Use of vitamin d3 in the preparation of a medicament for treating or ameliorating ulcerative colitis

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to application of vitamin D3 in preparation of medicine for treating or relieving ulcerative colitis. The application provides a new application of vitamin D3 as a single effective component in preparation of medicine for treating or relieving ulcerative colitis, and pharmacodynamic effects thereof are verified through a DSS-induced UC mouse model system. Experiments show that 0.005 mu g / g of vitamin D3 can significantly reduce a disease activity index (DAI) of a model mouse and improve colonic tissue pathological damage. Vitamin D3 has high safety, small toxic and side effects, strong accessibility, good patient compliance and other outstanding advantages as a natural product, and provides a safe and effective new selection for clinical treatment of ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the application of vitamin D3 in the preparation of drugs for treating or alleviating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific, relapsing inflammatory disease of the colon and rectum with an incompletely understood etiology. It primarily affects the colonic mucosa and submucosa, clinically manifesting as abdominal pain, diarrhea, and bloody, mucous stools. UC often recurs, has a protracted course, severely impacting patients' quality of life, and carries a progressive and cancerous risk. Globally, the incidence and prevalence of UC show significant regional differences and are continuously rising: the prevalence in North America and Europe remains stable at 0.5%–1.0%, while newly industrialized countries in Asia, Latin America, and Africa are experiencing a 5-10 fold increase in UC incidence. It is estimated that by 2025, the number of people diagnosed with UC in seven major global pharmaceutical markets will approach 3.3 million. The inflammation of UC mainly affects the colonic and rectal epithelium, and its clinical course alternates between active and remission phases. Patients suffer a long-term physical and psychological burden from the disease, highlighting the urgent need for safe and effective treatment methods.

[0003] Currently, clinical treatments for ulcerative colitis (UC) mainly include 5-aminosalicylic acid derivatives (5-ASA), glucocorticoids, immunosuppressants, biologics (such as anti-TNF-α monoclonal antibodies), and small molecule drugs. However, all of these drugs have limitations to varying degrees: long-term use of glucocorticoids and immunosuppressants can lead to adverse reactions such as increased risk of infection, decreased bone density, and metabolic disorders; while biologics provide an important option for refractory UC, they suffer from high treatment costs, primary or secondary drug resistance in some patients, and immune-related adverse events; furthermore, current treatment regimens are still unable to achieve long-term stable remission of UC, and poor patient compliance is a prominent clinical problem. More importantly, most of the existing drugs focus on simple anti-inflammatory or immunosuppressive therapy, failing to effectively target the core pathological link of intestinal mucosal barrier damage in UC, making it difficult to fundamentally repair the damaged intestinal barrier function, resulting in a persistently high relapse rate after drug withdrawal.

[0004] Therefore, there is an urgent need to develop a novel UC treatment drug that can simultaneously ensure safety, efficacy, and mucosal barrier repair function. Summary of the Invention

[0005] Based on this, the present invention provides the use of vitamin D3 in the preparation of drugs for treating or relieving ulcerative colitis.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides the use of vitamin D3 in the preparation of drugs for treating or alleviating ulcerative colitis.

[0007] Preferably, in the above applications, the ulcerative colitis is ulcerative colitis induced by sodium dextran sulfate.

[0008] Preferably, in the above applications, the drug is a drug that increases the expression level of the colonic epithelial tight junction protein ZO-1.

[0009] Preferably, in the above applications, the drug is a drug that reduces the level of inflammatory factors in colon tissue.

[0010] More preferably, in the above applications, the inflammatory factors are IL-6, IL-1β and / or TNF-α.

[0011] Preferably, in the above applications, the effective dose of vitamin D3 is 0.005 μg / g.

[0012] Preferably, in the above applications, the dosage form of the drug is an oral preparation, a targeted preparation, a sustained-release preparation, a controlled-release preparation, an injection, or a preparation administered via other routes of administration.

[0013] More preferably, in the above applications, the oral preparation is a capsule, granule, powder, or pill.

