Use of polyvinyl alcohol in the preparation of drugs related to the treatment of ulcerative colitis

Abstract

The present invention discloses the application of polyvinyl alcohol in the preparation of drugs related to the treatment of ulcerative colitis, belonging to the field of pharmaceutical technology. In the present invention, a DSS-induced ulcerative colitis model was established to observe the therapeutic effects of PVA aqueous solutions at different concentrations on the model mice. The degree of damage, inflammation and remission of the mouse colon tissue were evaluated by recording the changes in mouse body weight, clinical disease activity index, colon length, histopathology and inflammatory factor levels; and further, mouse colon organoids were cultured to observe the effects of PVA aqueous solutions at different concentrations on the number of organoids in vitro. The present invention finds that the PVA aqueous solution can relieve ulcerative colitis, improve tissue damage and symptoms, and its mechanism may be related to the promotion of intestinal stem cell expansion by the PVA aqueous solution, and then the promotion of intestinal mucosal damage repair, providing new ideas and methods for the clinical treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of polyvinyl alcohol in the preparation of drugs related to the treatment of ulcerative colitis. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Ulcerative colitis (UC) is a clinically refractory disease mainly characterized by persistent damage to the colonic epithelial mucosa. The main clinical manifestations are abdominal pain, diarrhea, mucopurulent bloody stools, etc. UC can occur in all age groups, is difficult to cure, and the incidence rate shows an increasing trend year by year. It has been listed as one of the modern refractory diseases by the World Health Organization (WHO). UC is difficult to cure, often prone to recurrence, and has a tendency to canceration. Currently, there is no specific therapy. Drugs for the treatment of ulcerative colitis include broad-spectrum anti-inflammatory drugs such as sulfasalazine and adrenal cortical hormones, as well as immunosuppressants and immunomodulators. Currently, there is no specific drug for the treatment of ulcerative colitis. Therefore, exploring new therapeutic drugs has important clinical value for blocking the progression of ulcerative colitis. Summary of the Invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide the application of polyvinyl alcohol in the preparation of drugs related to the treatment of ulcerative colitis. The present invention has found through experiments that an aqueous solution of polyvinyl alcohol (PVA) can relieve ulcerative colitis, improve tissue damage and symptoms, providing new ideas and methods for the clinical treatment of ulcerative colitis.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] On the one hand, an application of polyvinyl alcohol in the preparation of drugs related to the treatment of ulcerative colitis.

[0007] Preferably, the polyvinyl alcohol is an aqueous solution of polyvinyl alcohol, and the concentration is 1 mg / ml - 3 mg / ml.

[0008] Preferably, the administration method of the drug is enema or injection.

[0009] Preferably, the dosage form of the drug is one of injection, capsule, tablet, powder, granule, pill, microcapsule microsphere preparation, suppository, ointment, spray or targeted preparation.

[0010] Preferably, the application of polyvinyl alcohol in the preparation of drugs for promoting the amplification of intestinal stem cells.

[0011] Preferably, the application of polyvinyl alcohol in the preparation of a drug for promoting the expansion of the number of colon organoids.

[0012] Preferably, the application of polyvinyl alcohol in the preparation of a drug for inhibiting the release of inflammatory factors.

[0013] More preferably, the inflammatory factors include CD45, CD68, and MPO.

[0014] Preferably, the application of polyvinyl alcohol in the preparation of a drug for restoring intestinal mucosal injury.

[0015] On the other hand, a medicament, the medicament includes polyvinyl alcohol, and polyvinyl alcohol is used as the sole active ingredient.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The present invention discovers that the PVA aqueous solution can relieve ulcerative colitis, improve tissue damage and symptoms. Its mechanism may be related to the promotion of intestinal stem cell expansion by the PVA aqueous solution, and then the promotion of intestinal mucosal injury repair, providing new ideas and methods for the clinical treatment of ulcerative colitis.

