Application of combination of mesenchymal stem cells and mesalazine in preparation of anti-IBD drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of mesenchymal stem cells combined with mesalazine in preparation of an anti-IBD (infectious bursal disease) medicine. The mesenchymal stem cells are mesenchymal stem cells from human umbilical cord; the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD65, and low express CD34, CD45 and HLA-DR (Human Leukocyte Antigen-DR). The research finds that the hUC-MSCs has a remarkable treatment effect on a chronic IBD mouse model when being singly used or combined with 5-ASA for treatment, the hUC-MSCs and drug combination can restore the intestinal microecological balance by adjusting the composition of intestinal flora so as to improve the symptoms of IBD, and the hUC-MSCs and the drug combination have a synergistic effect when being combined with 5-ASA for treatment. The invention provides a new thought and experimental basis for clinical treatment of IBD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of mesenchymal stem cells combined with mesalazine in the preparation of anti-IBD drugs. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract mediated by nonspecific immunity. It is characterized by chronic inflammation and ulceration of the intestines, and primarily includes two types: Crohn's disease (CD) and ulcerative colitis (UC). Currently, the etiology of IBD is not fully understood. Some studies suggest that individual genetic factors, environmental factors, immune factors, gut microbiota factors, and other factors all contribute to the development of IBD. In recent years, the incidence of IBD has been rising globally, becoming a significant public health issue affecting human health.

[0003] Traditional treatments for IBD primarily include budesonide, azathioprine, and mesalazine (5-ASA). Budesonide provides rapid relief for mild IBD cases, offering short-term relief, but it cannot provide long-term relief. Furthermore, some patients develop dependence on budesonide, and long-term use (over 12 weeks) can cause side effects such as osteoporosis and cataracts. Mesalazine is the first-line treatment for mild to moderate ulcerative colitis (UC), effectively inhibiting the synthesis of prostaglandins and the formation of leukotrienes, thus significantly suppressing inflammation of the intestinal mucosa; however, its effectiveness is limited in severe cases. Azathioprine treats IBD by inhibiting lymphocyte activation and proliferation; it has some effect on both CD and UC, but azathioprine is highly toxic, easily causing side effects such as nausea, pancreatitis, and liver and kidney damage. Therefore, there is an urgent need to find safer and less toxic treatments for IBD.

[0004] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a type of pluripotent stem cells with self-renewal, multi-lineage differentiation, immunomodulation, and tissue repair capabilities, and have become a research hotspot in regenerative medicine and immunotherapy. Existing research indicates that hUC-MSCs play a significant role in stem cell therapy clinical trials, second only to bone marrow-derived stem cells (BMMSCs), accounting for 20.17% of all clinical trials. Compared to other sources, hUC-MSCs have significant advantages: First, obtaining ADMSCs and BMMSCs requires invasive procedures, posing a potential risk of infection; while hUC-MSCs are derived from postpartum umbilical cord tissue (medical waste), making the acquisition process simple and safe, and effectively avoiding ethical controversies. This unique source characteristic gives hUC-MSCs special value in clinical applications, and hUC-MSCs also proliferate faster, have stronger immunosuppressive functions, and lower immunogenicity than the other two types.

[0005] Some researchers have already used hUC-MSCs in the treatment of IBD. For example, patent CN112655702B discloses an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis. This preparation includes umbilical cord mesenchymal stem cells and a solution for umbilical cord mesenchymal stem cells, wherein the solution for umbilical cord mesenchymal stem cells is composed of clinical-grade DMSO, human serum albumin, low molecular weight dextran, trehalose, melatonin, and a compound electrolyte solution. This umbilical cord mesenchymal stem cell preparation can effectively correct the immune dysregulation in patients with ulcerative colitis, prevent the continued damage of the intestine by autoimmune disorders, and simultaneously secrete many cell growth factors and repair factors, promoting the healing of ulcers in the diseased intestinal segments, thus achieving the treatment of ulcerative colitis. However, although hUC-MSCs have shown good application potential in the treatment of IBD, their mechanism of action has not been fully elucidated, and there are limitations such as unstable efficacy, large individual differences, or insufficient durability of efficacy.

[0006] Therefore, more research is needed to verify the safety and efficacy of hUC-MSCs in the treatment of IBD; and it is necessary to conduct in-depth research on the mechanism of action of hUC-MSCs in the treatment of IBD, so as to provide new ideas for the treatment of IBD. Summary of the Invention

[0007] In view of this, one of the objectives of this invention is to provide the use of a pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease (IBD). This invention is the first to propose a strategy of combining hUC-MSCs with mesalazine for the treatment of IBD, and has found that the combination of hUC-MSCs and mesalazine is more effective than either hUC-MSCs or mesalazine alone in treating IBD, with both exhibiting a synergistic effect. This invention provides a completely new approach to the treatment of inflammatory bowel disease.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The use of a pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease, said pharmaceutical composition comprising human umbilical cord mesenchymal stem cells and mesalazine.

[0010] Preferably, the human umbilical cord mesenchymal stem cells highly express CD73, CD90, and CD65, and lowly express CD34, CD45, and HLA-DR.

[0011] Preferably, the positive rates of CD90, CD73 and CD65 in the hUC-MSCs are ≥95%, and the positive rates of CD34, CD45 and HLA-DR are ≤5%.

[0012] These hUC-MSCs have the potential to differentiate into osteogenic, adipogenic, and chondrogenic forms, and can target intestinal injury sites after transplantation.

[0013] As a preferred embodiment, the human umbilical cord mesenchymal stem cells are prepared using the following method:

[0014] Take the umbilical cord from a healthy newborn, rinse it with physiological saline under sterile conditions, separate the umbilical artery and vein, retain Wharton's jelly and cut it into 1mm pieces. 3 Small pieces. Place the tissue pieces on culture dishes, add α-MEM medium containing 10% fetal bovine serum, and incubate at 37°C and 5% CO2. Let stand for 48 hours, and change half of the medium on the 5th day. When the cells have crawled out of the tissue pieces and reached 70%-80% confluence, passage them by trypsin digestion.

[0015] Preferably, the inflammatory bowel disease includes one or more of Crohn's disease and ulcerative colitis.

[0016] Preferably, the inflammatory bowel disease is accompanied by weight loss, diarrhea, rectal bleeding, shortened colon, colonic mucosal damage, intestinal inflammation, impaired intestinal barrier function and / or intestinal flora imbalance.

[0017] Preferably, the pharmaceutical composition is used to inhibit colonic shortening;

[0018] And / or the pharmaceutical composition is used to inhibit weight loss caused by inflammatory bowel disease;

[0019] And / or the pharmaceutical composition thereof is used to inhibit rectal bleeding caused by inflammatory bowel disease;

[0020] And / or the pharmaceutical composition is used to reduce MPO activity in colonic tissue;

[0021] And / or the pharmaceutical composition is used to promote the proliferation of intestinal epithelial cells;

[0022] And / or the pharmaceutical composition thereof is used to promote colonic mucosal angiogenesis and improve intestinal epithelial barrier function;

[0023] And / or the pharmaceutical composition is used to repair intestinal barrier function;

[0024] And / or the pharmaceutical composition is used to inhibit intestinal inflammation;

[0025] And / or the pharmaceutical composition thereof is used to inhibit the activation of the NF-κB / SATAT3 signaling pathway;

[0026] And / or the pharmaceutical composition is used to regulate the gut microbiota.

[0027] Preferably, the promotion of intestinal epithelial cell proliferation includes promoting Ki67 expression.

[0028] Preferably, the promotion of colonic mucosal angiogenesis and improvement of intestinal epithelial barrier function includes promoting the expression of eNOS and occludin.

[0029] Preferably, the inhibition of intestinal inflammation includes inhibiting the expression of pro-inflammatory factors TNF-α, IL-6 and IL-1β, and promoting the expression of anti-inflammatory factors TGF-β and IL-10.

[0030] Preferably, the intestinal barrier repair function includes promoting the expression of claudin-3 and ZO-1 in colonic tissue to repair the tight junction structure of the intestine, and reducing serum LPS levels to improve intestinal barrier permeability.

[0031] Preferably, the inhibition of NF-κB / SATAT3 signaling pathway activation includes inhibiting the phosphorylation levels of IκBα, p50, p65, and STAT3, and reducing the expression levels of MYD88, TLR4, p65, and p50.

[0032] Preferably, the regulation of gut microbiota includes improving gut microbiota imbalance in IBD, restoring gut microbiota diversity, and regulating the abundance of beneficial bacteria, specifically: increasing Firmicutes and decreasing Bacteroidetes and Proteobacteria; increasing Trichophyceae and Akkermaniaceae and decreasing Bacteroidetes and Lactobacillusaceae; increasing Akkermania, Trichophycetes, and Lactobacillus and decreasing Muribacterium_unclassified.

[0033] A second objective of this invention is to provide a pharmaceutical composition for treating inflammatory bowel disease.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] A pharmaceutical composition for treating inflammatory bowel disease, the pharmaceutical composition comprising human umbilical cord mesenchymal stem cells and mesalazine.

[0036] Preferably, the pharmaceutical composition is a combination drug; or the pharmaceutical composition is prepared in the form of a compound drug in any pharmaceutically acceptable dosage form.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention is the first to propose a combined treatment strategy of "stem cell repair + anti-inflammatory drugs" for IBD, and demonstrates that hUC-MSCs and 5-ASA have a synergistic effect in the combined treatment of IBD. Details are as follows:

[0039] 1) Treatment with hUC-MSCs combined with 5-ASA can significantly improve DSS-induced mouse IBD model, including: restoring body weight, reducing DAI score, restoring colon length, reducing colonic mucosal damage, reducing MPO activity, and increasing the expression levels of Ki67, eNOS and occludin. Moreover, the combined administration is superior to hUC-MSCs alone in all indicators.

[0040] 2) hUC-MSCs combined with 5-ASA can alleviate systemic inflammatory responses by repairing intestinal barrier function and reducing intestinal permeability. Data show that the combined treatment can significantly increase the expression levels of claudin-3 and ZO-1 in a DSS-induced mouse IBD model, reduce serum LPS levels, and improve colonic mucosal damage, with better effects than hUC-MSCs alone.

[0041] 3) hUC-MSCs combined with 5-ASA regulate cytokine balance, restore intestinal immune response balance, reduce inflammation, and promote tissue repair. Data showed that the combined treatment significantly reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and increased the levels of anti-inflammatory factors (TGF-β and IL-10) in a DSS-induced mouse IBD model, and the combined treatment group was superior to the hUC-MSCs group in all indicators.

[0042] 4) hUC-MSCs combined with 5-ASA exert their therapeutic effect by regulating the NF-κB / STAT3 signaling pathway and affecting the expression of downstream genes. Data showed that after treatment with hUC-MSCs alone and in combination, the expression levels of p-IκBα, p-p50, p-p65, and p-STAT3 proteins, as well as the expression levels of MYD88, TLR4, p65, and p50 genes in the colon were reduced, and the changes in the expression levels of NF-κB / STAT3 signaling pathway-related proteins and genes were more significant in the combination treatment group.

[0043] 5) hUC-MSCs combined with 5-ASA improve IBD by regulating the gut microbiota. Studies have shown that hUC-MSCs alone or in combination with 5-ASA treatment can significantly increase the species richness and evenness of the gut microbiota in IBD mice, restore gut microbiota diversity, and regulate the abundance of specific bacterial groups. Furthermore, the combination treatment group is superior to the hUC-MSCs group in all indicators.

[0044] The above findings confirm that hUC-MSCs and 5-ASA have a synergistic effect in the combined treatment of IBD. This invention provides new ideas and experimental evidence for the clinical treatment of IBD, and has significant theoretical and practical implications. Attached Figure Description

[0045] Figure 1 This is a diagram showing the growth status of hUC-MSCs, where, Figure 1 -A growth status diagram with a cell density of 60%; Figure 1 -B represents the growth state at 100% cell density; the scale bar is 50 μm.

[0046] Figure 2 The image shows the results of flow cytometry detection of surface markers in hUC-MSCs. Figure 2 -A is the flow cytometry detection result of CD90; Figure 2 -B is the flow cytometry detection result of CD73; Figure 2 -C is the flow cytometry detection result of CD65; Figure 2 -D is the flow cytometry detection result of CD34; Figure 2 -E is the flow cytometry detection result of CD45; Figure 2 -F represents the flow cytometry results of HLA-DR;

[0047] Figure 3 Images showing the staining results of hUC-MSCs in adipogenesis, osteoogenesis, and chondrogenesis. Figure 3 -A shows the results of adipogenic staining of hUC-MSCs; Figure 3 -B shows the osteogenic staining results of hUC-MSCs; Figure 3 -C shows the results of hUC-MSCs chondrogenic staining; scale bar is 50μm.