[0014] The beneficial effects of this invention include: This invention provides a novel application of vitamin D3 as a single active ingredient in the preparation of drugs for treating or alleviating ulcerative colitis. Its pharmacodynamic effects were verified using a DSS-induced UC mouse model system. Experiments showed that 0.005 μg / g of vitamin D3 significantly reduced the disease activity index (DAI) in model mice and improved colonic tissue pathological damage. Its core mechanism of action lies in specifically upregulating the expression of the intestinal mucosal tight junction protein ZO-1 to repair the intestinal mechanical barrier, while effectively inhibiting the release of local colonic pro-inflammatory factors IL-6, IL-1β, and TNF-α. It synergistically exerts its therapeutic effect on UC through two key pathways: barrier repair and inflammation regulation. Furthermore, vitamin D3, as a natural product, has significant advantages such as high safety, low toxicity, high accessibility, and good patient compliance, providing a safe and effective new option for the clinical treatment of ulcerative colitis. Attached Figure Description

[0015] Figure 1 The graph shows the Disease Activity Index (DAI) scores of mice in each group. Figure 2 Images of intestinal tissue stained with hematoxylin and eosin (HE) in mice from each group; Figure 3 Immunohistochemical (IHC) staining images of tight junction protein (ZO-1) in the intestinal tissue of mice in each group; Figure 4 The relative expression levels of tight junction protein (ZO-1) in the intestinal tissue of mice in each group were determined by immunohistochemistry. Figure 5 A schematic diagram showing the enzyme-linked immunosorbent assay (ELISA) results of inflammatory factors interleukin (IL)-6, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the colon tissue of mice in each group; Figures 1 to 4 In the table, A represents the normal group, B represents the model group, C represents the vitamin D3 negative control group, and D represents the vitamin D3 treatment group. Detailed Implementation

[0016] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0017] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0018] Current mainstream clinical treatments for ulcerative colitis (UC) include 5-aminosalicylic acid, glucocorticoids, immunosuppressants, and anti-TNF-α biologics, but all have significant drawbacks: long-term use of hormones and immunosuppressants can easily lead to osteoporosis, metabolic disorders, and increased susceptibility to infection; monoclonal antibody drugs are expensive and prone to primary / secondary drug resistance; existing regimens only suppress inflammation and cannot repair the damaged colonic epithelial barrier, resulting in recurrent disease and a persistently high risk of cancer.

[0019] Based on this, the present invention can adopt the following technical solution: This invention provides the use of vitamin D3 in the preparation of drugs for treating or alleviating ulcerative colitis.

[0020] It should be noted that vitamin D3 is also known as cholecalciferol (structural formula as follows: Vitamin D3 (1,25-dihydroxyvitamin D3) is a fat-soluble vitamin derived from cholesterol. It is metabolized in the body into its active form, 1,25-dihydroxyvitamin D3, where it exerts its biological functions. Traditionally, vitamin D3 is thought to primarily regulate calcium and phosphorus metabolism and maintain bone health. However, recent studies have increasingly demonstrated its broad immunomodulatory and anti-inflammatory activities. Current technologies primarily utilize vitamin D3 as a dietary supplement or as an adjunct in combination therapy (such as mesalazine), focusing mainly on structural modifications of vitamin D compounds or specific formulations. They also tend to use vitamin D indiscriminately as a broad-spectrum anti-inflammatory agent for intestinal inflammation, without delving into its application as a single effective ingredient to specifically repair the intestinal mucosal tight junction barrier and regulate ulcerative colitis (UC)-related inflammatory signaling pathways to alleviate UC symptoms. Furthermore, they do not provide precise treatment plans for the core pathological aspects of UC patients, such as recurrent exacerbations, mucosal barrier damage, and imbalances in inflammatory factors. In short, there are currently no reports of using vitamin D3 alone to prepare drugs specifically for the treatment or relief of ulcerative colitis.

[0021] In some specific examples, in the above applications, the ulcerative colitis is ulcerative colitis induced by sodium dextran sulfate.

[0022] It should be noted that sodium dextran sulfate (DSS)-induced UC is an industry-recognized standardized animal model of acute ulcerative colitis. After modeling, mice exhibit weight loss, bloody stools, colonic crypt destruction, and extensive inflammatory cell infiltration. The pathological phenotype and inflammatory pathways highly match those of human clinical UC, making it a standard model for evaluating intestinal mucosal repair and anti-inflammatory efficacy. In this embodiment, a model was constructed using 3% DSS in the mice with free access to water for 7 days. Four parallel control groups were set up: a normal control group, a model group, a solvent-negative control group, and a VD3-treated group. The DAI score and colonic HE pathological sections directly confirmed that a dose of 0.005 μg / g of VD3 significantly reduced disease activity and alleviated mucosal ulceration and inflammatory infiltration in the model mice. This provides standardized animal experimental support for the efficacy of VD3 in alleviating human ulcerative colitis, and the efficacy conclusions can be extrapolated to clinical UC patient intervention scenarios.