[0018] The present invention establishes a DSS-induced ulcerative colitis model, observes the therapeutic effects of PVA aqueous solutions at different concentrations on model mice, and evaluates the degree of colon tissue damage, inflammation, and remission in mice by recording changes in mouse body weight, clinical disease activity index, colon length, histopathology, and inflammatory factor levels; and further uses mouse colon organoid culture to observe the effects of PVA aqueous solutions at different concentrations on the number of organoids in vitro. The results show that DSS can cause weight loss, colon tissue damage, and aggravated colon inflammation in mice. Treatment with PVA aqueous solutions (1 mg / ml and 3 mg / ml) can significantly relieve weight loss in model mice (p < 0.05), and significantly improve tissue damage (p < 0.05), and reduce the levels of inflammatory factors in intestinal mucosal tissues. In vitro organoid culture also confirms that the PVA aqueous solution can significantly promote the expansion of the number of mouse colon organoids (p < 0.05). Description of the Drawings

[0019] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0020] Figure 1 It is the curve of the change in the body weight of the mice in Example 1 of the present invention;

[0021] Figure 2 It is the histological score of the mice in Example 1 of the present invention;

[0022] Figure 3 Score of CD45 in mice in Example 1 of the present invention;

[0023] Figure 4 Score of ki67 in mice in Example 1 of the present invention;

[0024] Figure 5 Score of CD68 in mice in Example 1 of the present invention;

[0025] Figure 6 Score of MPO in mice in Example 1 of the present invention;

[0026] Figure 7 Pictures of mouse colon organoids cultured on day 0 and day 1 in Example 1 of the present invention;

[0027] Figure 8 Pictures of mouse colon organoids cultured on day 2 and day 3 in Example 1 of the present invention;

[0028] Figure 9 Pictures of mouse colon organoids cultured on day 4 and day 5 in Example 1 of the present invention;

[0029] Figure 10 Proliferation quantity graph of mouse colon organoids in Example 1 of the present invention. Detailed implementation manners

[0030] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0033] Example 1 Application of polyvinyl alcohol in the preparation of drugs related to the treatment of ulcerative colitis

[0034] 1. Establish a treatment model:

[0035] Fifty-four 6-8-week-old male C57BL / 6J mice were selected as experimental subjects and randomly divided into 9 groups. All mice were allowed to acclimatize to the environment for one week and were fed with Co660 mouse maintenance feed and pure water under the conditions of a room temperature of 25°C and good ventilation. After the experiment started, an animal model was established. The feeding environment remained unchanged. Six of the groups of mice were randomly selected, and the prepared 2.5% DSS solution was used to replace pure water to feed the mice to induce ulcerative colitis. Each mouse drank approximately 8 ml of water per day on average. The DSS solution was replenished in a timely manner, and the bedding was changed daily. The mice were fed for one week.

[0036] 2. Treatment with aqueous PVA solutions of different concentrations

[0037] The 3 groups of mice without an established animal model were used as the control group. The mice in the 3 control groups were respectively enema-treated with pure water + normal saline, 0.3 mg / ml aqueous PVA solution, and 3 mg / ml aqueous PVA solution once a day, 0.2 ml each time. For the 6 groups of mice with an established animal model, 0.2 ml of aqueous PVA solutions of different concentrations (0.1 mg / ml, 0.3 mg / ml, 1 mg / ml, 3 mg / ml, 10 mg / ml) were used to perform enema treatment on 5 animal model groups (i.e., 5 treatment groups) every day to observe the treatment effect, and 0.2 ml of normal saline was used to perform enema treatment on the remaining 1 animal model group (i.e., the non-treatment group) to observe the treatment effect. The enema was performed once a day.

[0038] Starting from the first day of establishing the animal model, each mouse was weighed and recorded. The body weight was retained to 2 decimal places. After enema, the mouse was placed in an empty cage. After it defecated, the feces were observed and the diarrhea and fecal occult blood conditions were recorded. The bleeding condition could be detected with a fecal occult blood test strip, and the clinical disease activity index was evaluated.

[0039] The rules for evaluating the clinical disease activity index are as follows:

[0040] Weight loss (no change = 0 points; <5% = 1 point; 6 - 10% = 2 points; 11 - 20% = 3 points; >20% = 4 points);

[0041] Stool (normal = 0 points; soft, good shape = 1 point; soft without pellets = 2 points; diarrhea = 4 points);

[0042] Bleeding (no blood = 0 points; blood visible in the rectum = 1 point; massive bleeding in the rectum = 2 points; blood visible subcutaneously = 4 points).