[0048] Figure 4 A schematic diagram illustrating the establishment of a chronic IBD mouse model;

[0049] Figure 5 The figure shows the experimental results of IBD chronic colitis mice. Figure 5 -A shows the percentage change in weight for the Control group and the DSS group; Figure 5 -B is a graph showing the changes in disease activity index in the Control group and the DSS group; Figure 5-C represents colon images of the Control group and DSS group; Figure 5 -D shows the colonic changes in the Control group and DSS group; Figure 5 -E shows the results of HE staining evaluation of the severity of colonic mucosal damage in the Control group and DSS group, with a scale bar of 100 μm; Figure 5 -F represents the pathological histological scores of the Control group and the DSS group; each group has at least three replicates, and the significance analysis of the results is based on the Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0050] Figure 6 A schematic diagram showing the grouping and treatment of mice with IBD treated with hUC-MSCs;

[0051] Figure 7 The image shows the results of in vivo animal bioluminescence imaging detection of near-infrared fluorescent surface dye-labeled hUC-MSCs in mice;

[0052] Figure 8 The figure shows the evaluation results of the therapeutic effect of hUC-MSCs on IBD mice. Figure 8 -A is a graph showing the percentage change in body weight after treatment in the Control group, DSS group, DSS+hUC-MSCs group, DSS+5-ASA group, and DSS+hUC-MSCs+5-ASA combined treatment group; Figure 8 -B is a graph showing the changes in disease activity index after treatment in each group; Figure 8 -C shows photographs of the colon after treatment in each group; Figure 8 -D is a graph showing the changes in colon length after treatment in each group; Figure 8 -E is a graph showing the severity of colonic mucosal damage after treatment in each group, stained with HE. The scale bar is 100 μm. Figure 8 -F represents the histopathological scores of each group after treatment; each group has at least three replicates, and the significance analysis is based on the Student's t-test; *P<0.05, **p<0.01, ***p<0.001;

[0053] Figure 9 The figure shows the evaluation results of the therapeutic effect of hUC-MSCs on IBD mice. Figure 9 -A is a graph showing the changes in MPO activity after treatment in the Control group, DSS group, DSS+hUC-MSCs group, DSS+5-ASA group, and DSS+hUC-MSCs+5-ASA combined administration group; Figure 9 -B is a graph showing the expression of eNOS and occludin proteins in each group after treatment, as detected by Western blot. Figure 9 -C is a graph showing the distribution of Ki67 in the colon after treatment in each group, as detected by immunohistochemistry;Figure 9 -D is Figure 9 -C quantization plot; scale bar 100μm; at least three replicates per group; significance analysis based on Student's t-test; *P<0.05, **p<0.01, ***p<0.001;

[0054] Figure 10 The figure shows the experimental results related to the investigation of the effect of hUC-MSCs treatment on the repair of intestinal barrier function in IBD mice. Figure 10 -A is a graph showing the expression level of claudin-3 in the colon after treatment by immunofluorescence staining in the Control group, DSS group, DSS+hUC-MSCs group, DSS+5-ASA group, and DSS+hUC-MSCs+5-ASA combined drug administration group. The scale bar is 100μm. Figure 10 -B is a graph showing the expression level of ZO-1 in the colon after treatment in each group by immunofluorescence staining, with a scale bar of 100μm; Figure 10 -C is a graph showing the expression levels of claudin-3 and ZO-1 proteins in the colon after treatment in each group, as detected by Western blot. Figure 10 -D is a graph showing the LPS levels in serum after treatment in each group as detected by ELISA; each group has at least three replicates, and the significance analysis of the results is based on the Student's t-test; *P<0.05, **p<0.01, ***p<0.001;

[0055] Figure 11 The figure shows the experimental results related to hUC-MSCs regulating cytokines to restore the balance of intestinal immune response. Figure 11 -A is a graph showing the serum expression levels of TNF-α, IL-6, and IL-1β after treatment in the Control group, DSS group, DSS+hUC-MSCs group, DSS+5-ASA group, and DSS+hUC-MSCs+5-ASA combined drug administration group, as detected by ELISA. Figure 11 -B is a graph showing the expression levels of TNF-α, IL-6, and IL-1β in the colon after treatment in each group, as detected by Western blot. Figure 11 -C is a graph showing the expression levels of TGF-β and IL-10 in each group detected by ELISA; Figure 11 -D is a graph showing the expression levels of TGF-β and IL-10 in the colon after treatment in each group, detected by Western blot; each group had at least three replicates, and the significance analysis of the results was based on the Student's t-test; *P<0.05, **p<0.01, ***p<0.001;

[0056] Figure 12 The figure shows the experimental results related to the effect of hUC-MSCs on the expression of NF-κB signaling pathway proteins in colitis mice. Figure 12-A is a graph showing the expression levels of p65, p-p65, STAT3, and p-STAT3 in the colon after treatment by Western blot in the Control group, DSS group, DSS+hUC-MSCs group, DSS+5-ASA group, and DSS+hUC-MSCs+5-ASA combined drug administration group. Figure 12 -B is a graph showing the expression levels of IκBα, p-IκBα, p50, and p-p50 in the colon after treatment in each group using Western blot. Each group had at least three replicates. The significance of the results was analyzed based on the Student's t-test, with *P<0.05, **p<0.01, and ***p<0.001.

[0057] Figure 13 The figure shows the effect of hUC-MSCs on gene expression in the NF-κB signaling pathway in colitis mice. Figure 13 -A is a graph showing the expression level of the MYD88 gene in the colon after treatment by Q-PCR in the Control group, DSS group, hUC-MSCs group, 5-ASA group, and hUC-MSCs+5-ASA combined drug administration group. Figure 13 -B is a graph showing the TLR4 gene expression level in the colon of each group after treatment, detected by Q-PCR. Figure 13 -C is a graph showing the p65 gene expression level in the colon of each group after treatment, detected by Q-PCR. Figure 13 -D is a graph showing the p50 gene expression level in the colon after treatment in each group detected by Q-PCR; each group has at least three replicates, and the significance analysis of the results is based on the Student's t-test; *P<0.05, **p<0.01, ***p<0.001;

[0058] Figure 14 α-Diversity Dilution Curves were obtained from 16S rDNA sequencing;

[0059] Figure 15 The results of the α-diversity analysis of the pielou.e index for each group are shown in the figure; each group was repeated six times, and the significance analysis of the results was based on the Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0060] Figure 16 The results of the α-diversity analysis of Shannon index for each group are shown in the figure; each group was repeated six times, and the significance analysis of the results was based on the Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0061] Figure 17 The results of the α-diversity analysis of Simpson index for each group are shown in the figure; each group was repeated six times, and the significance analysis of the results was based on the Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0062] Figure 18 The figure shows the effect of hUC-MSCs treatment on gut microbiota diversity in mice with DSS-induced colitis. Figure 18 -A is a graph showing the results of the β-diversity analysis using the PCA model; Figure 18 -B is the result of the β diversity analysis of the PCoA model; Figure 18 -C represents the β-diversity analysis results of the NMDS model; Figure 18 -D represents the ASVs results obtained after noise reduction; each group has six replicates, and the significance analysis of the results is based on the Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0063] Figure 19 The plots were used to assess the differences in population and abundance among the 20 species of *Phaseolus*. Each group was replicated six times. The significance of the results was analyzed based on the Student's t-test. *P<0.05, **p<0.01, ***p<0.001.

[0064] Figure 20 The plot was used to assess the differences in population and abundance among the top 30 species; six replicates per group, and the significance analysis was based on Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0065] Figure 21 A plot to assess the differences in population and abundance among the top 30 species; six replicates per group, significance analysis based on Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0066] Figure 22 Figure showing the grouping and treatment of mice in a fecal microbial transplantation experiment with hUC-MSCs;

[0067] Figure 23 The figure shows the experimental results of fecal microbiota transplantation from mice treated with hUC-MSCs suppressing colitis.

[0068] Figure 23 -A is a graph showing the percentage change in body weight in the FMT (Control) group, FMT (DSS) group, FMT (hUC-MSCs) group, and FMT (DSS+hUC-MSCs+5-ASA) combined administration group; Figure 23 -B is a graph showing the changes in disease activity index for each group; Figure 23 -C represents colon photos of each group; Figure 23 -D represents the changes in colon length in each group; each group has at least three replicates, and the significance analysis of the results is based on the Student's t-test, *P<0.05, **p<0.01, ***p<0.001;

[0069] Figure 24 The figure shows the experimental results of fecal microbiota transplantation from mice treated with hUC-MSCs suppressing colitis. Figure 24 -A is a graph showing the severity of colonic mucosal damage after drug administration in each group, stained with HE. The scale bar is 100 μm. Figure 24 -B is a graph showing the histopathological scores for each group; Figure 24 -C is a graph showing the changes in MPO activity in each group; Figure 24 -D is a graph showing the distribution of Ki67 in the colon in each group as detected by immunohistochemistry, with a scale bar of 100 μm; Figure 24 -E is Figure 24 -D result quantization plot; Figure 24 -F is a graph showing the expression of eNOS and occludin proteins in each group detected by Western blot, with a scale bar of 100 μm; each group has at least three replicates, and the significance analysis of the results is based on Student's t-test, *P<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0070] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0071] This invention establishes a DSS-induced IBD mouse model and demonstrates that hUC-MSCs, combined with conventional drugs (5-ASA), have a certain therapeutic effect on chronic IBD mouse models, manifested in: improved clinical symptoms, repair of intestinal barrier function, regulation of cytokine balance, and the NF-κB / STAT3 signaling pathway. Furthermore, the combined treatment can restore the diversity of gut microbiota in IBD mice and regulate the abundance of beneficial bacteria. This invention also confirms through fecal microbiota transplantation that the combined treatment can improve colitis by regulating gut microbiota. This invention provides a new theoretical basis for the treatment of IBD with hUC-MSCs. A detailed summary and discussion follow:

[0072] 1. Culture and identification of hUC-MSCs

[0073] Example 1 of this invention aims to establish a stable hUC-MSCs isolation and culture system and verify its stem cell characteristics through systematic identification methods, laying the foundation for subsequent research. Fresh human umbilical cord samples were obtained from the obstetrics department of a local hospital, with approval from the ethics committee and informed consent from the donors. After passage, the cells exhibited uniform morphology and rapid proliferation. The low immunogenicity of hUC-MSCs is another major advantage for their clinical application. Studies have shown that hUC-MSCs do not express or express low levels of major histocompatibility complex class II molecules and co-stimulatory molecules, enabling them to evade recognition and attack by the immune system. This characteristic not only benefits allogeneic transplantation but may also alleviate the inflammatory symptoms of IBD by regulating the immune response. Flow cytometry was used to detect and analyze the expression of surface markers. hUC-MSCs highly expressed mesenchymal stem cell markers CD73, CD90, and CD65, while the expression rates of hematopoietic stem cell markers CD34 and CD45 and the major histocompatibility complex HLA-DR were extremely low, meeting the mesenchymal stem cell identification criteria established by the International Society for Cell Therapy (ISCT). This indicates that they have low immunogenicity, which provides a possibility for allogeneic transplantation. After culture and staining with osteogenic induction medium, numerous red calcium nodules and a large number of red lipid droplets were observed, and the extracellular matrix was rich in proteoglycans. Multi-lineage differentiation experiments further confirmed that hUC-MSCs possess the potential to differentiate into osteogenic, adipogenic, and chondrogenic groups, providing important evidence for their subsequent applications.

[0074] 2. Establishment of a chronic IBD mouse model

[0075] Example 2 of this invention aims to establish a stable chronic IBD mouse model by cyclically administering DSS solution, providing a reliable experimental platform for subsequent studies on the therapeutic effects of hUC-MSCs. During DSS administration, the model group mice exhibited significant weight loss, reduced activity, and dull, frizzy fur; some mice also experienced diarrhea and bloody stools. The DAI score gradually increased during DSS administration; it decreased during the recovery period but increased again in the next cycle, exhibiting typical characteristics of chronic disease. The colon length of the model group mice was significantly shorter than that of the control group, indicating that DSS induces significant inflammatory and fibrotic changes in the colon. HE staining results showed that the colonic mucosa structure of the model group mice was damaged, with disordered or absent crypt structures, reduced goblet cells, extensive inflammatory cell infiltration in the lamina propria, and ulcer formation in some areas. The histopathological score was significantly higher than that of the control group.

[0076] During model establishment, we observed that the DSS-induced inflammatory response exhibited typical chronic disease characteristics. Each DSS dosing cycle was accompanied by significant clinical symptoms and histopathological changes, which were alleviated but not fully resolved during the recovery period. This relapsing-remission course is highly similar to the clinical course of human IBD, making this model particularly suitable for studying the pathogenesis and long-term treatment effects of chronic IBD. The DAI score is an important indicator for assessing the severity of IBD models. In this study, the DAI scores of the model group mice were significantly elevated and fluctuated with the progress of DSS dosing cycles, consistent with the changes in disease activity in clinical IBD patients. Shortening of colon length is another important feature of chronic IBD, reflecting the tissue remodeling and fibrosis process caused by long-term inflammatory response. Histopathological examination further confirmed the reliability of the model. The colon tissue of the model group mice showed typical IBD pathological features, including mucosal structure destruction, crypt structure changes, and inflammatory cell infiltration. These pathological changes are highly similar to the colon biopsy results of human IBD patients, and the establishment of this model provides an ideal experimental platform for subsequent research on the therapeutic effects of hUC-MSCs.

[0077] 3. Investigation on homing ability and therapeutic effect of hUC-MSCs in IBD mice

[0078] (1) Investigation on homing ability of hUC-MSCs in IBD mice treated with hUC-MSCs

[0079] The therapeutic effect of hUC-MSCs is closely related to their homing ability in vivo. Homing ability refers to the ability of stem cells to migrate and colonize at the site of injury, which is a key prerequisite for their therapeutic effect. To investigate the homing ability of hUC-MSCs in an IBD mouse model, this invention systematically evaluated the homing ability of hUC-MSCs in an IBD mouse model using near-infrared fluorescence labeling and in vivo imaging techniques, providing direct evidence for the study of the mechanism of hUC-MSCs in treating IBD. On day 1 post-transplantation, hUC-MSCs were mainly distributed in the lungs, liver, and spleen, but on day 5, they began to show a tendency to migrate to the intestinal injury site. The signal intensity in the intestinal region was significantly higher than that in other organs, but the overall signal intensity decreased compared to day 1; on day 9, only a weak fluorescence signal was detected at the intestinal injury site. The results in this section indicate that hUC-MSCs can target the intestinal injury site after transplantation, demonstrating a certain homing ability.