[0023] In some specific examples, in the above applications, the drug is a drug that increases the expression level of the colonic epithelial tight junction protein ZO-1.

[0024] It is important to note that ZO-1 is a key tight junction protein in colonic epithelial cells, maintaining the integrity of the intestinal barrier. The core initiation step in UC pathogenesis is the inflammation-mediated degradation of large amounts of ZO-1, leading to widening of the interepithelial spaces. This allows intestinal antigens and flora to invade the lamina propria, continuously amplifying inflammation and becoming the fundamental cause of recurrent UC. Existing anti-inflammatory drugs only suppress immune inflammation and cannot repair the damaged barrier. This invention, through immunohistochemical (IHC) staining of ZO-1 in colonic tissue, confirms that in the DSS model group, ZO-1 staining was weak and discontinuous. After administration of 0.005 μg / g VD3, continuous brown-yellow positive staining of the epithelial cell membrane and a significant increase in expression levels were observed. VD3 activates the VDR receptor in the intestinal epithelium, upregulating ZO-1 gene expression at the transcriptional level, repairing the intestinal mechanical barrier, blocking the source of leakage, and fundamentally reducing the probability of UC recurrence. This achieves both symptomatic and root-cause treatment, unlike traditional drugs that only treat inflammation.

[0025] In some specific examples, in the above applications, the drug is a drug that reduces the level of inflammatory factors in colonic tissue.

[0026] It should be noted that excessive activation of macrophages and dendritic cells within ulcerative colitis lesions leads to the release of large amounts of pro-inflammatory factors, continuously exacerbating mucosal erosion and bleeding. Existing drugs only block inflammation along a single pathway, resulting in limited inhibitory effects. This invention uses ELISA to quantitatively detect inflammatory factors in colonic homogenate. Experimental results show that VD3 can broadly inhibit multiple inflammatory signaling pathways (NLRP6 inflammasome, TRPV1-MAPK), comprehensively downregulate various pro-inflammatory mediators in colonic tissue, reduce local congestion, ulceration, and other acute-phase pathological damage, rapidly improve typical UC clinical symptoms such as diarrhea and mucus-containing bloody stools, and synergistically exert therapeutic effects with the barrier repair function of ZO-1.

[0027] In some specific examples, the inflammatory factors described above are IL-6, IL-1β, and / or TNF-α.

[0028] It should be noted that IL-1β, IL-6, and TNF-α are the three major pro-inflammatory factors that are key markers for assessing the severity of inflammation in ulcerative colitis (UC). TNF-α initiates epithelial apoptosis, IL-1β amplifies local infiltration, and IL-6 maintains chronic persistent inflammation. All three are significantly highly expressed in the colonic tissue of UC patients in the DSS model and clinically active phase, and are also the main targets of existing monoclonal antibody drugs. The ELISA quantitative data in this example show that compared with the DSS model group, the concentrations of IL-6, IL-1β, and TNF-α in the colonic homogenate of the VD3 treatment group were significantly reduced (P < 0.05). The three core pro-inflammatory factors were simultaneously inhibited, reducing acute and chronic inflammatory damage in the colon through multiple targets. The efficacy indicators are clearly quantified, and the experimental data provide sufficient support.

[0029] In some specific examples, the effective dose of vitamin D3 in the above applications is 0.005 μg / g.

[0030] It should be noted that the above dosage is the standardized weight-based dosage for intraperitoneal administration to mice, and the optimal safe effective concentration was determined through multi-gradient pharmacodynamic pre-experiment screening. Below 0.005 μg / g, the activation level of VDR in vivo is insufficient, and the upregulation of ZO-1 and the anti-inflammatory effect are significantly weakened; above this dosage, there is no significant gain in anti-inflammation and barrier repair, but there is a potential risk of increased blood calcium and increased renal metabolic burden. At a dosage of 0.005 μg / g, significant intestinal barrier repair and inflammation suppression can be achieved simultaneously without significant toxic side effects. This is the optimal range for balancing efficacy and safety and can be used as a standard dosage for human formulation conversion and animal efficacy evaluation. The physicochemical standards of VD3 used in this invention are also specified: white powder, melting point 83℃~86℃, chemical structure of standard cholecalciferol I, stable raw material purity, and good reproducibility of efficacy.

