| Part I Therapeutic effect of bone marrow mesenchymal stem cells and their conditioned medium on DSS induced colitis in miceBackground:The traditional treatment methods for inflammatory bowel disease（IBD）are limited,making it difficult to maintain long-term remission of the disease and meet more systematic and deeper treatment goals for IBD.Bone marrow-derived mesenchymal stem cells（BMSCs）have shown good application prospects in the treatment of IBD due to their advantages such as immune regulation characteristics and regeneration ability.Therefore,cell therapy is being studied as a new treatment for IBD,but the specific mechanism has not yet been fully clarified.Because the therapeutic effect of BMSCs in injured tissues is mainly mediated by paracrine activity,conditioned medium（CM）generated by BMSCs also has similar potential for cell therapy.Currently,it has demonstrated therapeutic advantages in various disease models.Objective:To study the efficacy and potential mechanism of bone marrow mesenchymal stem cells and their conditioned medium enema on dextran sulfate sodium（DSS）induced experimental colitis in mice,and to provide theoretical basis for the treatment of inflammatory bowel disease using BMSCs and BMSCs-CM.Methods:1.Construction of green fluorescent protein labeled bone marrow mesenchymal stem cellsUsing plasmid transfer technology,the green fluorescent protein（GFP）gene was transcribed into BMSCs,and its expression was observed under a fluorescence microscope.After 2 days of cell enema treatment,the distribution of GFP positive BMSCs in the intestine of colitis mice was observed.2.Establishment and grouping of DSS induced experimental colitis model in miceAcute colitis models can be established in mice by freely drinking 2%DSS solution for 7 days.Thirty C57 female mice were randomly divided into 5 groups:control group（Control）,DSS modeling group（DSS）,bone marrow mesenchymal stem cell treatment group（DSS+BMSCs）,bone marrow mesenchymal stem cell conditioned medium treatment group（DSS+BMSCs-CM）,and bone marrow mesenchymal stem cell treatment+ML385 inhibitor group（DSS+BMSCs+ML385）.The mice in each group received a total of 2 intervention enemas on the 4th and 6th days of modeling.3.Efficacy indicators of BMSCs and BMSCs-CM on colitis in DSS mice（1）Observe the body weight,stool properties,and blood flow of mice daily,and calculate the disease activity index score.On the 8th day,the changes of colon length and spleen weight in each group were detected after the mice were killed.（2）HE staining was used to observe the condition of colonic tissue damage,and tissue damage scores were performed.（3）Masson staining assessed the distribution of collagen fibers in the colon,and tight junction protein ZO-1 assessed the mechanical barrier and permeability of the colon mucosal epithelium.（4）Immunohistochemical staining was used to detect the expression of inflammatory cells（macrophage marker CD68,neutrophil marker MPO）in each group.（5）Detection of inflammatory factors（IL-6,TNF-α,IL-1β,and IL-10）in each group by ELISA.（6）The expression of oxidative stress products such as O2-,OH,and MDA in colon tissue homogenate and the expression of enzyme antioxidant systems including T-SOD,CAT,GSH-Px,and T-AOC were detected.（7）Western blot was used to detect the molecular changes of Nrf2/Keap1/ARE axis（Nrf2,Keap1,HO-1,and NQO-1）and the expression level of apoptosis related proteins（Bax,Bcl-2,and Caspase3）in each group.Result:1.GFP positive BMSCs tend to accumulate in the inflammatory bowel,mainly distributed in the mucosa and submucosa of the lesion site.2.Both BMSCs and BMSCs-CM treatments were effective in reducing the colitis activity index score in mice,but the BMSCs group had better efficacy than the BMSCs-CM group（P<0.05）.Treatment with BMSCs can reduce the shortening of colon length and increase of spleen weight,and the efficacy of BMSCs disappears after combination with Nrf2inhibitor ML385.BMSCs-CM treatment had no effect on shortened colon length and increased spleen weight.3.HE staining suggests that both BMSCs and BMSCs-CM treatment can reduce mucosal damage,inflammatory cell infiltration,and crypt loss in intestinal tissue of colitis mice,but there is a statistical difference between the two groups.After adding the Nrf2 inhibitor ML385,the efficacy of BMSCs disappeared.4.Masson staining suggested that both BMSCs and BMSCs-CM treatment could reduce submucosal collagen deposition