[0043] After 6 days of feeding and at the end of the experiment, the mice were dissected, and the colon (taken from the ileocecal valve to the anus, and the pubic symphysis could be incised) was removed, measured, and recorded. The colon lesions were cut and immersed in neutral formalin solution, and then embedded and sectioned for preparation. The degree of tissue damage was evaluated through pathological sections (HE). The evaluation content and rules are as follows:

[0044] a. Degree of tissue damage score:

[0045] 0 = None or few inflammatory cells in the lamina propria; 1 = Increased inflammatory cells in the lamina propria; 2 = Spread of inflammatory cells to the submucosa; 3 = Transmural extension of inflammatory cell infiltration.

[0046] b. Depth of injury score:

[0047] 0 = No mucosal injury; 1 = Lymphoepithelial lesion; 2 = Mucosal erosion or ulcer; 3 = Transmural injury.

[0048] c. Crypt injury score:

[0049] 0 = None; 1 = Less than 5% involved; 2 = 33% injury; 3 = 66% injury; 4 = Disappearance of the normal structure of the entire crypt and epithelium.

[0050] d. Percentage of area involved score:

[0051] 1 = 1 - 25%; 2 = 26 - 50%; 3 = 51 - 75%; 4 = 76 - 100%.

[0052] Subsequently, immunohistochemical detection (i.e., immunohistochemical staining) was performed for CD45, CD68, MPO, and Ki-67, and the evaluation method was as follows:

[0053] Scored in 4 grades according to the cell staining intensity. No positive staining (negative) was counted as 0 points, light yellow (weak positive) was counted as 1 point, brownish yellow (positive) was counted as 2 points, and brownish brown (strong positive) was counted as 3 points;

[0054] Scored in 4 grades according to the percentage of positive cells. ≤25% was counted as 1 point, 26% - 50% was counted as 2 points, 51% - 75% was counted as 3 points, and >75% was counted as 4 points. The final score was obtained by multiplying the two scores.

[0055] Calculate the number of positive cells: Count the number of positive cells in 5 randomly selected fields under a 20-fold microscope and calculate the average value.

[0056] The higher the scores of CD45, CD68, and MPO, the higher the degree of inflammation. Ki-67 can reflect the proliferative situation of the mucosal epithelium. Comprehensively judge the damage and remission conditions of the colonic mucosa.

[0057] Such as Figures 1-6As shown, DSS can cause weight loss in mice, damage to colon tissue, and exacerbation of colon inflammation. Treatment with PVA aqueous solutions (1 mg / ml and 3 mg / ml) can significantly alleviate weight loss in model mice (p<0.05), and significantly improve intestinal mucosal damage, with a significant reduction in tissue scores (p<0.05). The levels of inflammatory factors CD45, CD68, and MPO in the intestinal mucosal tissue are significantly reduced. However, Ki-67 is significantly increased, indicating promotion of intestinal mucosal epithelial proliferation. In summary, PVA aqueous solution significantly alleviates ulcerative colitis and inhibits inflammation.

[0058] 3. Proliferation of mouse colon organoids

[0059] 1) Take normal mice without any treatment, kill them, wipe the abdomen with an alcohol cotton ball, quickly open the abdominal cavity, pour in ice-cold sterile PBS, take the colon into PBS, and transfer it to a sterile operating table.

[0060] 2) Place the colon in a petri dish and immerse it in PBS (try to keep the intestinal tissue in ice-cold sterile PBS throughout the following steps); clean the adipose tissue and mesenteric tissue outside the intestine, and flush out the food residues in the intestine with a needle or pipette tip.

[0061] 3) Longitudinally cut open the colon and wash the colon with PBS until clean.

[0062] 4) Cut the colon into small segments of 2-4 mm, transfer them to a 50 ml centrifuge tube, add 30 ml of PBS, pipette and discard the supernatant, repeat three to five times until the supernatant is clear and transparent.