[0031] In some specific examples, in the above applications, the dosage form of the drug is an oral preparation, capsule, granule, powder, pill, targeted preparation, sustained-release preparation, controlled-release preparation, injection, or preparation administered via other routes.

[0032] It should be noted that vitamin D3 is a lipid-soluble active ingredient with good compatibility with various pharmaceutical excipients, allowing for the development of corresponding dosage forms based on the different stages and locations of ulcerative colitis. Specifically, oral formulations (capsules, granules, powders, pills) have simple manufacturing processes, high patient compliance, and are suitable for maintaining remission in mild UC; targeted formulations can avoid gastric acid and small intestinal breakdown, releasing high concentrations of vitamin D3 at specific points in the colon, resulting in stronger local barrier repair and anti-inflammatory effects, and lower systemic calcium side effects, making them suitable for moderate to severe active UC; sustained-release / controlled-release formulations provide uniform and long-lasting drug release, maintaining a stable effective concentration in the body, reducing the frequency of administration, and are suitable for long-term maintenance therapy; injectable formulations (intraperitoneal / subcutaneous / intravenous) are suitable for severe UC, patients with oral malabsorption, and patients with intestinal intolerance, rapidly achieving effective blood drug concentrations; multiple dosage forms cover all clinical scenarios in the acute phase, remission phase, and mild / severe cases, with low difficulty in developing industrialized formulations and adaptability to different dosing needs.

[0033] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0034] Example 1: Effects of Vitamin D3 on Disease Activity and Colonic Histopathological Morphology in DSS-Induced Ulcerative Colitis Model Mice (1) Laboratory animals and their housing conditions Thirty-two healthy male C57BL / 6 mice, 6 weeks old and weighing 18g–22g, were selected. Four mice were housed per cage in standard polycarbonate cages with corncob bedding, under standard environmental conditions: 12h:12h light / dark cycle, temperature 23℃, and relative humidity 50%. Standard rodent food and water were provided freely. All mice were acclimatized to the new animal facility for at least 7 days before the experiment began.

[0035] (2) Construction of ulcerative colitis model and grouping of drugs for administration 1) Thirty-two mice were randomly divided into four groups of eight mice each: Group A: Normal group (normal control); Group B: DSS-induced ulcerative colitis model group (model group); Group C: Vitamin D3 negative control group (solvent control group); Group D: Vitamin D3 treatment group (0.005 μg / g).

[0036] Except for the normal control group, all other groups were given free access to 3% dextran sulfate sodium (DSS) solution (prepared with sterile drinking water as the solvent) for 7 consecutive days to establish an acute ulcerative colitis model. Mice in the normal control group had free access to sterile drinking water throughout the modeling period. After 7 days, the DSS-induced ulcerative colitis model was successfully established.

[0037] 2) One week before the start of modeling in the model group (i.e., 7 days after the end of acclimatization and before DSS administration), the solvent control group and the vitamin D3 treatment group began receiving medication. Specific administration details for each group are as follows: The solvent control group received an equal volume of drug carrier (sterile corn oil) via intraperitoneal injection. The vitamin D3 treatment group received vitamin D3 at a concentration of 0.005 μg / g (prepared with sterile corn oil as a solvent) via intraperitoneal injection on days 1, 3, 5, and 7 before modeling. The normal group and the model group were given an equal volume of drug carrier (sterile corn oil) via intraperitoneal injection as a control.

[0038] All groups were administered the medication for two weeks (including a pre-administration period of one week before modeling and seven days during the modeling period).

[0039] During the experiment, the weight changes, fecal characteristics and bloody stools of mice in each group were observed and recorded daily, and scores were calculated according to the Disease Activity Index (DAI) scoring criteria.

[0040] (3) Disease Activity Index (DAI) score The DAI score is based on the following criteria: (1) Weight loss rate: 0 points, no weight loss; 1 point, weight loss of 1% to 5%; 2 points, weight loss of 5% to 10%; 3 points, weight loss of 10% to 15%; 4 points, weight loss of >15%. (2) Stool characteristics: 0 points, normal; 2 points, loose stool; 4 points, diarrhea. (3) Blood in stool: 0 points, normal; 2 points, positive for occult blood; 4 points, visible blood in stool. DAI score = (weight loss score + stool characteristics score + blood in stool score) / 3.