[0080] (2) Investigation on the therapeutic effect of hUC-MSCs on IBD mice

[0081] 5-ASA, a classic drug for IBD treatment, possesses anti-inflammatory and immunomodulatory effects and is widely used in clinical practice. This invention uses 5-ASA treatment as a positive control, allowing for indirect clarification of the therapeutic effect of mesenchymal stem cells (MSCs) by observing indicators compared to the positive control group. Example 3 of this invention confirms that hUC-MSCs, alone or in combination with 5-ASA, significantly improves DSS-induced mouse IBD models, manifested as weight recovery, decreased DAI score, restored colon length, reduced colonic mucosal damage, decreased MPO activity, and increased expression of Ki67, eNOS, and occludin. The combined treatment group showed superior results compared to the hUC-MSCs group in all indicators, suggesting a synergistic effect between 5-ASA and hUC-MSCs. This finding provides a new approach for the clinical treatment of IBD, namely, a combined strategy of "stem cell repair + anti-inflammatory drugs."

[0082] (3) Investigation on the effect of hUC-MSCs treatment on the repair of intestinal barrier function in IBD mice

[0083] The pathogenesis of IBD is complex, involving the interaction of multiple factors such as genetics, environment, immunity, and microorganisms. Disruption of the intestinal barrier function is a crucial step in the occurrence and development of IBD. The intestinal barrier is composed of intestinal epithelial cells and their tight junction proteins; impaired function leads to increased intestinal permeability, allowing bacteria and their metabolites to translocate into the bloodstream, triggering a systemic inflammatory response. This invention establishes a DSS-induced mouse IBD model to explore the restorative effect of hUC-MSCs on intestinal barrier function, providing new insights and experimental evidence for the clinical treatment of IBD. 5-ASA, as a classic anti-inflammatory drug, can inhibit the release of inflammatory mediators and reduce the inflammatory response; while hUC-MSCs further promote inflammation resolution and tissue repair through immunomodulation and tissue repair. The combined application of both can exert therapeutic effects through different pathways, achieving better therapeutic outcomes. The experimental results of Example 3 of this invention show that hUC-MSCs and the combined drug significantly increased the expression levels of claudin-3 and ZO-1 in the DSS-induced mouse IBD model, reduced serum LPS levels, and improved colonic mucosal damage. These results suggest that hUC-MSCs and their combination therapy can alleviate systemic inflammatory responses by repairing intestinal barrier function and reducing intestinal permeability.

[0084] (4) Effects of hUC-MSCs treatment on cytokines in the intestinal immune response of IBD mice

[0085] Cytokines (including pro-inflammatory and anti-inflammatory factors) are key effector molecules in changes in intestinal immune homeostasis. Pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) play important roles in the inflammatory response of IBD, while anti-inflammatory factors such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) have an inhibitory effect on the inflammatory response. The experimental results of Example 3 of this invention confirm that treatment with hUC-MSCs alone or in combination with 5-ASA can significantly reduce the levels of pro-inflammatory cytokines and increase the levels of anti-inflammatory factors in a DSS-induced mouse IBD model. The combined treatment group was superior to the hUC-MSCs group in all indicators, suggesting that the combined application of 5-ASA and hUC-MSCs has a synergistic effect. These results suggest that hUC-MSCs and combined treatment can restore the balance of intestinal immune response, reduce inflammation, and promote tissue repair by regulating the balance of cytokines.

[0086] (5) Effects of hUC-MSCs treatment on the NF-κB signaling pathway in IBD mice

[0087] The NF-κB signaling pathway plays a crucial role in the inflammatory response of IBD. NF-κB is a multipotent protein molecule involved in regulating the gene expression of various factors and plays an important role in immune regulation, inflammation, stress response, and apoptosis. Inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-10 can mediate inflammatory responses by activating the NF-κB pathway. Previous studies have demonstrated that hUC-MSCs treatment significantly altered colonic cytokines in DSS-induced colitis mice, with a significant decrease in pro-inflammatory cytokine expression and a significant increase in anti-inflammatory cytokine expression. Therefore, this invention further investigates how hUC-MSCs treatment affects the NF-κB signaling pathway in IBD mice. The results confirm that hUC-MSCs treatment, alone or in combination with 5-ASA, can inhibit the phosphorylation levels of signaling pathway-related proteins and reduce the expression levels of related genes.

[0088] 4. Effects of hUC-MSCs treatment on the diversity and composition of gut microbiota in IBD mice

[0089] Gut microbiota dysbiosis is a key characteristic of IBD. The gut microbiota plays a crucial role in the occurrence and development of IBD through various mechanisms, including regulating immune responses and maintaining intestinal barrier function. 16S rDNA sequencing was used to investigate the effects of hUC-MSCs treatment on the diversity and composition of the gut microbiota in IBD mice. Alpha diversity indices (Chao1, Shannon, and Simpson indices) were used to assess species richness and evenness within the samples. hUC-MSCs treatment significantly increased the species richness and evenness of the gut microbiota in IBD mice, restoring gut microbiota diversity. hUC-MSCs treatment significantly altered the composition of the gut microbiota in IBD mice, bringing it closer to a normal state. At the phylum, family, and genus levels, hUC-MSCs treatment significantly increased the abundance of beneficial bacteria genera and decreased the abundance of opportunistic pathogenic bacteria genera. The combined treatment group showed superior results compared to the hUC-MSCs group in all indicators, suggesting a synergistic effect between 5-ASA and hUC-MSCs. Example 3 of this invention further confirms that treatment with hUC-MSCs alone or in combination with 5-ASA can significantly increase the species richness and evenness of the gut microbiota in IBD mice, restore gut microbiota diversity, and regulate the abundance of specific microbiota.

[0090] 5. To investigate whether gut microbiota participates in the therapeutic effect of hUC-MSCs on IBD mice.

[0091] Fecal microbiota transplantation (FMT) is a treatment method that restores gut microbiota balance by transplanting fecal microbiota from a healthy donor into a patient. This invention utilizes an antibiotic-induced gut microbiota depletion (ABX) mouse model and, in conjunction with FMT technology, investigates whether gut microbiota participates in the therapeutic effect of hUC-MSCs on IBD mice and its underlying mechanism. The study found that the gut microbiota treated with hUC-MSCs significantly alleviated colitis symptoms, and the microbiota in the group treated with 5-ASA showed a stronger therapeutic effect. This provides direct evidence for elucidating the microbiota-dependent mechanism of hUC-MSCs in treating IBD.

[0092] In summary, this invention established an internationally standardized hUC-MSCs culture system, demonstrating its low immunogenicity and multi-lineage differentiation potential. A mouse model simulating the chronic course of human IBD was successfully constructed using DSS cyclic drug administration. hUC-MSCs exhibit specific homing ability to the inflamed gut; they repair intestinal barrier function by upregulating tight junction proteins; they regulate the NF-κB signaling pathway and cytokines to reshape immune homeostasis; and FMT experiments confirmed their therapeutic effect through regulation of the gut microbiota. The study also revealed the synergistic effect between 5-ASA and hUC-MSCs. This invention provides experimental evidence for clinical combination therapy and has guiding significance for promoting the clinical application of stem cells.

[0093] In the embodiments of the present invention, the preparation method of human umbilical cord mesenchymal stem cells is as follows:

[0094] Take the umbilical cord of a healthy newborn, rinse it with physiological saline under sterile conditions, strip the umbilical artery and vein, retain Wharton's jelly and cut it into 1 mm 3 small pieces. Attach the tissue pieces to a culture dish, add α-MEM medium containing 10% fetal bovine serum, and culture at 37 °C and 5% CO2. Let it stand still within 48 hours and change half of the medium on the 5th day. When the cells crawl out of the tissue pieces and reach 70%-80% confluence, digest and passage them with trypsin.

[0095] Example 1. Culture and identification of hUC-MSCs

[0096] I. Experimental materials, reagents and instruments

[0097] (1) Experimental materials

[0098] The human umbilical cord mesenchymal stem cells (hUC-MSCs) used in this example were derived from umbilical cord tissue provided by the Ninth People's Hospital of Chongqing, passed the review of the Medical Ethics Committee of the Ninth People's Hospital of Chongqing (No.: 2024-Ke w (Ethical Review)-001), and were cultured and passaged by this laboratory.

[0099] (2) Preparation of main solutions

[0100] 1) 1×Phosphate Buffered Saline (PBS): Take 10 g of 10×PBS powder; measure an appropriate amount of double-distilled water to the 10 L scale line, add the PBS powder and dissolve it fully. Divide the prepared solution into glass containers with a specification of 400 mL. Then place the divided solutions in a high-pressure steam sterilizer and sterilize them at 121 °C for 30 minutes. After the sterilization procedure is completed, let the containers cool naturally at room temperature, and finally transfer them to a 4 °C refrigeration equipment for storage and standby.

[0101] 2) Cell cryopreservation solution: DMSO:FBS = 1:9, freshly prepared and used immediately.

[0102] 3) 75% ethanol: Prepared from 175 mL of absolute ethanol and 125 mL of double-distilled water, and stored in a -4 °C refrigerator after preparation.

[0103] 4) 4% Paraformaldehyde (PFA): Take 4 g of PFA powder and 100 mL of 1×PBS and place them in a beaker. Seal the beaker and heat it in a 60 °C constant temperature water bath until the content is completely dissolved, and then let it cool naturally at room temperature. After the solution temperature is balanced, adjust it to 7.3 using a pH meter, and finally transfer it to 4 °C for refrigerated storage.

[0104] 5) The preparation method of Solution A of the adipogenic induction differentiation medium for human related stem cells is as follows:

[0105] a. Serum pretreatment: Serum pretreatment is required before the experiment. Superior fetal bovine serum was thawed slowly at 4°C.

[0106] b. Additive AI (AI) culture medium for inducing adipogenesis and differentiation of human stem cells should be refrigerated before use. Additive A-II (A-II) culture medium for inducing adipogenesis and differentiation of human stem cells needs to be thawed at room temperature. Before mixing, invert the reagent tube several times and centrifuge at 3000 rpm for 30 seconds to allow the reagent to precipitate fully.

[0107] c. After disinfecting all reagent surfaces with 75% ethanol, transfer them to a clean bench for further processing.

[0108] d. Add the pretreated serum and additives AI and A-II sequentially. In the basal culture medium.

[0109] e. Rinse the inner walls of each reagent container with a small amount of basal culture medium to ensure that all active ingredients are transferred to the culture medium.

[0110] f. The prepared culture medium should be sealed with sealing film, wrapped with aluminum foil to protect it from light, and clearly labeled with the name of the culture medium and the preparation date.

[0111] 6) The preparation method of human-related stem cell adipogenic differentiation induction medium B is as follows:

[0112] a. Before use, remove the contents of the reagent kit. Human-related stem cell adipose-induced differentiation additive B (hereinafter referred to as "additive B") was thawed in a 4°C refrigerator to allow it to mix thoroughly.

[0113] b. The serum needs to be thawed slowly at 4°C for 6 hours in advance; Additive B for human stem cell adipogenic differentiation (additive B) needs to be thawed at 4°C and thoroughly mixed before use.

[0114] c. Centrifuge additive B for 30 seconds (3000 rpm) to ensure complete precipitation of the reagent; after sterilizing all reagent packaging with 75% ethanol, open and use it in a clean bench.

[0115] d. Add the pretreated serum and additive B to In the basal medium; rinse the reagent container with an appropriate amount of basal medium to ensure complete transfer of components.

[0116] e. After tightening the cap, mix gently; seal with sealing film, then package in light-proof aluminum foil, and label with the culture medium name and preparation date.

[0117] 7) Complete Osteogenic Culture Medium: Before use, remove OriCell advanced fetal bovine serum from the kit and thaw at 4°C. Remove OriCell Human Associated Stem Cell Osteogenic Induced Differentiation Additive from the kit and thaw at 4°C. Carefully invert the test tube to mix thoroughly.

[0118] (8) Complete chondrogenic culture medium:

[0119] a. Before the experiment, OriCell advanced fetal bovine serum was placed in a refrigerated environment at 4°C for slow thawing; simultaneously, OriCell human stem cell osteogenic differentiation induction additive was taken out, thawed at 4°C, and then gently inverted and mixed 5 times.

[0120] b. Thoroughly disinfect all reagent surfaces with 75% ethanol, and complete reagent opening and transfer operations in a clean bench.

[0121] c. Add the pretreated serum and additives sequentially to OriCell basal medium, and rinse each reagent container with 5 mL of basal medium to ensure complete transfer.

[0122] d. After tightening the cap, gently mix for 30 seconds; seal with sealing film, then package in light-proof aluminum foil with clear labeling.

[0123] II. Experimental Methods

[0124] 1. Cell Culture

[0125] (1) Cell passage

[0126] 1) Observe the state and density of cells using an optical microscope. If the cell density reaches 80-90% and the cells are in good condition, they can be passaged. First, use a Pasteur pipette to remove the old culture medium, then add 5 ml of 1×PBS, gently shake the culture dish, use a Pasteur pipette to remove the PBS, and wash away any remaining culture medium on the cell surface.

[0127] 2) During cell digestion, add 1 ml of trypsin to the culture dish using a pipette, then place the culture dish in a 37°C carbon dioxide incubator and wait for the cells to digest. The digestion time is approximately 3 minutes.

[0128] 3) Observe the cell digestion under a microscope. The digestion process is complete when the cells are found to have completely detached from the bottom of the culture dish. Then add complete culture medium to the culture dish to complete the digestion. Transfer the cell suspension to a 5 ml centrifuge tube and centrifuge at 800 rpm for 5 minutes.

[0129] 4) Before starting the experiment, prepare the culture dishes and add an appropriate amount of complete culture medium to each dish. After centrifugation, use a pipette to aspirate the supernatant, then add 3 ml of complete culture medium. Gently pipette the cell suspension to suspend the cell particles, and then transfer the cell suspension to the culture dish containing the culture medium.

[0130] (2) Cell cryopreservation

[0131] 1) Observe the cells under a microscope when they have grown to 80-90% and freeze them for cell digestion.

