-fungal and autoimmune responses. Studies have found that in the inflamed intestinal mucosal tissue of UC patients, the infiltration of Th1 and Th17 cells is significantly increased, and the secretion of inflammatory factors such as IFN-γ, IL-17A and TNF-α is increased. This over-activated Th1 / Th17 cell response leads to the continuous aggravation of the inflammatory response of the intestinal mucosa and continuous damage to the intestinal tissue.

[0036] In some embodiments, the dosage form of the drug includes oral preparations and enemas.

[0037] In some embodiments, the oral preparations include tablets, granules, capsules and oral solutions.

[0038] In some embodiments, the enema formulation includes enema solutions and suppositories.

[0039] The present application also provides a drug for treating ulcerative colitis, which comprises polydatin and a pharmaceutically acceptable carrier.

[0040] The present invention constructs two colitis models that are recognized both at home and abroad and can better simulate the clinical characteristics and pathophysiological characteristics of ulcerative colitis: the DSS-induced chronic colitis model and the CD45RB high CD4 + The T cell transplantation-induced chronic colitis model confirmed that polydatin can effectively relieve symptoms such as weight loss and bloody stools in mice, reduce disease activity score (DAI), protect the integrity of the intestinal epithelial barrier, and alleviate colon pathological damage. At the same time, it inhibits Th1 / Th17 cell infiltration, reduces the secretion of inflammatory factors and alleviates oxidative stress response, thereby achieving the effect of relieving colon inflammation. This provides new ideas for the clinical treatment strategy of ulcerative colitis and has broad application prospects.

[0041] Example 1: The alleviating effect of polydatin on chronic experimental colitis induced by DSS in mice

[0042] 1. Materials and Methods

[0043] (1) Animals: Male C57BL / 6 mice (8 weeks old, 23-25 ​​g) were purchased from Shanghai Slake Experimental Animal Co., Ltd. After 7 days of adaptive feeding, the animals were randomly divided into three groups according to their body weight: control group (Control), DSS model group (DSS+Water) and polydatin treatment group (DSS+Polydatin), with 10 mice in each group.

[0044] (2) Modeling: Figure 1 As shown in Figure A, the model was established on day 0. The control group had free drinking water. The model group and the treatment group had drinking water replaced with 2% DSS aqueous solution for 7 consecutive days, followed by regular drinking water for 14 days. This cycle lasted for 21 days and was repeated for two cycles. Then, 2% DSS aqueous solution was given for another 7 days. On day 49, CO 2 All mice were killed under anesthesia. During the modeling period, the weight, defecation and blood in the feces of the mice were observed and recorded.

[0045] (3) Intervention: Purified polydatin was produced by ChemFaces, Wuhan, China, and dissolved in 0.5% dimethyl sulfoxide (DMSO) with a final concentration of 4.5 g / mL. The oral dose was 45 g / kg with a volume of 0.01 mL / g. From day 0, the polydatin treatment group was given 45 g / kg of polydatin solution by oral gavage daily, and the control group and the model group were given an equal volume of 0.5% DMSO.

[0046] (4) Material: CO 2 The mice were anesthetized and killed, the colon was removed, the intestinal contents were removed, and 5-10 mm complete circular colon tissue was cut with ophthalmic scissors and placed in 10% paraformaldehyde tissue fixative to denature and coagulate the proteins of the tissue and cells for HE staining. The remaining colon tissue was quickly placed in a 50 mL centrifuge tube containing 25 mL PBS and placed on ice for extraction of intestinal mucosal lamina propria mononuclear cells (LPMC) for flow cytometric analysis.

[0047] (5) Histopathological scoring: The colon tissues were fixed with 10% paraformaldehyde for 24 hours and then embedded in paraffin. Serial sections were cut and stained with hematoxylin-eosin (HE). The structural integrity of the colon tissue and the infiltration of immune cells were observed under an upright optical microscope, and the histopathological scoring was performed according to Table 1.