[0041] Data analysis was performed using GraphPad Prism 9.0 statistical software. DAI scores are expressed as mean ± standard deviation (Mean ± SD). Since DAI scores were obtained from repeated measurements at multiple time points after modeling in the same batch of mice, a two-way repeated measures ANOVA was used for the overall analysis. "Group" represented the between-group factor, and "time" represented the within-group factor, while also examining the group × time interaction effect. If the Mauchly test of sphericity showed a violation of the sphericity hypothesis (P < 0.05), the Greenhouse-Geisser method was used to adjust the degrees of freedom.

[0042] When the group-time interaction effect or main effect was statistically significant, further post-hoc multiple comparisons were performed: one-way ANOVA was used for comparisons between groups at each time point; Tukey's HSD method was used for pairwise comparisons between all groups, or Dunnett's method was used to compare the vitamin D3 treatment group, vitamin D3 negative control group, and model group respectively; Bonferroni correction was used for post-hoc comparisons of repeated measures data. If the data did not conform to normal distribution or homogeneity of variance, the Kruskal-Wallis H test was used, and Dunn's multiple comparison test was used post-hoc. All statistical tests were two-tailed tests, and P < 0.05 was considered statistically significant. The specific values ​​(mean ± standard deviation) of the DAI scores of each group from day 0 to day 7 are shown in Table 1 below.

[0043] Table 1. Specific numerical values ​​(mean ± standard deviation) of DAI scores for each group from day 0 to day 7.

[0044] The results are as follows Figure 1As shown, the DAI score of mice in the normal group (Group A) remained at a low level (close to 0) throughout the experiment. Compared with the normal group, the DAI score of mice in the model group (Group B) gradually increased from day 4 of DSS administration, reaching a peak on day 7, with a statistically significant difference (P < 0.01), indicating that the DSS-induced UC model was successfully established. The solvent control group (Group C) showed no significant difference in DAI score compared with the model group (P > 0.05), indicating that the solvent control had no effect on disease progression. Compared with the model group, the DAI score of mice in the vitamin D3 treatment group (Group D, 0.005 μg / g) was significantly reduced from day 5 of administration (P < 0.05), suggesting that 0.005 μg / g vitamin D3 can effectively alleviate the disease activity of DSS-induced UC mice.

[0045] (4) Colon histopathological examination (HE staining) After the last administration, mice in each group were fasted for 12 hours but allowed free access to water. They were then anesthetized and euthanized, and colonic tissue was collected from each group. Distal colonic tissue, approximately 0.5 cm to 1.0 cm in length, was gently rinsed with pre-cooled phosphate-buffered saline (PBS, pH 7.4) to remove intestinal contents and fixed in 4% paraformaldehyde solution for 24 h. The fixed colonic tissue was dehydrated with a gradient of ethanol, embedded in paraffin, and then prepared into 4 μm thick serial sections. After dewaxing with xylene and rehydration with a gradient of ethanol, the sections were stained with hematoxylin and eosin (HE). The staining procedure was as follows: hematoxylin staining for 5 min, rinsing with running water for 5 min; differentiation with 1% hydrochloric acid ethanol for 3 s, rinsing with running water for 5 min; eosin staining for 2 min, rinsing with running water for 5 min; dehydration with a gradient of ethanol, clearing with xylene, and mounting with neutral resin. Histopathological changes in the colonic tissue were observed under an optical microscope and scored according to a colonic histopathological grading system.

[0046] The grading criteria for colonic histopathology are as follows: (1) Inflammation score: 0 points, normal; 1 point, mild focal infiltration of the lamina propria; 2 points, mild infiltration of the lamina propria, multifocal, mild glandular separation; 3 points, infiltration with multifocal mild edema; 4 points, mixed infiltration of the submucosa and lamina propria, extensive glandular separation, enlarged lymphoid follicles, and edema.

[0047] (2) Epithelial score: 0 points, normal; 1 point, focal mucosal hyperplasia; 2 points, multifocal hyperplasia of villous epithelial cells and goblet cells; 3 points, local diffuse damage or reduction of epithelial cells; 4 points, local diffuse mucosal ulcer.

[0048] (3) Glandular score: 0 points, normal; 1 point, rare glandular dilatation; 2 points, multifocal glandular dilatation; 3 points, multifocal glandular dilatation with abscess, occasionally with glandular reduction; 4 points, localized widespread glandular reduction.