[0132] 2) After centrifugation, aspirate the supernatant, add 1 ml of cell cryopreservation solution, gently pipette, and inject 500 μL of cell suspension into each cryopreservation tube;

[0133] 3) Place the preservation tube in a freezer and then transfer it to a -80°C freezer to allow the cells to cool gradually.

[0134] 4) After 48 hours, the cells were transferred to a liquid nitrogen tank for long-term preservation.

[0135] 2. Identification of cell surface differentiation antigens (flow cytometry)

[0136] (1) Cell treatment: Wash the cell suspension three times with sterile physiological saline to thoroughly remove culture medium residues and digestive enzymes. After measuring cell density using a hemocytometer, aliquot the cell suspension into 1.5 mL centrifuge tubes. After washing three times with PBS, take 1.5 mL of cell suspension, add 50 μL of PBS buffer and 5 μL of fluorescently labeled antibody (PE or FITC-labeled CD90, CD73, CD105, CD34, HLA-DR, or CD45 antibody), and incubate in the dark for 30 minutes.

[0137] (2) Sample fixation and detection: The labeled cells were transferred to a dedicated flow cytometry tube, and 400 μL of 4% paraformaldehyde fixative was added to each tube for fixation. At the same time, a blank control group without antibody was set up. All samples were immediately tested after preparation.

[0138] 3. Adipogenic differentiation

[0139] (1) Add 1 mL of 0.1% gelatin solution to each well of a 6-well plate, place it in a 37°C, 5% CO2 incubator for 30 minutes, remove the gelatin solution and set aside.

[0140] (2) hUC-MSCs were loaded with 2×10 4 cells / cm 2The culture medium was inoculated into pretreated 6-well plates at a density of 10%, and 2 mL of complete culture medium containing 10% FBS was added to each well. The plates were then incubated under standard culture conditions (37°C, 5% CO2).

[0141] (3) When the cell fusion rate reaches 100%, replace it with 2 mL of adipogenic differentiation-inducing medium A.

[0142] (4) After 72 hours of induction, replace with 2 mL of lipid-inducing differentiation medium B, and after 24 hours, replace with medium A again. Repeat the above alternating induction process.

[0143] (5) Observe the cell status daily using an inverted microscope. If cells show signs of shrinkage or death in culture medium A, immediately replace with culture medium B for recovery.

[0144] (6) When obvious lipid droplet accumulation occurs in the cells, induction can be terminated and Oil Red O staining can be performed for identification.

[0145] 4. Oil Red O staining analysis

[0146] (1) After the lipid induction is completed, the induction medium is discarded, and the cell monolayer is washed three times with pre-cooled PBS buffer (pH 7.4). 2 mL of 4% paraformaldehyde fixative is added to each well and fixed at 25°C for 30 minutes.

[0147] (2) Take Oil Red O stock solution and deionized water at a volume ratio of 3:2, shake well and mix thoroughly, centrifuge at 250×g for 5 minutes, collect the supernatant as staining working solution, add 2mL PBS buffer to cover the cell layer, and immediately observe and record the lipid droplet staining under an inverted microscope.

[0148] (3) After the culture plate has been stained, it can be sealed and stored in a refrigerator at 4°C for a short period of time.

[0149] 5. Osteogenic induction differentiation

[0150] (1) Take a 6-well culture plate, add 1 mL of 0.1% gelatin solution to each well, place it in a clean bench and let it stand for 30 minutes for coating, remove the gelatin solution and set aside.

[0151] (2) Induced human stem cells were injected at a rate of 2 × 10⁻⁶ 4 cells / cm 2 The inoculation density was inoculated into the pretreated 6-well plates, and 2 mL of complete culture medium containing 10% fetal bovine serum was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator.

[0152] (3) When the cell fusion reaches about 70%, replace it with 2 mL of OriCell human stem cell osteogenic induction differentiation medium, maintain the culture conditions unchanged, and replace it with fresh osteogenic induction differentiation medium every 72 hours.

[0153] (4) After continuous induction culture for 2-4 weeks, the osteogenic differentiation effect can be identified by alizarin red staining based on changes in cell morphology (formation of matrix mineralization nodules).

[0154] 6. Alizarin Red Staining Analysis

[0155] (1) After osteogenic induction was completed, the induction medium was first aspirated, and the cells were gently washed three times with pre-cooled PBS buffer (pH 7.4). 2 mL of 4% paraformaldehyde fixative was added to each well, and the cells were fixed at 25°C for 30 minutes. After discarding the fixative, the cells were thoroughly washed three more times with PBS.

[0156] (2) Add 2 mL of freshly prepared alizarin red staining working solution to each well, incubate at 25°C in the dark for 10 minutes, remove the staining solution, wash three times with PBS buffer to terminate the staining reaction, and add 2 mL of PBS to cover the cell layer.

[0157] (3) Observe the staining of calcium nodules under a microscope. After staining, the culture plate can be sealed and stored in a refrigerator at 4°C for 14 days.

[0158] 7. Chondrogenic induction differentiation

[0159] (1) Take 3×10 5 Collect cells by centrifuging at 250×g for 5 minutes, discard the supernatant, add 0.5mL of chondrogenic differentiation premix to resuspend the cells, and centrifuge at 150×g for 5 minutes to complete the washing.

[0160] (2) Add OriCell human stem cell chondrogenic differentiation additive I to the premix at a ratio of 10 μL additive I / mL premix, gently vortex for 30 seconds, resuspend the cell pellet in this complete culture medium, and centrifuge at 150×g for 5 minutes to form cell clumps.

[0161] (3) Slightly loosen the centrifuge tube cap to maintain gas exchange. Place the centrifuge tube containing cell clumps vertically in a 37°C, 5% CO2 incubator. After the cells aggregate into spheres, gently tap the bottom of the tube to detach the cartilage spheres from the tube wall and suspend them in the culture medium.

[0162] (4) From the date of inoculation, replace 0.5 mL of fresh cartilage induction complete culture medium every 72 hours and continue to induce until the cartilage tissue is fully differentiated.

[0163] 8. Staining of cartilage bulb sections

[0164] (1) Fixation: After rinsing with PBS, the induced cartilage balls were immersed in 4% paraformaldehyde fixative and fixed at 25°C for 30 minutes.

[0165] (2) Dehydration: Dehydration was carried out sequentially with the following series of ethanol, each time for 30 minutes:

[0166] 50% ethanol → 70% ethanol → 80% ethanol → 95% ethanol → Anhydrous ethanol (I) → Anhydrous ethanol (II)

[0167] (3) Transparent: Soak in a 1:1 xylene / anhydrous ethanol mixture for 2 hours, soak in pure xylene (I) for 1.5 hours, and soak in pure xylene (II) for 1 hour.

[0168] (4) Wax impregnation: 1:1 xylene / paraffin mixture, impregnated at 40℃ for 40 minutes; pure paraffin (I), impregnated at 55℃ for 30 minutes; pure paraffin (II), impregnated at 55℃ for 30 minutes.

[0169] (5) Embedding: The tissue was placed in an embedding cassette and infused with paraffin. After cooling and solidification, the paraffin block was trimmed. Continuous sections were made using a rotary microtome with a thickness set to 3 μm.

[0170] (6) Slicing: Continuous slicing, each slice is about 3 micrometers thick.

[0171] (7) Adhesive sectioning: Place the cartilage section on a glass slide coated with adhesive and dry it in an oven at 35°C.

[0172] (8) Dewaxing: Soak the slices in xylene for 15 minutes, then replace with new xylene and soak for another 10 minutes. Soak the slices in a 1:1 mixture of xylene and anhydrous ethanol for 10 minutes. Soak the slices in 95%, 85%, 70% and 50% alcohol concentrations for 10 minutes in sequence, and then air dry.

[0173] (9) Staining: Add alexandrite blue staining solution to cover the tissue, stain in a humid chamber at 37°C for 60 minutes, rinse with running water for 5 minutes, and observe under a microscope to evaluate the proteoglycan staining effect.

[0174] III. Experimental Results

[0175] (1) Culture of hUC-MSCs

[0176] The results are as follows Figure 1 As shown, after resuscitation and passage culture, the hUC-MSCs gradually became uniform in morphology, exhibiting a typical spindle shape, which is consistent with the morphological characteristics of hUC-MSCs.

[0177] (2) Phenotypic identification of hUC-MSCs

[0178] Surface markers of hUC-MSCs were analyzed by flow cytometry. Results are as follows: Figure 2 As shown, hUC-MSCs highly expressed the classic mesenchymal stem cell markers CD90, CD73, and CD65 with positive rates as high as 99.8%, 98.9%, and 96.9%, respectively, while the positive rates of hematopoietic and immune cell-related markers CD34, CD45, and HLA-DR were only 0.37%, 0.048%, and 2.46%, respectively. This confirms the mesenchymal stem cell characteristics of hUC-MSCs, with low immunogenicity, making them suitable for allogeneic transplantation.

[0179] (3) hUC-MSCs induce differentiation

[0180] The results are as follows Figure 3 As shown, hUC-MSCs exhibited significant multi-lineage differentiation potential under specific induction conditions. After 14 days of induction in adipogenic differentiation medium, Oil Red O staining revealed a large accumulation of lipid droplets in the cytoplasm, confirming successful differentiation into adipocytes. After 14 days of culture in osteogenic differentiation medium, Alizarin Red staining detected calcium salt deposition, indicating that the cells had differentiated towards osteogenic differentiation. Furthermore, after 21 days of induction in chondrogenic differentiation medium, the formation of typical chondroid structures was observed, and Alixin Blue staining showed positive expression of acidic mucopolysaccharides in the cartilage tissue, further confirming successful chondrogenic differentiation. These experimental results fully demonstrate the strong proliferation and differentiation capacity of hUC-MSCs, providing a reliable basis for their application in subsequent experiments.

[0181] IV. Summary and Discussion

[0182] This invention uses human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) as the research object. hUC-MSCs have advantages such as wide availability, ease of acquisition, low immunogenicity, strong proliferative capacity, and significant multi-lineage differentiation potential, making them ideal seed cells for tissue engineering and regenerative medicine. Recent studies have shown that hUC-MSCs have immunomodulatory, tissue repair-promoting, and inflammatory-inhibiting effects, demonstrating great potential in the treatment of inflammatory diseases such as IBD. The cultured cells exhibit typical mesenchymal stem cell morphology, namely long spindle-shaped and whorled growth, with vigorous proliferation capacity. Cell morphology and proliferation rate remain stable after passage. P3 generation hUC-MSCs cryopreserved in liquid nitrogen showed a cell viability of over 90% after thawing, with no significant changes in cell morphology or proliferation capacity. Flow cytometry results showed that the cells highly expressed CD90, CD73, and CD65, positive markers for mesenchymal stem cells, and lowly expressed CD34, CD45, and HLA-DR, markers for hematopoietic stem cells and endothelial cells. Both the positive and negative rates met the identification criteria for mesenchymal stem cells set by the International Society for Cell Therapy (ISCT). Under specific induction conditions, the cells successfully differentiated into osteoblasts, chondrocytes, and adipocytes, expressing corresponding specific markers, indicating their multi-lineage differentiation potential. This experiment successfully cultured hUC-MSCs that met the standards, providing a reliable cell source for subsequent research.

[0183] Example 2. Establishment of a chronic IBD mouse model

[0184] I. Key Experimental Materials and Reagents

[0185] (1) Experimental materials

[0186] The C57BL / 6 mice (male, 8 weeks old) used in this animal experiment were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., and have passed the experimental animal welfare ethics review of Southwest University (SWU_LAC2025020072). The AIN-93G purified mouse food used in this experiment was purchased from Daizi Biotechnology Co., Ltd.

[0187] (2) Preparation of main solutions

[0188] (1) 3% DSS drinking water: Weigh 6g of DSS powder and pour it into 200mL of sterile water, then stir and mix well.

[0189] (2) 4% PFA solution: 40g paraformaldehyde, 800mL 1×PBS, put in an oven, dissolve and cool to room temperature, adjust the pH to 7, and then make up to volume with 1×PBS.

[0190] (3) 1% ammonia water: 17.85 mL of 28% ammonia water, diluted to volume with double-distilled water.

[0191] II. Experimental Methods

[0192] (1) Establishment of a chronic IBD mouse model

[0193] To establish a stable mouse model of chronic inflammatory bowel disease (IBD), this study used the dextran sulfate sodium (DSS) induction method. The specific steps are as follows: Male C57BL / 6 mice were selected as experimental subjects. Mice were given free access to a 3% DSS solution for 5 days. After a 10-day interval, this cycle of "continuously administering DSS solution for 5 days every 5 days" was repeated four times. See details below. Figure 4 .

[0194] (2) Blood collection after enucleation of mouse eyeballs

[0195] 1) After the modeling is completed, remove the eyeballs of the mice to collect blood, and hold the mice to make the eyeballs protrude.

[0196] 2) Use small tweezers to remove the protruding eyeball, then immediately tilt the eye socket down to let the blood drip into a 1.5mL Ep tube.

[0197] 3) After letting the blood stand at room temperature for about 1 hour, centrifuge at 3000 rpm and 20°C for 15 minutes; use a pipette to transfer the supernatant serum into a new 1.5 mL Eppendorf tube and store at -80°C.

[0198] (3) HE staining

[0199] 1) After blood collection, the mouse is in a state of hemorrhagic shock. Quickly cut open the abdomen to open the abdominal cavity, cut off the entire colon to the anus, and gently rinse it in physiological saline to remove feces.

[0200] 2) Fixation: Fix the tissue in 4% PFA for 24 hours; the remaining portion is loaded into Eppendorf tubes and stored in liquid nitrogen tubes.