[0048] Table 1 Scoring criteria for mouse colitis tissue pathology

[0049]

[0050]

[0051] (6) Flow cytometry analysis: Prepare LPMC suspension and separate CD4 + T cells, 2×10 6 The cells were transferred to a 24-well cell culture plate, and anti-mouse CD28 antibody (2 μg / mL), PMA (50 ng / mL), and Ionomycin (750 ng / mL) were added and incubated in a 37°C primary cell culture incubator for 2 hours. Brefeldin A (BFA, 3 μg / mL) was then added and cultured for another 3 hours, and intracellular staining was performed according to the flow cytometry antibody staining procedure of BD Bioscience, USA.

[0052] (7) Data and statistical analysis: GraphPad Prism 10 was used to perform statistical analysis, and t-test was used to determine differences. p < 0.05 was considered statistically significant.

[0053] 2. Experimental results

[0054] (1) Polydatin effectively alleviates the clinical symptoms of DSS-induced chronic colitis in mice and reduces intestinal pathological damage. Figure 1 As shown, polydatin significantly reduced the weight loss of mice. HE staining showed that the colon tissue structure of mice in the model group was significantly damaged, with a large number of inflammatory cells infiltrating. However, the colon tissue structure of mice in the polydatin treatment group was more complete, with reduced cell infiltration, and the histopathological score was significantly lower than that in the DSS modeling group.

[0055] (2) Polydatin reduces the infiltration of Th1 / Th17 cells in intestinal mucosal tissue.

[0056] like Figure 2 As shown, the intestinal mucosal IFN-γ of mice in the DSS model group + and IL-17A + CD4 + The proportion of T cells (Th1 and Th17) increased significantly, and polydatin treatment significantly alleviated the over-activation of Th1 / Th17 cells in intestinal mucosal tissue.

[0057] Example 2: Effect of Polydatin on CD45RB high CD4 + Relief of chronic experimental colitis induced by T cell transplantation

[0058] 1. Materials and Methods

[0059] (1) Animals: Male C57BL / 6 mice (6 weeks old, 18-22 g) were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Rag1 - / - Mice were purchased from Shanghai Model Organisms Technology Co., Ltd. After 7 days of adaptive feeding, the animals were randomly divided into 4 groups according to their body weight: blank control group (Control), drug control group (PBS+Polydatin), model group (CD45RB hi CD4+T cells+Water) and polydatin treatment group (CD45RB hi CD4+T cells+Polydatin), 10 mice in each group.

[0060] (2) Modeling: Magnetic beads were used to separate high-purity CD4 + T cells were then flow cytometry sorted for CD25-CD45RB high CD4+T cells, 5×10 5 / rag1 was injected intravenously into the model group and polydatin treatment group - / - The blank and drug control groups were injected with the same volume of PBS. The beginning of modeling was defined as day 0. After 8 weeks, CO 2 Sacrifice by anesthesia.

[0061] (3) Intervention: Starting from the fourth week of modeling, the drug control group and the polydatin treatment group were given polydatin solution 45 g / kg by intragastric administration every day, and the blank control group and the model group were given an equal volume of 0.5% DMSO.

[0062] (4) Record: Starting from day 0, the weight, stool characteristics, and blood in stool of the mice were recorded every day, and the disease activity index (DAI, DAI = (weight loss score + stool characteristics score + blood in stool score) / 3) was calculated according to Table 2.

[0063] Table 2 Disease activity scoring criteria

[0064] score Weight loss (%) Stool characteristics Occult blood in stool / blood in stool 0 0 normal normal 1 1-5 Loose —— 2 5-10 —— Occult blood positive 3 10-15 Loose stools —— 4 >15 —— Naked bloody stool

[0065] (5) Real-time fluorescence quantitative PCR (qRT-PCR): A mouse intestinal mucosal sample of about 5 mm was collected and placed in a cryovial, which was quickly stored in a dry ice or liquid nitrogen tank. Total RNA was extracted using the phenol-chloroform method, reverse transcribed using the 5× ALL-In-One MasterMix kit, and then quantitatively analyzed using the Takara fluorescence quantitative PCR kit.