[0049] (4) Depth of lesion score: 0 points, none; 1 point, mucosal layer; 2 points, mucosal layer and submucosa; 3 points, transmural.

[0050] (5) Score of the degree of impact of the lesion: 0 points, none; 1 point, very mild (≤10%); 2 points, mild (10%~25%); 3 points, moderate (26%~50%); 4 points, severe (>50%).

[0051] The sum of the scores for the five dimensions mentioned above is the total score for colonic histopathology. The total score ranges from 0 to 19 points, with a higher score indicating more severe damage to the colonic tissue.

[0052] HE staining results are as follows Figure 2 As shown. Specifically: In the normal group (Group A), the colonic mucosal epithelium of mice was intact, the glands were neatly arranged, the crypt structure was clear, goblet cells were abundant, and no or only a very small number of inflammatory cells were seen in the lamina propria. There was no edema in the submucosa, and the histological morphology was normal. The histopathological grading of the colonic tissue was as follows: inflammation 0 points, epithelium 0 points, glands 0 points, depth 0 points, degree of influence 0 points, for a total score of 0 points.

[0053] In the model group (Group B), the colonic mucosal epithelium of mice showed significant damage, with widespread mucosal ulceration, severe destruction and large-area loss of crypt structures, extensive reduction of glands accompanied by multifocal glandular dilation and abscess formation. Numerous mixed inflammatory cells (neutrophils, lymphocytes, macrophages, etc.) infiltrated the lamina propria and submucosa, accompanied by widespread glandular separation and enlargement of lymphoid follicles. Submucosal edema was significant, and the lesions involved the entire thickness of the colon, exhibiting transmural inflammation. The histopathological grading of the colon was as follows: inflammation 3 points, epithelium 3 points, glands 4 points, depth 2 points, and degree of impact 4 points, for a total score of 16 points. Compared with the normal group, the total histopathological score of the colon in the model group was significantly higher (P<0.01), indicating that the DSS-induced UC model mice experienced severe pathological damage to the colonic tissue, and the model was successfully established.

[0054] The colonic tissue structure of mice in the solvent control group (Group C) was similar to that of the normal group, with normal histological morphology. The total score for colonic tissue pathological grading was 0. This indicates that vitamin D3 solvent alone had no significant effect on healthy mice. Compared with the model group, the integrity of the colonic mucosal epithelium of mice in the vitamin D3 treatment group (Group D, 0.005 μg / g) was significantly restored, the ulcer area was significantly reduced, the crypt structure became more regular, the glands were arranged in an orderly manner, the inflammatory cell infiltration was significantly reduced, only mild focal inflammatory cell infiltration was seen in the lamina propria, and the submucosal edema was basically subsided, with the lesions mainly limited to the mucosal layer. The colonic tissue pathological grading score was: inflammation 1 point, epithelium 0 points, glands 0 points, depth 1 point, and degree of impact 1 point, for a total score of 3 points. Compared with the model group, the total score of colonic tissue pathology in the vitamin D3 treatment group was significantly reduced (P<0.01), and the scores of each dimension were significantly reduced, suggesting that vitamin D3 can effectively improve the colonic tissue pathological damage in mice with DSS-induced ulcerative colitis.

[0055] The histopathological grading scores of the colon tissue of mice in each group are summarized in Table 2 below.

[0056] Table 2. Histopathological grading of colon tissue in mice of each group

[0057] The above results indicate that the total histopathological score of the colon tissue in the DSS-induced UC model mice was as high as 16 points, characterized by severe colonic damage with widespread glandular reduction (4 points), severe lesion impact (4 points), inflammatory infiltration with multifocal mild edema (3 points), and localized diffuse epithelial damage (3 points). The total score in the vitamin D3 treatment group (Group D) decreased to 3 points, showing only mild focal inflammatory infiltration in the lamina propria (1 point), lesion involving the mucosal layer (1 point), and very mild lesion impact (1 point), with both epithelium and glands returning to normal (0 points). This suggests that 0.005 μg / g vitamin D3 can significantly improve the histopathological morphological damage of the colon tissue in DSS-induced UC model mice, effectively reversing colonic inflammation and mucosal barrier disruption.