[0201] 3) Dehydration: Remove the tissue, wash it three times with 1×PBS for 10 min each time, and then place it in a gradient of alcohol with concentrations of 70%, 80%, 90%, 100%, and 100% for dehydration, changing the solution every 30 min.

[0202] 4) Transparency: Immerse the tissue in a 1:1 xylene:anhydrous ethanol solution for 5 minutes, then transfer it to a xylene solution and immerse for 2 minutes. Note that the tissue should not be too transparent, otherwise it will easily break.

[0203] 5) Immersion in paraffin: Immerse the tissue in melted paraffin overnight. Select an appropriate temperature based on the melting point of the paraffin, generally not higher than 65℃.

[0204] 6) Embedding: Place the tissue in the embedding mold, add enough paraffin, and cool it to room temperature on a cold plate.

[0205] 7) Sectioning: The completely cooled and solidified paraffin block is cut into 5μm thin sections using a microtome. The sections are placed in a 44℃ spreader to allow them to spread out, and then the sections are picked up with a glass slide.

[0206] 8) Baking the slides: Place the dried glass slides in a 65℃ oven and bake for 2 hours.

[0207] 9) Dewaxing and hydration: Dewax the paraffin sections in xylene for 10 minutes, then soak them in a 1:1 solution of xylene and anhydrous ethanol for 5 minutes; then soak them in 100%, 90%, 80%, and 70% alcohol for 5 minutes each, and finally wash them three times with distilled water for 5 minutes each time.

[0208] 10) Staining: Place the sections in hematoxylin staining solution for 15 min, rinse with tap water, differentiate with 3% hydrochloric acid solution for 30 s, wash with distilled water for 5 min, then place in eosin solution for 3 min, and rinse with tap water three times.

[0209] 11) Dehydration and clearing: Soak the slices in 90% and 95% alcohol for 10 seconds each, and 100% alcohol for 2 minutes. Then soak the slices in xylene for 5 minutes.

[0210] 12) Mounting: After the sections have dried, mount them with neutral resin, observe under a microscope, take photos and record the results.

[0211] (4) Model Evaluation

[0212] 1) Disease Activity Index (DAI) score: During the modeling period, the mice were weighed daily, their fecal morphology was observed, and fecal blood was detected to score the disease activity index. The DAI scoring criteria are shown in Table 1.

[0213] Table 1. DAI Scoring Criteria

[0214]

[0215] 2) Histopathological scoring: After modeling, the colon tissue was stained with hematoxylin and eosin (HE) to assess the severity of colonic mucosal damage, and histopathological scoring was performed simultaneously. The histopathological scoring criteria are shown in Table 2.

[0216] Table 2. Histopathological Scoring

[0217]

[0218] (5) Statistical Analysis

[0219] All experiments were repeated three times, and data were analyzed using GraphpadPrism 8 software. All quantitative data are expressed as mean ± standard deviation. Significance analysis was performed based on Student's t-test; *p<0.05, **p<0.01, ***p<0.001.

[0220] III. Experimental Results

[0221] like Figure 5 -A and Figure 5 As shown in Figure B, the DSS-induced chronic IBD model mice exhibited typical disease characteristics. During the modeling process, body weight fluctuated and decreased, remaining at a low level, and intermittent bloody stools occurred. Disease activity index scores were calculated based on mouse body weight, fecal morphology, and bloody stool occurrence; the disease activity index scores of the model group mice were significantly higher than those of the control group.

[0222] like Figure 5 -C and Figure 5 As shown in Figure D, the colon length of mice in the model group was significantly shorter compared to the control group. HE staining results are as follows... Figure 5 As shown in Figure -E, there was extensive loss of colonic crypt structures, with abundant inflammatory cell infiltration throughout the entire intestinal wall and severe damage to goblet cells. Some samples exhibited full-thickness fibrin exudation, intestinal wall swelling due to cell proliferation, and intestinal luminal narrowing, with complete destruction of the epithelial structure and disordered tissue layers. Based on these pathological changes, the histopathological score of the model group mice was significantly higher than that of the control group. (See details...) Figure 5 -F indicates that a chronic IBD mouse model has been successfully established.

[0223] IV. Summary and Discussion

[0224] The DSS-induced IBD model is simple to operate, highly reproducible, and low in cost. It can effectively simulate the typical clinical features and phenotypes of IBD, including weight loss, diarrhea, rectal bleeding, colonic shortening, and colonic mucosal damage, making it a commonly used animal model for studying IBD treatment methods. This invention successfully established a chronic IBD mouse model using the DSS-induced method, providing a reliable animal model for studying the therapeutic effects and mechanisms of hUC-MSCs on DSS-induced inflammatory bowel disease. Mice were allowed free access to 3% DSS solution for 5 days, followed by a 10-day interval, and then repeated four times in a cycle of "5 days of continuous DSS solution administration every 5 days." Mouse weight, fecal morphology, and rectal bleeding were monitored daily to score the disease activity index. Compared to the control group, the model group mice showed significantly lower body weight, shorter colon length, loose or watery stools, significant rectal bleeding, and a significantly higher disease activity index score. After modeling, colonic tissue was stained with hematoxylin and eosin (HE) to assess the severity of colonic mucosal damage, and histopathological scoring was performed. The colonic mucosa structure of the model group mice was damaged, the crypt structure disappeared, the number of goblet cells decreased, the infiltration of inflammatory cells was obvious, and the histopathological score was significantly higher than that of the control group.

[0225] Example 3. Investigation on the homing ability and efficacy of hUC-MSCs in treating IBD mice

[0226] I. Key Reagents

[0227] (1) Preparation method of 5-ASA solution (75 mg / kg)

[0228] Accurately weigh 1.5 mg of 5-ASA using a balance, dissolve it in 1 mL of PBS buffer to prepare a 1.5 mg / mL solution. Filter the solution through a 0.22 μm sterile filter, aliquot the filtered solution into sterile tubes, and store at 4°C protected from light.

[0229] (2) Preparation method of main reagents for Western blotting detection of protein expression

[0230] 10% Ammonium Persulfate (AP) Solution: Place 1.0 g of AP in a container, add 10 mL of double-distilled water, and dissolve thoroughly using a vortex mixer. Filter the solution through a 0.45 μm filter membrane and store at 4°C until use.

[0231] 10% Sodium Dodecyl Sulfate (SDS) Solution: Accurately weigh 10.0 g of SDS powder, pour it into a glass flask, add 100 mL of double-distilled water, stir thoroughly until completely dissolved, and store at room temperature until needed.

[0232] 5×SDS-PAGE electrophoresis buffer: Take 5.0g SDS, 94.0g Glycine, and 15.1g Tris, add distilled water, and mix thoroughly until completely dissolved; then continue to add an appropriate amount of distilled water to make the total volume reach 1 liter, and store at room temperature for later use.

[0233] 1× Transfer Buffer: Take 4.35g Glycine, 8.70g Tris, and 0.55g SDS. Mix with double-distilled water and the sample, and use a magnetic stirrer to completely dissolve them. Then, bring the volume to 1.2L and store at room temperature for later use. Before use, prepare the transfer buffer and methanol at a ratio of 4:1 and store at 4°C.

[0234] 5×TBST Wash Buffer: Take 44g NaCl, 100mL 1.0M Tris-HCl (pH=8.0), and 2.5mL Tween-20, add double-distilled water, and stir thoroughly until completely dissolved. Then, add an appropriate amount of double-distilled water to bring the total volume to 1 liter, and store at room temperature for later use. Before use, dilute the 5×TBST wash buffer to 1×TBST.

[0235] The preparation system for SDS-PAGE protein gel is shown in Table 3:

[0236] Table 3. Formulation system of SDS-PAGE protein gel

[0237] Experimental materials 10% resolving gel (mL) 12% resolving gel (mL) Concentrated gel (mL) Double distilled water 1.8 1.6 1.4 30% acrylamide 1.7 2.0 0.38 1.5 M Tris-HCl (pH = 8.8) 1.3 1.3 -- 1.0 M Tris-HCl (pH = 6.8) -- -- 0.25 10% AP 0.05 0.05 0.02 10% SDS 0.05 0.05 0.02 TEMED 0.002 0.002 0.002

[0238] (3) Preparation methods of main reagents for immunohistochemical staining

[0239] 4% PFA solution: 40g paraformaldehyde, 800mL 1×PBS, place in a constant temperature drying oven, dissolve and cool, then adjust the pH to 7, and finally add 1×PBS to make up to the final volume.

[0240] Citric acid-sodium citrate buffer: 3.42g sodium citrate·2H2O, 0.4g citric acid, diluted to volume with double-distilled water.

[0241] Immunohistochemical blocking solution: 500 μL goat serum, diluted with 1×PBS.

[0242] 1% ammonia solution: 17.85 mL 28% ammonia solution, diluted to volume with double-distilled water.

[0243] II. Experimental Methods

[0244] 1. hUC-MSCs treatment of IBD mice

[0245] (1) Grouping and treatment of experimental animals

[0246] Grouping and treatment of laboratory animals, such asFigure 6 As shown. After modeling, IBD mice were randomly divided into four groups: DSS group, DSS+hUC-MSCs group, DSS+5-ASA group, and DSS+hUC-MSCs+5-ASA combined administration group, with 6 mice in each group; a normal control group of 8 mice was also included. The DSS+hUC-MSCs group and the DSS+hUC-MSCs+5-ASA combined administration group were intraperitoneally injected with hUC-MSCs (1×10⁻⁶ mg / kg) according to the mice's body weight (5 mg / kg). 7 Mice in the DSS+5-ASA group and the DSS+hUC-MSCs+5-ASA combined administration group were given 75 mg / kg of 5-ASA by gavage for 7 days. Mice in the DSS group were injected intraperitoneally with an equal volume of physiological saline.

[0247] (2) Intraperitoneal injection

[0248] 1) After resuscitating hUC-MSCs, the cells were washed twice with sterile saline and then resuspended in saline. Aggregates were removed by filtration through a cell filter, and cell counts were performed. The final cell density was adjusted to 1×10⁻⁶. 7 Units / mL are available for use.

[0249] 2) Gently lift the mouse by pinching the skin behind its ears and the back of its neck with your thumb and forefinger, positioning it in a supine position with its head slightly lower than its body. Disinfect the abdominal skin by wiping it with a 75% alcohol swab. Choose the lower abdomen as the injection site, approximately 1-2 mm to the left or right of the midline. Insert the needle obliquely into the subcutaneous tissue, advancing it parallel to the midline for about 3 mm. Adjust the angle to 45° and slowly penetrate the abdominal muscle layer. You can sense changes in resistance during needle insertion; the resistance decreases sharply when the needle tip breaks through the muscle layer.

[0250] 3) Connect the syringe and slowly inject the cell suspension, avoiding rapid injection that could cause tissue damage. Immediately after removing the needle, gently press the injection site with a sterile dry cotton ball for 5-10 seconds to prevent reflux or leakage of the suspension.

[0251] (3) Gavage

[0252] 1) Animal weighing: Weigh the mice using a balance and calculate the volume of medication administered (based on body weight and dosage).

[0253] 2) Fixing the animal: Grab the mouse's tail with your right hand and let its front paws grip the cage cover. Pinch the skin on the animal's back with your left thumb and forefinger to fix its head and keep the mouse's body in a straight line.

[0254] 3) Insert the gavage needle: Insert the gavage needle into the corner of the mouse's mouth, slide it backward along the palate, and enter the esophagus. Be careful not to insert it into the trachea.

[0255] 4) Injecting the drug: Slowly push the drug solution into the gavage syringe, observing to ensure that the mouse does not choke. After the injection is complete, remove the gavage syringe.

[0256] 5) Animal observation: After administration, observe whether the mice have any abnormal reactions (such as difficulty breathing, coughing, etc.).

[0257] (4) Blood collection and HE staining after eyeball removal

[0258] The specific steps are the same as those described in Example 2, which describes the method for removing mouse eyeballs, collecting blood, and staining with hematoxylin and eosin (HE).

[0259] (5) Evaluation of treatment effect

[0260] The specific steps are as described in the model evaluation method in Example 2.

[0261] 2. In vivo animal imaging

[0262] (1) hUC-MSCs staining

[0263] 1) Add DID dye working solution to hUC-MSCs suspension.

[0264] 2) After adding the dye working solution, tighten the centrifuge tube, vortex mix for 5 minutes, and then incubate at 37°C for 20 minutes.

[0265] 3) Add 10 mL of 1×PBS to the incubated hUC-MSCs-dye complex and mix well.

[0266] 4) Take 200 μL of 1×PBS to resuspend the precipitate. The precipitate is the stained hUC-MSCs.

[0267] (2) In vivo animal imaging detection

[0268] Twenty minutes after intraperitoneal injection of labeled hUC-MSCs, the abdominal hair was shaved off and time-course epifluorescence imaging was performed using IVIS spectroscopy (Perkin Elmer) to detect the distribution of starter cells in mice. Real-time imaging was performed on days 3 and 9.

[0269] 3. Degree of mucosal neutrophil infiltration and MPO activity detection

[0270] 1) According to the instructions of the myeloperoxidase (MPO) activity assay kit, the ratio of tissue sample to mass is 1:5. Add the mass (g) of reagent II to the volume (mL) of reagent II, homogenize in an ice bath, centrifuge at 12000g for 10 minutes at 4℃, remove the precipitate, collect the supernatant and place it on ice.

[0271] 2) Heat the microplate reader for 30 minutes, set the wavelength to 460 nm, and then zero it with distilled water.

[0272] 3) Add samples to 1.5 mL Eppendorf tubes in the order shown in Table 4.

[0273] Table 4. Sampling Procedure

[0274]

[0275]

[0276] After mixing, react at 60℃ for 10 min. Take 300 μL of the reaction solution into a 96-well plate and measure the absorbance at 460 nm in each well. Record these values ​​as Ameasurement and Acontrol, respectively. Calculate ΔA = Ameasurement - Acontrol. One control well should be set up for each measurement well.