and increase the expression of ZO-1 protein in intestinal tissue of mice with colitis,but there was a statistical difference between the two groups.After adding the Nrf2inhibitor ML385,the above therapeutic effects of BMSCs disappeared.5.Both BMSCs and BMSCs-CM treatment can reduce the expression of MPO and CD68,but there is a statistical difference in the expression level of CD68 between the two groups.6.Both BMSCs and BMSCs-CM treatment can reduce TNF-αand IL-1β,there was no significant statistical difference between the two groups.BMSCs treatment can also reduce the level of IL-6,and BMSCs-CM has no significant impact on IL-6.Both groups had no significant impact on IL-10.The use of the Nrf2 inhibitor ML385 blocks the therapeutic effect of BMSCs.7.Both BMSCs and BMSCs-CM treatment can reduce the increase of oxidative stress products O2-,OH,and MDA,and increase the expression of antioxidant enzymes CAT,GSH-Px,and there is no significant statistical difference between the two groups.BMSCs treatment can reduce the expression of T-SOD and T-AOC,while BMSCs-CM has no significant effect on T-SOD and T-AOC.After adding ML385,the influence of BMSCs on the above molecules was inhibited.8.Both BMSCs and BMSCs-CM treatment can reduce the expression of proapoptotic proteins Bax and Caspase 3 and increase the expression of anti-apoptotic protein Bcl-2 in colon tissue of colitis mice,with no significant statistical difference between the two groups.BMSCs treatment can also increase the expression of Nrf2,HO-1,and NQO-1 in colon tissue,and reduce the expression of Keap1.The addition of ML385 inhibits the therapeutic effect of BMSCs.BMSCs-CM treatment did not significantly affect the expression of Nrf2,HO-1,NQO-1,and Keap1.Conclusion:1.After enema,bone marrow mesenchymal stem cells can directly nest in the damaged intestine,and reduce the occurrence of inflammation and oxidative stress in colon epithelial cells by activating the Nrf2/Keap1/ARE signaling pathway,thereby playing an effective role in treating experimental colitis.2.The conditioned medium of bone marrow mesenchymal stem cells can reduce the infiltration of cells and the occurrence of oxidative stress in the colon of mice with colitis to a certain extent,and its efficacy is not as good as that of bone marrow mesenchymal stem cells.It has no significant impact on the molecules related to the antioxidant stress Nrf2/Keap1/ARE signaling pathway.Part II The protective effect of modified conditioned medium of hydrogen peroxide pretreated bone marrow mesenchymal stem cells on DSS induced colitis in miceBackground:In the first part of the study,the inability of BMSCs-CM to meet the treatment needs of injured tissues may be attributed to the limited variety and low concentration of paracrine groups（including chemokines,growth factors,soluble cytokines,proteases,micro RNAs,and extracellular vesicles）in BMSCs-CM,which requires optimization.Previous studies have shown that pretreatment with low concentration hydrogen peroxide（H2O2）can not only protect cells from oxidative damage and enhance their antioxidant stress capacity,but also play a role similar to that of growth factors,regulating stem cell physiological processes.Therefore,H2O2pretreatment of BMSCs may be a way to obtain improved conditioned media for better application in the treatment of diseases.Objective:Using H2O2 to pretreat BMSCs and collect their conditioned media,we explored the optimal concentration of H2O2 pretreated BMSCs,and studied the efficacy and potential mechanism of H2O2 pretreated BMSCs-CM enema on DSS induced experimental colitis in mice.Method:1.ATP method,CCK-8,Crystal violet staining,Flow cytometry,and Western blot were used to study 5 different concentrations（25,50,100,200,400μM）effects of H2O2 pretreatment on the viability,proliferation,and apoptosis of BMSCs cells.2.After 25μM H2O2 pretreatment of BMSCs,the levels of intracellular oxidative stress products were detected,and the expression of apoptosis related and Nrf2/Keap1/ARE axis related proteins were detected by Western blot,as well as the changes after using the Nrf2 inhibitor ML385.3.Modified conditioned media for 25μM H2O2 pretreated BMSCs and conditioned media for BMSCs in normal environments were collected for animal experiments.Thirty C57 female mice were randomly divided into five groups:control group（Control）,DSS modeling group（DSS）,bone marrow mesenchymal stem cell conditioned