[0058] (5) Immunohistochemical (IHC) detection of tight junction protein ZO-1 in colonic tissue Zonula occludens-1 (ZO-1) is a member of the membrane-associated guanylate kinase (MAGUK) family with a molecular weight of approximately 220 kD. It is an important component of tight junction proteins, mainly distributed between intestinal epithelial cells, located on the apical side of the cell membrane, and plays a key role in maintaining the integrity and permeability of the intestinal mucosal mechanical barrier.

[0059] This experiment used immunohistochemistry to detect the expression level of ZO-1 in the colon tissue of mice in each group. Paraffin-embedded colon tissue was used to prepare 4 μm thick serial sections. After dewaxing with xylene and rehydration with graded ethanol, the sections were placed in 0.01 M citrate buffer (pH 6.0) and subjected to antigen retrieval using microwave heating (95℃, 15 min). After cooling, the sections were incubated with 3% H2O2 for 10 min to block endogenous peroxidase activity, and washed three times with PBS. 5% normal goat serum blocking solution was added, and the sections were blocked at room temperature for 30 min. The blocking solution was discarded, and rabbit anti-mouse ZO-1 primary antibody (dilution ratio 1:200) was added, and the sections were incubated overnight at 4℃. The next day, after washing three times with PBS, horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added, and the sections were incubated at 37℃ for 30 min. After washing with PBS, the sections were developed with DAB chromogenic solution, counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Images were observed and acquired under an optical microscope, and the percentage of positive ZO-1 area in the colonic mucosa of each group was determined using ImageJ image analysis software. The percentage of positive area in the normal group (Group A) was set as 1, and the remaining groups were normalized to calculate the relative expression level.

[0060] The results are as follows Figure 3 and Figure 4As shown, ZO-1 exhibits strong positive expression in normal colonic tissue, appearing as a continuous brownish-yellow positive staining along the intestinal epithelial cell membrane, with high staining intensity and a wide positive area. The percentage of positive area in the normal group (Group A) was 0.292%. Compared with the normal group (Group A), the positive expression intensity of ZO-1 in the colonic mucosal epithelial cells of the model group (Group B) mice was significantly weakened, with the brownish-yellow staining changing from continuous distribution to discontinuous or even absent states, and the percentage of positive area was 0.156%. Normalized with Group A as 1, the relative expression level in Group B was 0.53 (i.e., 53.4%), significantly lower than that in the normal group (P<0.01), indicating that the DSS-induced ulcerative colitis model mice have severe damage to the tight junction structure of the intestinal mucosa. The percentage of positive area in the solvent control group (Group C) was 0.289%, and the normalized relative expression level was 0.99 (i.e., 99.0%), which was not significantly different from the normal group (P>0.05), indicating that the solvent control had no effect on ZO-1 expression. Compared with the model group, the vitamin D3 treatment group (Group D, 0.005 μg / g) showed a significant increase in the positive expression intensity of ZO-1 in the colonic mucosal epithelial cells of mice, and the positive brown-yellow staining was restored to a relatively continuous cell membrane distribution, with a positive area percentage of 0.243% and a normalized relative expression level of 0.83 (i.e., 83.2%). Compared with the model group, the percentage of ZO-1 positive area in the vitamin D3 treatment group was significantly increased (P<0.05). These results indicate that vitamin D3 can effectively repair the mechanical barrier damage of the intestinal mucosa in ulcerative colitis model mice and restore the normal expression and distribution of the tight junction protein ZO-1.

[0061] Example 2: Effects of Vitamin D3 on the Levels of Inflammatory Factors in Colonic Tissue of DSS-Induced Ulcerative Colitis Model Mice (1) Experimental animals, model construction and grouping of drugs The experimental animals, model construction methods, grouping and dosing regimens are the same as in Example 1.

[0062] (2) Collection and processing of colon tissue samples After the last administration, mice in each group were fasted for 12 hours but allowed free access to water. They were then anesthetized and euthanized. Colon tissue was collected from each group, and an appropriate amount (approximately 20 mg–50 mg) was weighed and placed in pre-chilled phosphate-buffered saline (PBS, pH 7.4). The tissue was thoroughly washed to remove residual blood. The tissue was then minced and placed in homogenization tubes. Pre-chilled PBS (containing protease inhibitors) was added at a weight-to-volume ratio of 1:9. Homogenization was performed using a tissue homogenizer under ice bath conditions until the tissue was completely ruptured. The homogenate was centrifuged at 4°C and 3000 rpm for 15 min. The supernatant was collected, aliquoted, and stored at -80°C for later use.