[0277] 4) MPO activity calculation

[0278] Based on sample protein concentration: MPO activity (U / mgprot) = ΔA ÷ (exd) × Vtotal ÷ (Cpr x Vsample) ÷ T = 3.053 × ΔA + Cpr

[0279] 4. Immunohistochemical staining method

[0280] (1) Baking treatment: Place the paraffin slices in a 60℃ constant temperature oven and bake for 2 hours to enhance tissue adhesion.

[0281] (2) Dewaxing and gradient hydration

[0282] Dewaxing: Immerse the sections in xylene for 10 minutes to thoroughly remove paraffin. Transition treatment: Transfer to a 1:1 xylene / anhydrous ethanol mixture and soak for 5 minutes. Gradient ethanol hydration: Sequentially hydrate with 100%, 90%, 80%, and 70% ethanol for 5 minutes each, and finally rinse three times with distilled water for 5 minutes each time.

[0283] (3) Antigen retrieval

[0284] Microwave thermal retrieval was performed using citrate buffer (pH 6.0): The buffer was heated to boiling in the microwave, and the slides were placed in the microwave and kept boiling for 8 minutes. The slides were then allowed to cool naturally to room temperature and rinsed three times with double-distilled water for 5 minutes each time.

[0285] (4) Immunohistochemical staining

[0286] Blocking: Cover the tissue with blocking solution and incubate at room temperature for 2 hours, then wash three times with PBS (3 minutes each time). Primary antibody incubation: After removing PBS, add primary antibody and incubate overnight at 4°C. Secondary antibody treatment: Wash three times with PBS (5 minutes each time), add polymerase enhancer, react at room temperature for 30 minutes, then wash three more times with PBS (3 minutes each time). Enzyme-labeled polymerase incubation: Add HRP-labeled secondary antibody, incubate at room temperature for 30 minutes, then wash three times with PBS (5 minutes each time). DAB colorimetry: Add fresh DAB substrate and monitor the colorimetric reaction under a microscope.

[0287] (5) Re-staining and differentiation

[0288] Stain with hematoxylin for 5 minutes, then differentiate with 0.1% hydrochloric acid ethanol for a few seconds. Rinse with running water to regain blue color, then rinse with distilled water.

[0289] (6) Dehydrated and transparent

[0290] Gradient dehydration with mounting: 90% ethanol, 95% ethanol for 10 seconds each, then 100% ethanol for 5 minutes. Clearing: Immersion in xylene for 5 minutes. Mounting: Mount with neutral resin, observe and acquire images under an optical microscope.

[0291] 5. Western blot detection of protein expression

[0292] (1) Protein sample preparation

[0293] 1) Preparation of lysis working buffer: Take 1 mL of pre-chilled lysis buffer, add 10 μL of phosphatase inhibitor, PMSF, and protease inhibitor sequentially, and mix well on ice. 2) Tissue processing: Weigh 100-200 mg of fresh tissue, flash-freeze in liquid nitrogen, grind into powder, and transfer to the lysis buffer. 3) Lyse on ice for 50 minutes, vortexing for 30 seconds every 10 minutes. 4) Centrifuge at 12000 × g for 20 minutes at 4℃, and centrifuge the supernatant again. 5) Aliquot and store at -80℃; determine protein concentration using the BCA method.

[0294] (2) Electrophoretic analysis

[0295] 1) Prepare an SDS-PAGE gel of appropriate concentration according to the molecular weight of the target protein. 2) Mix the protein sample with the loading buffer in the correct ratio and denature at 100°C for 10 minutes. 3) Load 10 μL (containing 40 μg of protein) into each well and add the pre-stained marker. Electrophoresis conditions: constant voltage 100V, until bromophenol blue reaches the bottom of the gel.

[0296] (3) Transfer operation

[0297] 1) Activate the PVDF membrane with methanol for 2 minutes. 2) Transfer sandwich assembly (negative electrode → filter paper → membrane → gel → filter paper → positive electrode). 3) Wet transfer conditions: 25V constant voltage, 1.2A current, transfer time 30 minutes.

[0298] (4) Immunological testing

[0299] 1) Blocking: Block with 5% skim milk at room temperature for 2 hours. 2) Primary antibody incubation: After rinsing with TBST, incubate overnight at 4°C. 3) Secondary antibody incubation: Incubate at room temperature for 2 hours. 4) Development: Detect using an ECL chemiluminescence system.

[0300] 6. ELISA experiment

[0301] 1) Pre-experiment preparation: The reagent plate should be equilibrated at room temperature for 20 minutes before use. Unused strips should be sealed and stored at 4℃. 2) Sample loading procedure: Standard curve preparation: Add 50 μL of serially concentrated standards to each standard well. Sample processing: Mix 10 μL of the test sample with 40 μL of sample diluent and add to the detection well. Set up blank control wells, adding only an equal volume of buffer. 3) Immunoassay: Except for the blank wells, add 100 μL of HRP-labeled secondary antibody working solution to each well. After sealing, incubate at 37℃ for 1 hour. 4) Washing process: Discard the liquid in the wells. Add 300 μL of washing buffer to each well, let stand for 3 minutes, then shake off the excess. Repeat 5 times to complete the washing. 5) Colorimetric reaction: Add 50 μL each of equal volumes of TMB substrate solution A and solution B sequentially. Incubate at 37℃ in the dark for 20 minutes. 6) Termination and reading: Add stop solution to each well to terminate the reaction. Immediately read the absorbance of each well at 450 nm using a microplate reader. 7) Result calculation: Plot a standard curve with the concentration of the standard as the x-axis and the corresponding OD value as the y-axis, and calculate the sample concentration through the regression equation.

[0302] 7. Real-time quantitative PCR experiment

[0303] (1) RNA extraction

[0304] 1) Take 100 mg of the tissue sample stored in liquid nitrogen and grind it into powder using a mortar and pestle. Transfer the powder to a 1.5 mL Eppendorf tube and add 1 mL of lysis buffer for lysis. The tissue homogenate should be thoroughly shaken and incubated at room temperature for 40 minutes to ensure complete lysis of ribosomes.

[0305] 2) Centrifuge the lysis buffer at 12,000 rpm for 5 minutes at 4°C, and then transfer the supernatant to a new RNase-free centrifuge tube.

[0306] 3) Add 0.2 mL of chloroform solution to each 1 mL of lysis buffer. Tightly cap the centrifuge tube, vortex for 20 seconds, and then incubate at room temperature for 3 minutes.

[0307] 4) Remove the supernatant and add 300 μL of chloroform solution. Shake for 29 seconds, then vortex for 20 seconds to ensure thorough mixing. Let stand on ice for 15 minutes, then centrifuge at 12000 rpm for 10 minutes.

[0308] 5) After centrifugation, the sample becomes three layers. Transfer the upper aqueous phase (containing RNA) to a new centrifuge tube, add 500 μL of isopropanol, shake until there is no oil, let stand on ice for 15 min, centrifuge at 12000 rpm for 20 min, and remove the supernatant.

[0309] 6) Add 1 mL of pre-cooled 75% ethanol, gently tap the white precipitate in the centrifuge tube to let it float up, let it stand at room temperature for about 2 minutes, centrifuge at 12000 rpm for 10 minutes, aspirate the supernatant, and let it air dry at room temperature.

[0310] 7) Add 40 μL of enzyme-free water to the tube and repeatedly blow and mix to accelerate dissolution;

[0311] 8) Measure the concentration of RNA, record and save it.

[0312] (2) Reverse transcription

[0313] According to the reverse transcription kit instructions, add 2 μg RNA, 3 μL 5×g DNA digesterMix, and enzyme-free water to a PCR tube to a final volume of 15 μL. Centrifuge to mix and place in a PCR instrument (42℃, 2 min). Remove from the instrument and add 5 μL of 4×Hifair III superMix plus, mix well, and place back in the PCR instrument. Set the reverse transcription reaction program to 25℃, 5 min; 55℃, 15 min; 85℃, 5 min; 12℃. The reverse transcription product can be used for q-PCR or stored at -80℃.

[0314] (3) Real-time quantitative PCR

[0315] The real-time quantitative PCR system consisted of 20 μL: 10 μL Mix, 7 μL double-distilled water, 2 μL cDNA, and 0.5 μL forward / reverse primers (primer sequences are shown in Table 5). The PCR instrument reaction program was: 95℃ for 20 s, 60℃ for 30 s, and 72℃ for 30 s (40 cycles). Afterwards, [the PCR was performed using...]. Data obtained from analysis using 96 software and the relative quantitative 2-ΔΔCt method.

[0316] Table 5. Primer sequence listing

[0317]

[0318] 8. Immunofluorescence staining experiment

[0319] 1) Section pretreatment: Tissue sections are baked at a constant temperature of 65℃ for 120 minutes to improve the adhesion between the tissue and the glass slide.

[0320] 2) Dewaxing and hydration with xylene: 3 times, 5 minutes each time; gradient ethanol hydration: 100% → 90% → 80% → 70%, 5 minutes per stage; rinsing with distilled water: 5 minutes × 3 times.

[0321] 3) Antigen retrieval treatment: Endogenous enzyme blockade: 3% H2O2 treatment at room temperature for 10 minutes. Heat-induced antigen retrieval: Microwave retrieval in pH 6.0 sodium citrate buffer for 5 minutes. Membrane permeability treatment: Incubation in 0.3% Triton X-100 at room temperature for 20 minutes.

[0322] 4) Immunostaining: Blocking: Block with 10% goat serum at room temperature for 120 minutes. Primary antibody: Incubate at 4°C for 16 hours (antibody diluted with 1% serum). Secondary antibody: Incubate at room temperature in the dark for 120 minutes.

[0323] 5) Counterstaining and mounting: Stain with Hoechst 33342 for 20 minutes; observe immediately after mounting with anti-quenching agent.

[0324] 9. Statistical Analysis

[0325] For specific steps, please refer to Example 2.

[0326] III. Experimental Results

[0327] (1) Investigation on homing ability of hUC-MSCs in IBD mice

[0328] The distribution and survival of near-infrared fluorescent dye-labeled hUC-MSCs in vivo can be monitored in real time using in vivo imaging technology. Experimental results are as follows: Figure 7 As shown, in both the control and treatment groups, the bioluminescent signal of labeled hUC-MSCs exhibited a time-dependent decreasing trend, and in vivo imaging revealed that hUC-MSCs could specifically home to the site of intestinal injury. The signal intensity of hUC-MSCs decreased significantly on day 5, and only a weak signal could be detected by day 9. This result confirms that transplanted hUC-MSCs have the ability to migrate directionally to the site of intestinal injury in vivo, but their survival time is limited.

[0329] (2) Evaluation of the therapeutic effect of hUC-MSCs on IBD mice

[0330] like Figure 8 -A~ Figure 8As shown in Figure -D, compared with the DSS model group, both the DSS+hUC-MSCs treatment group and the DSS+hUC-MSCs+5-ASA combination treatment group significantly improved DSS-induced colitis symptoms. Specifically, the trend of weight loss in mice was significantly alleviated, the Disease Activity Index (DAI) score was significantly reduced, and the degree of colonic length shortening was significantly improved. Among them, the combination treatment group showed the most significant effect, with better weight recovery, colonic length maintenance, and DAI score improvement than the hUC-MSCs monotherapy group. HE staining results are shown below. Figure 8 As shown in Figure -E, compared with the DSS model group, the colon tissue structure of mice in the hUC-MSCs treatment group and the combined treatment group was relatively intact, with milder mucosal damage and reduced mononuclear cell infiltration. Histopathological scores are as follows: Figure 8 As shown in Figure -F, the pathological scores in the hUC-MSCs treatment group were significantly lower than those in the DSS model group, while the pathological scores in the combination therapy group were further reduced, significantly better than those in the hUC-MSCs monotherapy group. This invention uses 5-ASA as a positive control, confirming the reliability of this experimental model. The efficacy of the hUC-MSCs monotherapy group was comparable to that of 5-ASA, while the combination therapy group showed a synergistic therapeutic effect, providing experimental evidence for the treatment of IBD with hUC-MSCs.

[0331] Myeloperoxidase (MPO) activity assays showed that, compared with the DSS model group, MPO levels were significantly lower in both the hUC-MSCs treatment group and the combination therapy group, with a more pronounced decrease in the combination therapy group. (See details...) Figure 9 -A.

[0332] The expression of Ki67, endothelial nitric oxide synthase (eNOS), and occludin was used to assess cell proliferation, colonic mucosal angiogenesis, and intestinal epithelial integrity, respectively. Immunohistochemical results are shown below. Figure 9 -C and Figure 9 As shown in Figure D, the number of Ki67-positive cells in the hUC-MSCs treatment group and the combined treatment group was significantly higher than that in the DSS model group, indicating that MSC treatment can promote the proliferation of intestinal epithelial cells. Western blot results are as follows: Figure 9 As shown in Figure B, compared with the DSS model group, the expression levels of endothelial nitric oxide synthase (eNOS) and occludin were significantly increased in the hUC-MSCs treatment group and the combined treatment group, indicating that hUC-MSCs treatment can promote colonic mucosal angiogenesis and improve intestinal epithelial barrier function, with the combined treatment group showing the most significant effect.