[0063] 5) Discard the upper layer of PBS, add 30 ml of cold 2 mM EDTA / PBS, seal with parafilm, and shake on a shaker at 4°C for 60 min at 100 rpm.

[0064] 6) Transfer the intestine to the operating table, let it stand, and remove the supernatant.

[0065] 7) Add 20 ml of PBS, pipette 50 times with a pipette, let it stand, transfer the supernatant to a new 50 ml centrifuge tube, make a mark, take 20 ul of the supernatant for microscopic examination, and repeat this step until most of the crypts are released.

[0066] 8) Select the tube with more and intact crypts, seal with parafilm, centrifuge at 4°C, 300 g for 5 min.

[0067] 9) Suspend the precipitate with 1 ml of ice-cold DMEM (1x double antibody), take 20 ul for microscopic examination, require 200-500 crypts per 50 ul of Matrigel, transfer a part according to the required number of crypts to a 15 ml centrifuge tube, seal, and centrifuge at low speed (4°C, 200 g, 3 min) to remove single cells.

[0068] 10) Discard the supernatant, resuspend the crypts with ice-cold DMEM suspension precipitate (1x double antibody), add an equal volume of Matrigel ( #356231) and mix well (Note: The new Matrigel needs to be thawed overnight at 4°C, and all pipette tips in contact with Matrigel need to be pre-cooled. Put the Matrigel in the 4°C refrigerator to melt in advance for each experiment, keep the tube vertically placed, and avoid repeated freezing and thawing).

[0069] 11) Add 50 μL / 40 μL / 10 μL of Matrigel to the pre-warmed 24 / 48 / 96-well plates (previously placed in a 37°C incubator). The Matrigel needs to be added to the middle wells to form hemispherical droplets. After inoculation, it should be quickly transferred to the incubator to solidify for 20 minutes (the solidification time should be appropriately extended in winter). Add 500 μL / 250 μL / 100 μL of mouse colon organoid expansion medium (biogeneousTM, #K2204-MC) to each well.

[0070] 12) Culture in the incubator. Discard the medium after one day, stimulate the crypts with 0 mg / mL, 1 mg / mL, and 3 mg / mL PVA (Sigma-Aldrich, #363146) solutions respectively, change the medium every 3 days, and dissolve the Matrigel with cell repair solution (Corning, #354253) on the 6th day to collect the organoids.

[0071] As Figures 7-10 shown, during the culture of mouse colon organoids, 1 mg / ml and 3 mg / ml PVA aqueous solutions significantly promoted the proliferation of mouse colon organoids, manifested as a significant increase in the number of organoids starting from the fourth and fifth days, while the change in the size of the organoids was not obvious. The results indicate that the PVA aqueous solution promotes the expansion of organoids. In summary, in vitro organoid culture confirms that the PVA aqueous solution can significantly promote the expansion of the number of mouse colon organoids (p < 0.05).

[0072] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of polyvinyl alcohol in the preparation of drugs for treating ulcerative colitis.

2. The use according to claim 1, wherein, the polyvinyl alcohol is an aqueous solution of polyvinyl alcohol with a concentration of 1 mg / ml - 3 mg / ml.

3. The use according to claim 1, wherein, the administration method of the drug is enema or injection.

4. The use according to claim 1, wherein, the dosage form of the drug is one of injection, capsule, tablet, powder, granule, pill, microcapsule microsphere preparation, suppository, ointment, spray or targeted preparation.

5. The use according to claim 1, wherein, the use of polyvinyl alcohol in the preparation of drugs for promoting the expansion of intestinal stem cells.

6. The use according to claim 5, wherein, the use of polyvinyl alcohol in the preparation of drugs for promoting the expansion of the number of colonic organoids.

7. The use according to claim 1, wherein, the use of polyvinyl alcohol in the preparation of drugs for inhibiting the release of inflammatory factors.

8. The use according to claim 7, wherein, the inflammatory factors include CD45, CD68, MPO.

9. The use according to claim 1, wherein, the use of polyvinyl alcohol in the preparation of drugs for restoring intestinal mucosal injury.

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