[0066] (6) Enzyme-linked immunosorbent assay (ELISA): 5 mm × 5 mm colon tissue was collected and placed in 1640 culture medium. After incubation at 37°C for 24 h, the supernatant was collected and the cytokine levels were detected using the Invitrogen ELISA kit.

[0067] (7) Histopathological scoring, flow cytometric analysis and statistical analysis were as described in Example 1.

[0068] 2. Experimental results

[0069] (1) Polydatin treatment effectively alleviates CD45RB high CD4 + T cell transplantation induced chronic colitis symptoms in mice and alleviated intestinal pathological damage.

[0070] As shown in Table 3 and Figure 3 As shown, polydatin significantly reduced the weight loss and colon length shortening of mice, and reduced the disease activity score. HE staining showed that the intestinal mucosal structure of mice in the model group was severely damaged, while polydatin significantly alleviated this situation and reduced the colon tissue pathology score.

[0071] Table 3 DAI scores, colon length and colon histopathological scores of mice

[0072] Grouping DAI Rating Colon length (cm) Histopathological scoring Blank control group 0.00±0.00 7.96±0.53 0.00±0.00 Drug control group 0.00±0.00 7.74±0.45 0.00±0.00 Model Group 2.56±0.54 6.72±0.83* 5.40±1.51 Polydatin treatment group <![CDATA[1.17±0.80 ### ]]> <![CDATA[7.45±0.51 # ]]> <![CDATA[3.10±0.99 ### ]]>

[0073] Note: DAI score is the value after 8 weeks of modeling. Data are rounded to 2 decimal places. In the same column of the table, compared with the Control group: *p<0.05, ***p<0.001; compared with the CD45RBhiCD4+T cells+Water group: ###p<0.001.

[0074] (2) Polydatin treatment alleviates the Th1 / Th17 cell immune response in intestinal mucosal tissue.

[0075] like Figure 4 As shown in the figure, compared with the model group, the intestinal mucosal IFN-γ of mice in the polydatin treatment group + IL-17A + TNF-α + CD4 + The proportion of T cells decreased significantly, while IL-10 + CD4 + The significant increase in T cells indicated that polydatin inhibited the proliferation and differentiation of intestinal mucosal Th1 / Th17 cells and inhibited CD4 + Proinflammatory responses of T cells.

[0076] (3) Polydatin treatment reduces the levels of intestinal inflammatory factors.

[0077] The levels of inflammatory factors in the intestinal tissue culture supernatant are shown in Table 4. qRT-PCR detection of total colon RNA (e.g. Figure 5 ) and intestinal tissue culture supernatant ELISA test (as shown Figure 6 The results showed that the levels of inflammatory factors IFN-γ, IL-1β, IL-17A and TNF-α in the polydatin treatment group were significantly lower than those in the model group, indicating that polydatin can improve the intestinal immune microenvironment.

[0078] Table 4 Levels of inflammatory factors in intestinal tissue culture supernatant

[0079]

[0080] Note: Data are rounded to 2 decimal places. In the same column of the table, compared with the CD45RBhiCD4+T cells+Water group: ###p<0.001.

[0081] (4) Polydatin activates the Nfe2l2 pathway and reduces oxidative stress.

[0082] Oxidative stress is an important factor that causes intestinal epithelial cell damage, promotes Th1 / Th17 cell differentiation and activation, and promotes the expression of pro-inflammatory cytokines, and Nfe2l2 plays an important role in the anti-oxidative stress response. Figure 7As shown in the results, the expression of Nfe2l2 and downstream antioxidant stress molecules Sod, Hmox1, and Nqo1 in the colon of mice treated with polydatin were upregulated, indicating that polydatin can activate the intestinal antioxidant stress response and improve the intestinal microenvironment.