[0333] (3) Investigation on the effect of hUC-MSCs treatment on the repair of intestinal barrier function in IBD mice

[0334] Gut microbiota and their metabolites, acting as antigens, can disrupt the integrity of the intestinal mucosal barrier, alter the abundance of opportunistic pathogens and dominant bacteria, thereby inducing autoimmune responses and the development of intestinal dysplasia (IBD). To investigate whether hUC-MSCs can repair intestinal barrier function in colitis mice, the expression levels of tight junction proteins (claudin-3) and Zonulaoccludens-1 (ZO-1) in the colon and the level of serum lipopolysaccharide (LPS) were measured. The results are as follows: Figure 10 As shown in the figure. Western blot and immunofluorescence staining results showed that, compared with the DSS model group, the expression levels of claudin-3 and ZO-1 in the colon tissue of mice in the hUC-MSCs treatment group and the combined treatment group were significantly increased. The increase in claudin-3 and ZO-1 expression levels was more pronounced in the combined treatment group, indicating that MSC treatment can effectively repair the tight junction structure of the intestine. See details... Figure 10 -A~ Figure 10 -C. ELISA results showed that, compared with the DSS model group, the serum LPS levels in mice treated with hUC-MSCs and in the combined treatment group were significantly reduced. (See details...) Figure 10 -D. Among them, the combined treatment group showed the greatest reduction in LPS levels, suggesting that hUC-MSCs treatment can effectively improve intestinal barrier permeability.

[0335] (4) Effects of hUC-MSCs treatment on intestinal immune response cytokines in IBD mice

[0336] Cytokines (including pro-inflammatory and anti-inflammatory factors) are key effector molecules in changes in intestinal immune homeostasis. Using ELISA to detect the levels of inflammatory factors in mouse serum, compared with the DSS group, the levels of pro-inflammatory factors tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the serum of mice in the hUC-MSCs group and the combined treatment group were decreased, with a more significant decrease in pro-inflammatory factors in the combined treatment group. Conversely, the expression levels of anti-inflammatory factors TGF-β and IL-10 were increased, with a more significant increase in pro-inflammatory factors in the combined treatment group. (See details...) Figure 11 -A and Figure 11 -C.

[0337] Western blot analysis of inflammatory factors in mouse colon tissue revealed changes in inflammatory factor expression similar to those in serum. The expression levels of TNF-α, IL-6, and IL-1β were decreased in the colon of mice in both the hUC-MSCs group and the combined treatment group. Furthermore, the expression of pro-inflammatory factors was even lower in the colon of the combined treatment group. Conversely, the expression levels of anti-inflammatory factors TGF-β and IL-10 increased, with a more pronounced increase in pro-inflammatory factors in the combined treatment group (see details). Figure 11 -B andFigure 11 -D.

[0338] (5) Effects of hUC-MSCs treatment on the NF-κB / STAT3 signaling pathway in IBD mouse models

[0339] NF-κB is a protein molecule with multipotent regulatory functions, participating in the regulation of gene expression of various factors and playing an important role in immune regulation, inflammation, stress response, and apoptosis. Western blot results are as follows... Figure 12 As shown, compared with the normal group, the phosphorylation levels of IκBα, p50, p65, and STAT3 were increased in the model group, while the phosphorylation levels were decreased in the hUC-MSCs treatment group and the combination therapy group compared with the model group.

[0340] The results of the Q-PCR experiment are as follows: Figure 13 As shown, compared with the DSS group mice, the expression levels of related genes (MYD88, TLR4, p65, p50) in the hUC-MSCs group and the combined drug administration group decreased, thereby achieving the effect of inhibiting the inflammatory response, indicating that the activation of the NF-κB / SATAT3 signaling pathway in colitis mice was inhibited.

[0341] IV. Summary and Discussion

[0342] This study established a DSS-induced IBD mouse model to examine the homing ability of hUC-MSCs in vivo and explore the therapeutic effect and potential mechanism of hUC-MSCs on IBD. The results showed that hUC-MSCs alone effectively alleviated clinical symptoms in IBD mice, improved colonic tissue pathological damage, repaired intestinal barrier function, inhibited inflammatory responses, and regulated the NF-κB / STAT3 signaling pathway. Furthermore, the combination of hUC-MSCs and mesalazine showed a more significant therapeutic effect, suggesting a possible synergistic effect.

[0343] In vivo bioluminescence imaging showed that hUC-MSCs could home to the colonic injury sites in IBD mice and survive locally. This indicates that hUC-MSCs have the ability to target inflammatory sites, providing important evidence for their application in IBD treatment. In both the hUC-MSCs monotherapy and combination therapy groups, the clinical symptoms and weight loss trends in IBD mice were significantly alleviated, the Disease Activity Index (DAI) score was significantly reduced, and the degree of colonic length shortening was significantly improved. Impaired intestinal barrier function is one of the important characteristics of IBD. Western blot and immunofluorescence staining results showed that the expression levels of colonic tight junction proteins claudin-3 and ZO-1 were significantly increased and serum LPS levels were significantly decreased in both the hUC-MSCs monotherapy and combination therapy groups. This indicates that hUC-MSCs can repair intestinal barrier function, reduce leakage of intestinal contents, and thus alleviate the inflammatory response. The expression levels of colonic tight junction proteins and serum LPS levels were further increased and decreased in the combination therapy group, suggesting that the combination therapy may have a synergistic effect. hUC-MSCs may alleviate inflammatory responses by promoting tight junction protein expression, repairing intestinal barrier function, and reducing intestinal leakage. Inflammation is a core pathological process in IBD. ELISA and Western blot results showed that the expression levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly reduced in serum and colon of mice treated with hUC-MSCs alone and in combination with other drugs, while the expression levels of anti-inflammatory cytokines TGF-β and IL-10 were significantly increased. This indicates that hUC-MSCs can regulate inflammatory responses, inhibit the expression of pro-inflammatory cytokines, and promote the expression of anti-inflammatory cytokines, thereby exerting an anti-inflammatory effect. The combined drug group showed a further decrease in the expression levels of pro-inflammatory cytokines and a further increase in the expression levels of anti-inflammatory cytokines, suggesting a possible synergistic effect. hUC-MSCs may suppress inflammatory responses by secreting anti-inflammatory cytokines, inhibiting the expression of pro-inflammatory cytokines, and regulating immune cell function. hUC-MSCs may also alleviate inflammatory responses by secreting IL-10 or TGF-β to inhibit the activation of macrophages and T cells. The NF-κB / STAT3 signaling pathway plays a crucial role in the pathogenesis of IBD. Western blot and Q-PCR results showed significant changes in the expression levels of p-IκBα, p-p50, p-p65, and p-STAT3 proteins, as well as the gene expression levels of MYD88, TLR4, p65, and p50 in the colon of mice treated with hUC-MSCs alone and in combination therapy. The changes in the expression levels of NF-κB / STAT3 signaling pathway-related proteins and genes were more pronounced in the combination therapy group, suggesting a possible synergistic effect of the combination therapy. hUC-MSCs may exert their therapeutic effect by regulating the NF-κB / STAT3 signaling pathway and influencing downstream gene expression.hUC-MSCs may reduce the expression of pro-inflammatory factors by inhibiting NF-κB activation, or promote the expression of anti-inflammatory factors by activating STAT3.

[0344] Example 4. Effects of hUC-MSCs treatment on the diversity and composition of gut microbiota in IBD mice

[0345] I. Experimental Methods

[0346] 1. Fecal collection

[0347] Fresh feces were collected from each group of mice. After wiping the mice's anus with alcohol, the fresh feces were collected with sterilized tweezers and placed in sterile test tubes. The feces were stored in a refrigerator at -80°C.

[0348] 2. 16S rDNA sequencing

[0349] 1) Sample processing and sequencing: Fecal genomic DNA was extracted using the TruSeq DNAPCR-Free library preparation kit. Specific primers were designed to amplify the V3-V4 variable region of the 16S rRNA gene, and sequencing libraries were constructed. The quality of the libraries was assessed using a Qubit quantitative PCR instrument and real-time quantitative PCR. Qualified libraries were sequenced at both ends using the NovaSeq 6000 sequencing platform.

[0350] 2) Data quality control and processing: Raw sequencing data (Raw reads) undergo the following processing steps: paired-end sequence assembly, quality filtering (Q20≥90%), chimeric sequence removal, to obtain high-quality valid data (Clean reads) for subsequent analysis.

[0351] 3) Bioinformatics analysis: The intestinal microbiota of each group was analyzed, the changes in the microbiota among the mice in each group were calculated and analyzed, and correlation analysis was performed.

[0352] 3. Analysis of gut microbiota diversity and composition

[0353] 1) Sequence Analysis and Denoising: The DADA2 algorithm was used to denoise the sequencing data, and a taxonomic unit matrix was constructed based on amplicon sequence variants (ASVs). Compared with traditional OTU clustering methods, ASV technology can provide higher resolution sequence variant information.

[0354] 2) α-Diversity analysis: The following indicators were calculated using the QIIME2 platform: Chao1 index (community richness), Shannon index (community diversity), and Simpson index (community evenness). Dilution curves were plotted to assess whether the sequencing depth was sufficient.

[0355] 3) β-diversity analysis: Calculate the unweighted UniFrac distance matrix and visualize intergroup differences using multivariate statistical methods: Principal Component Analysis (PCA), Principal Coordinate Analysis (PCoA), and Non-Metric Multidimensional Scaling (NMDS).

[0356] 4) Based on the ASV annotation results and the ASV abundance tables of each sample, obtain species abundance tables at the kingdom, phylum, class, order, family, genus and species levels, and conduct species composition analysis of different samples (groups) for different levels of species abundance tables.

[0357] 4. Statistical Analysis

[0358] Data analysis was performed using GraphPad Prism 5 statistical software. Independent samples t-tests were used for intergroup comparisons, with a p-value < 0.05 considered statistically significant. All statistical analyses were two-tailed tests, and data visualization was performed using GraphPad Prism 5 software.

[0359] II. Experimental Results

[0360] (1) Effects of hUC-MSCs treatment on gut microbiota diversity in IBD mice

[0361] Gut microbiota dysbiosis is a key characteristic of IBD. The gut microbiota plays a crucial role in the occurrence and development of IBD through various mechanisms, including regulating immune responses and maintaining intestinal barrier function. To investigate the effect of hUC-MSCs treatment on gut microbiota diversity in IBD mice, fecal samples from each group were sequenced using 16S rDNA. The α-diversity dilution curves are shown below. Figure 14 As shown, this indicates that after sequencing depth >40,000, the curve gradually flattens out, providing effective and reliable data for experimental analysis. Alpha diversity indices (Chao1, Shannon, and Simpson indices) are used to assess species richness and evenness within the sample. To represent species richness, bar charts of the observed_otus, Shannon, and Simpson indices were created. Figure 15-17 As shown, the species diversity in the control group was significantly higher than that in the model group, indicating that the gut microbiota of normal mice was richer than that of mice with colitis. The species diversity in the hUC-MSCs group and the combined treatment group was significantly higher than that in the model group, suggesting that hUC-MSCs transplantation altered the gut microbiota and increased its richness.

[0362] To assess the complexity of community composition and compare differences between groups, β-diversity was calculated. Results are as follows: Figure 18 -A~ Figure 18As shown in -C, three weighted PCA models, PCoA model, and NMDS visualized the differences between samples in complex multidimensional data. The results showed a significant separation of the gut microbiota structure between the normal group and colitis mice, and separation was also observed between the DSS group and the hUC-MSCs group, and between hUC-MSCs+5-ASA. However, the community composition was more similar between the normal group mice and the hUC-MSCs treatment group, and between the hUC-MSCs combined treatment group.

[0363] Furthermore, based on the ASVs obtained from noise reduction, common and unique ASVs among the groups were analyzed. The results are as follows: Figure 18 As shown in Figure -D, the number of ASV overlaps between the hUC-MSCs treatment group and the normal group was 1.9 times that of the model group and the normal group (117:61), and the number of ASV overlaps between the hUC-MSCs combined treatment group and the normal group was 3 times that of the model group and the normal group (175:57), indicating that hUC-MSCs can restore the dysregulated microbiota in colitis mice to some extent.

[0364] (3) Effects of hUC-MSCs treatment on gut microbiota composition in IBD mice

[0365] To identify which bacteria are altered by hUC-MSCs treatment and thus affect the progression of DSS-induced colitis, differences in the populations and abundance of the top 20 phyla, top 30 families, and top 30 genera were assessed among the four groups.

[0366] like Figure 19 As shown, at the phylum level, Firmicutes, Bacteroidetes, and Proteobacteria are the major phyla in the fecal microbiota of healthy mice. Compared with the control group, the Firmicutes phylum was reduced in the model group, while Bacteroidetes and Proteobacteria were increased. In the hUC-MSCs and combined treatment groups, Firmicutes increased, while Bacteroidetes and Proteobacteria decreased. Furthermore, the microbiota restoration was more significant in the combined treatment group.

[0367] like Figure 20 As shown, at the family level, Trichophyceae, Bacteroidetes, Lactobacillusaceae, and Akkermansiaceae are the major families in the fecal microbiota of healthy mice. Compared with the normal group, the model group mice showed a decrease in Trichophyceae and Akkermansiaceae, while Bacteroidetes and Lactobacillusaceae increased. In the hUC-MSCs or combination treatment groups, the mice showed an increase in Trichophyceae and Akkermansiaceae, while the Bacteroidetes and Lactobacillusaceae decreased. Furthermore, the microbiota recovery was more significant in the combination treatment group.

[0368] like Figure 21As shown, at the genus level, *Ackermania myxophilus*, *Trichophyton*, *Muribaculaceae_unclassified*, and *Lactobacillus* are the major phyla in the fecal microbiota of healthy mice. Compared with the normal group, the model group mice showed a decrease in *Ackermania myxophilus*, *Trichophyton*, and *Lactobacillus*, while an increase in *Muribaculaceae_unclassified*. In the hUC-MSCs or combination treatment groups, the levels of *Ackermania myxophilus*, *Trichophyton*, and *Lactobacillus* increased, while *Muribaculaceae_unclassified* decreased. Furthermore, the microbiota restoration was more pronounced in the combination treatment group.