[0063] (3) ELISA detection of inflammatory factors IL-6, IL-1β and TNF-α in mouse colon tissue The concentrations of inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the colon tissue of mice in each group were detected using enzyme-linked immunosorbent assay (ELISA). Specifically, the procedure was performed according to the instructions of the corresponding ELISA kit (BioQuanti-48T, detection range: 10 pg / mL-1600 pg / mL): 100 μL of colon tissue homogenate sample and standards were added to each well of a pre-coated antibody-impregnated ELISA plate and incubated at 37°C for 90 min; after discarding the liquid, biotinylated antibody working solution was added and incubated at 37°C for 60 min; after washing 5 times, enzyme conjugate working solution was added and incubated at 37°C in the dark for 30 min; after washing again, TMB chromogenic substrate was added and incubated at 37°C in the dark for 15 min; finally, stop solution was added, and the absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. The concentrations of IL-6, IL-1β, and TNF-α in each sample were calculated based on the standard curve, and the results are expressed in pg / mL. The specific concentrations (pg / mL, mean ± standard deviation) of IL-6, IL-1β, and TNF-α in the colon tissue of mice in each group are shown in Table 3 below.

[0064] Table 3. Specific concentrations of IL-6, IL-1β, and TNF-α in the colon tissue of mice in each group.

[0065] The bar chart results are as follows Figure 5 As shown, compared with the normal group (group A), the levels of IL-6, IL-1β, and TNF-α in the colon tissue of the model group (group B) mice were significantly increased, with statistically significant differences (P < 0.01), indicating a significant inflammatory response in the DSS-induced ulcerative colitis model mice. Compared with the normal group, the solvent control group (group C) showed no significant difference in the expression levels of the above three inflammatory factors (P > 0.05), indicating that the solvent control had no effect on inflammatory factors. Compared with the model group, the levels of IL-6, IL-1β, and TNF-α in the colon tissue of mice treated with vitamin D3 (Group D, 0.005 μg / g) were significantly reduced, with IL-6 decreasing by approximately 58.7%, IL-1β by approximately 61.0%, and TNF-α by approximately 67.1%, and the differences were statistically significant (P < 0.05). This suggests that 0.005 μg / g vitamin D3 can effectively inhibit the expression of inflammatory factors in the colon tissue of ulcerative colitis model mice, reduce local inflammatory response, and thus exert a relieving effect on ulcerative colitis.

[0066] The results of the above examples indicate that vitamin D3 (0.005 μg / g) can significantly improve the disease activity index (DAI) in DSS-induced ulcerative colitis model mice. Figure 1 ), improve colonic tissue pathological morphological damage ( Figure 2 ), upregulate the expression of the intestinal mucosal tight junction protein ZO-1 to repair intestinal barrier function ( Figure 3 and Figure 4 It effectively inhibits the expression levels of key inflammatory factors IL-6, IL-1β, and TNF-α in colon tissue. Figure 5 Vitamin D3 exerts its therapeutic or alleviating effects on ulcerative colitis through multiple pathways, including maintaining the integrity of the intestinal epithelial barrier and regulating the body's inflammatory response. The vitamin D3 provided by this invention has high safety and few toxic side effects, showing promising application prospects in the treatment or alleviation of ulcerative colitis, and can be used to prepare drugs for treating or alleviating ulcerative colitis.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of vitamin D3 in the preparation of drugs for treating or alleviating ulcerative colitis.

2. The application according to claim 1, characterized in that, The ulcerative colitis mentioned is ulcerative colitis induced by sodium dextran sulfate.

3. The application according to claim 1 or 2, characterized in that, The drug is a drug that increases the expression level of the colonic epithelial tight junction protein ZO-1.

4. The application according to claim 1 or 2, characterized in that, The drug is one that reduces the level of inflammatory factors in colon tissue.

5. The application according to claim 4, characterized in that, The inflammatory factors are IL-6, IL-1β and / or TNF-α.

6. The application according to claim 1, characterized in that, The effective dose of vitamin D3 is 0.005 μg / g.

7. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is an oral preparation, a targeted preparation, a sustained-release preparation, a controlled-release preparation, or an injection.

8. The application according to claim 7, wherein the oral preparation is a capsule, granule, powder, or pill.

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