[0083] Example 3: Polydatin inhibits the differentiation of Th1 / Th17 cells in peripheral blood of UC patients

[0084] 1. Materials and Methods

[0085] (1) Isolation, culture and differentiation of human peripheral blood CD4+ T cells: 5-10 mL of venous blood was collected from UC patients (10 cases) and healthy subjects (HC) using EDTA anticoagulant tubes. Peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium (Ficoll-Paque), and CD4+ T cells were then isolated using anti-human CD4 magnetic beads. + T cells. 0.5×10 per well 6 The separated cells were plated into a 24-well plate pre-coated with anti-human CD3 at a density of 10 cells / well. 2 μL of anti-human CD8 antibody was added to each well, followed by CD4 + The final volume of each well was 1 mL with complete T cell culture medium. Th1 / Th17 directional differentiation was induced according to the conditions in Table 5. After 5 days of culture, intracellular flow cytometry staining was performed.

[0086] Table 5 Th1 / Th17 cell differentiation induction conditions

[0087]

[0088]

[0089] (2) Intervention: 2 μM polydatin was added to the polydatin group, and an equal amount of DMSO was added to the control group (Medium).

[0090] 2. Experimental results

[0091] Peripheral blood CD4 + After T cells were cultured, the results were as follows Figure 8 Polydatin group IFN-γ + IL-17A + TNF-α + CD4 + The proportion of T cells decreased significantly, while IL-10 + CD4 +The proportion of T cells increased significantly, indicating that polydatin can prevent and treat UC by inhibiting the proliferation and differentiation of Th1 / Th17 cells and their pro-inflammatory response, indicating that polydatin has broad application potential as an active ingredient in the preparation of dietary supplements to prevent UC or in the preparation of drugs to treat UC.

[0092] The present application provides the use of polydatin in the preparation of a drug for treating ulcerative colitis. The present application confirms that polydatin can improve pathological damage to colon tissue, inhibit abnormally activated Th1 / Th17 immune responses in the intestine, significantly reduce the expression of proinflammatory factors, and reduce oxidative stress, thereby effectively alleviating the symptoms of ulcerative colitis and solving the problem of how to safely and effectively treat ulcerative colitis. The present application also provides a drug for treating ulcerative colitis, the drug comprising polydatin and a pharmaceutically acceptable carrier, and polydatin is prepared into a drug for treating ulcerative colitis, which has broad clinical application prospects.

[0093] The specific implementations are only a few examples under the general concept of this application and do not constitute a limitation on the protection scope of this application. For those skilled in the art, any other implementations expanded from the scheme of this application without creative work belong to the protection scope of this application.

Claims

1. Application of polydatin in the preparation of drugs for the treatment of ulcerative colitis.

2. The use according to claim 1, characterized in that: The polydatin treats ulcerative colitis by inhibiting Th1 / Th17 cell immune response.

3. The use according to claim 1, characterized in that: The polydatin treats ulcerative colitis by reducing the levels of pro-inflammatory cytokines.

4. The use according to claim 3, characterized in that: The pro-inflammatory factors include IFN-γ, IL-1β, IL-17 and TNF-α.

5. The use according to claim 1, characterized in that: The polydatin treats ulcerative colitis by reducing the infiltration of Th1 / Th17 cells in intestinal mucosal tissue.

6. The use according to claim 1, characterized in that: The polydatin treats ulcerative colitis by activating Nfe2l2 and downstream signaling pathways to reduce oxidative stress.

7. The use according to claim 1, characterized in that: The dosage forms of the drug include oral preparations and enema.

8. The use according to claim 7, characterized in that: The oral preparations include tablets, granules, capsules and oral liquids.

9. The use according to claim 7, characterized in that: The enema includes enema solution and suppository.

10. A drug for treating ulcerative colitis, characterized in that: The medicine comprises polydatin and a pharmaceutically acceptable carrier.

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