[0369] IV. Summary and Discussion

[0370] Gut microbiota dysbiosis is one of the important characteristics of IBD. This study analyzed the effects of hUC-MSCs treatment on the diversity and composition of the gut microbiota in IBD mice using 16S rDNA sequencing technology. The results showed that hUC-MSCs treatment significantly improved gut microbiota dysbiosis in IBD mice, restored gut microbiota diversity, and regulated the abundance of specific bacterial groups. α-diversity analysis: α-diversity indices (Chao1, Shannon, and Simpson indices) were used to assess species richness and evenness within samples. Compared with the model group, the Chao1, Shannon, and Simpson indices of the gut microbiota in the hUC-MSCs treatment group were significantly increased, indicating that hUC-MSCs treatment significantly increased the species richness and evenness of the gut microbiota in IBD mice, restoring gut microbiota diversity. β-diversity analysis: β-diversity analysis was used to assess differences in community composition between samples. PCoA and NMDS analyses showed that the gut microbiota composition of mice in the hUC-MSCs treatment group was significantly different from that in the model group and closer to that of the normal control group, indicating that hUC-MSCs treatment can significantly alter the gut microbiota composition of IBD mice, bringing it closer to a normal state. Phylum-level microbiota composition changes: Experimental data showed that the gut microbiota structure of the model group mice underwent significant changes at the phylum level. Compared with the healthy control group, the relative abundance of Bacteroidetes in the model group was significantly decreased, while the proportion of Firmicutes was significantly increased. After hUC-MSCs intervention, this microbiota imbalance was significantly improved, with a recovery in the number of Bacteroidetes and a decrease in the proportion of Firmicutes, with the ratio approaching normal levels. Family-level microbiota characteristics changes: In family-level analysis, the model group exhibited obvious microbiota dysbiosis characteristics. Specifically, the abundance of beneficial bacteria such as Lachnospiraceae and Akkermansiaceae was significantly reduced, while the number of potentially pathogenic bacteria such as Bacteroidaceae and Lactobacillaceae was significantly increased. hUC-MSCs treatment effectively regulated this abnormal state, promoting the proliferation of beneficial bacteria while inhibiting the overgrowth of opportunistic pathogens. Genus-level flora distribution characteristics: Further analysis showed that at the genus level, the number of gut-protective genera such as Akkermansia, Lachnospira, and Lactobacillus was significantly reduced in the model group, while potentially harmful bacteria such as unclassified Muribaculaceae were significantly increased. hUC-MSCs treatment significantly restored the abundance of these beneficial genera while reducing the proportion of opportunistic pathogens. hUC-MSCs treatment significantly altered the gut microbiota composition of IBD mice, bringing it closer to a normal state.It can increase the abundance of beneficial bacteria, which can produce short-chain fatty acids (SCFAs), such as acetic acid, propionic acid, and butyric acid, and have anti-inflammatory effects, maintain intestinal barrier function, and regulate immune responses. It can also reduce the abundance of opportunistic pathogens, which may proliferate excessively when there is intestinal flora imbalance, producing toxins, damaging intestinal barrier function, and inducing inflammatory responses.

[0371] Example 5. Investigating whether gut microbiota participates in the therapeutic effect of hUC-MSCs on IBD mice.

[0372] I. Key Reagents

[0373] Broad-spectrum antibiotic mixture: (ABX: vancomycin 100 mg / kg; neomycin sulfate 200 mg / kg; metronidazole 200 mg / kg; ampicillin 200 mg / kg) (7 mL): Dissolve 2 mg vancomycin in 1 mL PBS buffer, 4 mg neomycin sulfate in 2 mL PBS buffer, 4 mg metronidazole in 2 mL PBS buffer, and 4 mg ampicillin in 2 mL PBS buffer. Combine 1 mL vancomycin solution, 2 mL neomycin sulfate solution, 2 mL metronidazole solution, and 2 mL ampicillin solution in a sterile centrifuge tube and filter the mixture using a 0.22 μm sterile filter.

[0374] Fecal gavage solution: Mix feces with sterile saline at a ratio of 1:5, adding 50 mL of saline to 10 g of feces. Stir thoroughly until a suspension is formed. Centrifuge at 3000 rpm for 10 minutes and collect the supernatant. Aliquot the supernatant into sterile centrifuge tubes and store at 4°C for a short period (not exceeding 24 hours), or freeze at -80°C after adding a cryoprotectant (such as glycerol).

[0375] The preparation of the remaining reagents is the same as in Examples 2 and 3.

[0376] II. Experimental Methods

[0377] (1) Fecal microbial transplantation of mice treated with hUC-MSCs

[0378] Wild-type mice were administered a broad-spectrum antibiotic (ABX) via gavage for five consecutive days to eliminate intestinal microbiota. Fecal filtrate from the control group, DSS group, DSS+hUC-MSCs group, and DSS+hUC-MSCs+5-ASA combined treatment group was transferred into ABX mice via gavage once daily for five days. After a three-day interval, the mice were given free access to 3% DSS for ten days, followed by three days of sterile water before sacrifice for sample collection. Experimental grouping and treatment are as follows. Figure 22 As shown.

[0379] (2) Other experiments

[0380] HE staining, disease activity index, pathological histological scores, and statistical analysis were all performed according to Example 2; the degree of mucosal neutrophil infiltration, MPO activity detection, immunohistochemical staining, and Western blotting for protein expression were all performed according to Example 3.

[0381] III. Experimental Results

[0382] To investigate whether gut microbiota participates in the ameliorative effect of hUC-MSCs on colitis, this study validated this effect using a fecal microbiota transplantation (FMT) experiment. Wild-type mice were administered a broad-spectrum antibiotic (ABX) via gavage for 5 days to eliminate gut microbiota. Subsequently, fecal suspensions from the control group, DSS group, hUC-MSCs group, and combined treatment group (DSS+hUC-MSCs+5-ASA) were transplanted into ABX mice via gastric administration once daily for 5 days. Three days after transplantation, mice were given free access to 3% DSS solution for 10 days. Three days after the modeling process was completed, mice were sacrificed and samples were collected for analysis. Results are as follows: Figure 23 As shown, compared with the FMT(DSS) group, the FMT(DSS+hUC-MSCs) group and the FMT(DSS+hUC-MSCs+5-ASA) group showed significantly reduced weight loss, significantly lower Disease Activity Index (DAI) scores, and significantly improved colon length shortening. Among them, the FMT(DSS+hUC-MSCs+5-ASA) group showed the most significant improvement.

[0383] HE staining results are as follows Figure 24 As shown in Figure -A. The results indicate that, compared to the FMT(DSS) group, the colonic tissue structure of mice in the FMT(DSS+hUC-MSCs) and FMT(DSS+hUC-MSCs+5-ASA) groups was relatively intact, with significantly reduced mucosal damage and a marked decrease in mononuclear cell infiltration. Histopathological scores are as follows: Figure 24 As shown in Figure -B, the scores of the FMT(DSS+hUC-MSCs) group and the FMT(DSS+hUC-MSCs+5-ASA) group were significantly lower than those of the FMT(DSS) group, with the combined treatment group showing more significant improvement. Myeloperoxidase (MPO) activity test results are as follows... Figure 24 As shown in Figure C, the MPO levels in the FMT(DSS+hUC-MSCs) group and the FMT(DSS+hUC-MSCs+5-ASA) group were significantly lower than those in the FMT(DSS) group, indicating that gut microbiota transplantation after hUC-MSCs treatment can effectively inhibit neutrophil infiltration and reduce the inflammatory response in the IBD mouse model.

[0384] Besides localized inflammation, changes in the appearance of the intestinal epithelium directly affect its integrity. Immunohistochemical results are as follows:Figure 24 -D and Figure 24 As shown in Figure -E, the number of Ki67-positive cells in the FMT(DSS+hUC-MSCs) group and the FMT(DSS+hUC-MSCs+5-ASA) group was significantly higher than that in the FMT(DSS) group. Western blot results are as follows... Figure 24 As shown in Figure -F, the expression levels of endothelial nitric oxide synthase (eNOS) and occludin in the FMT(DSS+hUC-MSCs) and FMT(DSS+hUC-MSCs+5-ASA) groups were significantly higher than those in the FMT(DSS) group. This indicates that gut microbiota transplantation after hUC-MSCs treatment can promote colonic mucosal angiogenesis and improve intestinal epithelial barrier function.

[0385] In summary, fecal microbiota transplantation from mice treated with hUC-MSCs suppressed colitis in IBD mice. hUC-MSCs and combination therapy exert their therapeutic effects by modulating the gut microbiota.

[0386] IV. Summary and Discussion

[0387] This experiment investigated whether gut microbiota participates in the therapeutic effect of hUC-MSCs on IBD mice by constructing an antibiotic-induced gut microbiota depletion (ABX) mouse model and combining it with fecal microbiota transplantation (FMT). The results showed that gut microbiota plays an important role in the treatment of IBD mice with hUC-MSCs, and hUC-MSCs may exert their therapeutic effect by regulating gut microbiota.

[0388] Compared with the DSS group, ABX mice in the FMT (DSS+hUC-MSCs) and FMT (DSS+hUC-MSCs+5-ASA) groups showed significantly reduced weight loss, significantly less shortening of colon length, improved fecal morphology, reduced rectal bleeding, and significantly lower Disease Activity Index (DAI) scores. HE staining results showed that, compared with the FMT (Control) and FMT (DSS) groups, ABX mice in the FMT (DSS+hUC-MSCs) and FMT (DSS+hUC-MSCs+5-ASA) groups exhibited significantly reduced colonic mucosal structural damage, decreased inflammatory cell infiltration, increased goblet cell count, and significantly lower histopathological scores. This indicates that the FMT (DSS+hUC-MSCs) and FMT (DSS+hUC-MSCs+5-ASA) groups can effectively repair colonic mucosal damage in ABX mice. MPO activity assays showed that, compared with the FMT(Control) and FMT(DSS) groups, the colonic MPO activity of ABX mice in the FMT(DSS+hUC-MSCs) and FMT(DSS+hUC-MSCs+5-ASA) groups was significantly reduced, indicating decreased neutrophil infiltration. Immunohistochemical assays showed that, compared with the FMT(Control) and FMT(DSS) groups, the colonic Ki67 expression level of ABX mice in the FMT(DSS+hUC-MSCs) and FMT(DSS+hUC-MSCs+5-ASA) groups was significantly increased, indicating enhanced cell proliferation. Western blot analysis showed that, compared with the FMT(Control) and FMT(DSS) groups, the colonic eNOS expression level of ABX mice in the FMT(DSS+hUC-MSCs) and FMT(DSS+hUC-MSCs+5-ASA) groups was significantly increased, indicating enhanced angiogenesis. Western blot results showed that, compared with the FMT (Control) group and the FMT (DSS) group, the expression level of occludin in ABX mice in the FMT (DSS+hUC-MSCs) group and the FMT (DSS+hUC-MSCs+5-ASA) group was significantly increased, indicating that the integrity of the intestinal epithelium was improved.

Claims

1. The use of a pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease, characterized in that, The pharmaceutical composition comprises human umbilical cord mesenchymal stem cells and mesalazine.

2. The application according to claim 1, characterized in that, The human umbilical cord mesenchymal stem cells highly express CD73, CD90, and CD65, and lowly express CD34, CD45, and HLA-DR.

3. The application according to claim 1, characterized in that, The inflammatory bowel disease includes one or more of Crohn's disease and ulcerative colitis.

4. The application according to claim 1, characterized in that, The inflammatory bowel disease is characterized by weight loss, diarrhea, rectal bleeding, shortened colon, colonic mucosal damage, intestinal inflammation, impaired intestinal barrier function, and / or intestinal flora imbalance.

5. The application according to claim 1, characterized in that, The pharmaceutical composition is used to inhibit colonic shortening; And / or the pharmaceutical composition is used to inhibit weight loss caused by inflammatory bowel disease; And / or the pharmaceutical composition thereof is used to inhibit rectal bleeding caused by inflammatory bowel disease; And / or the pharmaceutical composition is used to reduce MPO activity in colonic tissue; And / or the pharmaceutical composition is used to promote the proliferation of intestinal epithelial cells; And / or the pharmaceutical composition thereof is used to promote colonic mucosal angiogenesis and improve intestinal epithelial barrier function; And / or the pharmaceutical composition is used to repair intestinal barrier function; And / or the pharmaceutical composition is used to inhibit intestinal inflammation; And / or the pharmaceutical composition thereof is used to inhibit the activation of the NF-κB / SATAT3 signaling pathway; And / or the pharmaceutical composition is used to regulate the gut microbiota.

6. The application according to claim 5, characterized in that, The intestinal barrier repair function includes promoting the expression of claudin-3 and ZO-1 in colon tissue to repair the tight junction structure of the intestine, and reducing serum LPS levels to improve intestinal barrier permeability.

7. The application according to claim 5, characterized in that, The inhibition of NF-κB / SATAT3 signaling pathway activation includes inhibiting the phosphorylation levels of IκBα, p50, p65, and STAT3, and reducing the expression levels of MYD88, TLR4, p65, and p50.

8. The application according to claim 5, characterized in that, The regulation of gut microbiota includes improving gut microbiota dysbiosis in IBD, restoring gut microbiota diversity, and regulating the abundance of beneficial bacteria. Specifically, this involves increasing Firmicutes and decreasing Bacteroidetes and Proteobacteria; increasing Trichophyceae and Akkermaniaceae and decreasing Bacteroidetes and Lactobacillusaceae; and increasing Akkermania, Trichophycetes, and Lactobacillus and decreasing Muribactaceae_unclassified.

9. A pharmaceutical composition for treating inflammatory bowel disease, characterized in that, The pharmaceutical composition comprises human umbilical cord mesenchymal stem cells and mesalazine.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is for combination therapy; or the pharmaceutical composition is prepared in the form of a compound drug in any pharmaceutically acceptable dosage form.

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