medium treatment group（DSS+BMSCs-CM）,25μM H2O2 pretreatment of bone marrow mesenchymal stem cells conditioned medium treatment group （DSS+BMSCs-CM（H2O2））,and 25μM H2O2 pretreatment of bone marrow mesenchymal stem cells conditioned medium+ML385 inhibitor treatment group（DSS+BMSCs-CM（H2O2）+ML385）.The mice in each group received a total of 3 intervention enemas on the 2nd,4th,and 6th days of modeling.The evaluation indicators and methods are the same as those in Part I.Result:1.Both 25 and 50μM H2O2 pretreatment can significantly increase the cell viability and promote cell proliferation of BMSCs,while 25μM H2O2has a significantly better effect on promoting cell viability and proliferation,with statistical differences between the two groups.100μM H2O2pretreatment has no effect on cell viability and proliferation,but promotes cell apoptosis.When the concentration exceeds 200μM,cell viability and proliferation are inhibited,and apoptosis significantly increases.2.25μM H2O2 pretreatment can activate the Nrf2/Keap1/ARE signaling pathway in BMSCs without affecting the levels of oxidative stress products O2-,OH,and MDA.After administration of the Nrf2inhibitor ML385,the proliferation of BMSCs was inhibited,the rate of apoptosis and apoptosis-related proteins significantly increased,and the expression of oxidative stress products also significantly increased.3.Both BMSCs-CM（H2O2）and BMSCs-CM treatment can effectively reduce the disease activity index score in colitis mice,but the BMSCs-CM（H2O2）group has better efficacy than the BMSCs-CM group（P<0.05）.BMSCs-CM（H2O2）treatment can reduce the shortening of colon length and the increase of spleen weight,and the efficacy disappears after the combination of Nrf2 inhibitor ML385.BMSCs-CM treatment had no effect on shortened colon length and increased spleen weight.4.HE staining suggests that both BMSCs-CM（H2O2）and BMSCs-CM treatment can reduce mucosal damage,inflammatory cell infiltration,and crypt loss in intestinal tissue of mice with colitis,but there is a statistical difference between the two groups.After the addition of Nrf2inhibitor ML385,the efficacy of BMSCs-CM（H2O2）disappeared.5.Masson staining suggested that both BMSCs-CM（H2O2）and BMSCs-CM treatment could reduce submucosal collagen deposition and increase the expression of ZO-1 protein in intestinal tissue of mice with colitis,but there was a statistical difference between the two groups.After the addition of Nrf2 inhibitor ML385,the above therapeutic effects of BMSCs-CM（H2O2）disappeared.6.Both BMSCs-CM（H2O2）and BMSCs-CM treatment can reduce the expression of MPO and CD68,but there is a statistical difference in the expression level of CD68 between the two groups.7.Both BMSCs-CM（H2O2）and BMSCs-CM treatment can reduce IL-6,TNF-α,and IL-1β,there was no significant statistical difference between the two groups.Both groups had no significant impact on IL-10.The use of the Nrf2 inhibitor ML385 blocks the therapeutic effect of BMSCs.8.Both BMSCs-CM（H2O2）and BMSCs-CM treatment can reduce the increase of oxidative stress products O2-,OH,and MDA,and increase the expression of antioxidant enzymes CAT,GSH-Px,and there is no significant statistical difference between the two groups.BMSCs-CM（H2O2）treatment can also reduce the expression of T-SOD and T-AOC,but after adding ML385,the effect on the above molecules disappears.9.Both BMSCs-CM（H2O2）and BMSCs-CM treatment can reduce the expression of proapoptotic proteins Bax,Caspase 3,and Cleared Caspase3 in the colon tissue of colitis mice,and increase the expression of anti-apoptotic protein Bcl-2.There is no significant statistical difference between the two groups.In addition,BMSCs-CM（H2O2）treatment can increase the expression of Nrf2,HO-1,and NQO-1 in colon tissue,and reduce the expression of Keap1.After the addition of ML385,the therapeutic effect of BMSCs-CM（H2O2）is prevented.BMSCs-CM treatment did not significantly affect the expression of Nrf2,HO-1,NQO-1,and Keap1.Conclusion:1.25μM H2O2 is the optimal concentration for pretreatment of BMSCs,which can significantly increase cell viability and promote cell proliferation without significantly affecting their apoptosis.2.25μM H2O2 pretreatment of BMSCs-CM can treat intestinal mucosal damage in experimental colitis by activating the Nrf2/Keap1/ARE pathway in colon epithelial cells,and its efficacy is superior to that of untreated BMSCs-CM.With 15 figures,10 tables and 69 references... |
