Stem Cells Derived Microvesicles Attenuate Colitis And Associated Intestinal Fibrosis And The Synergistic Action Of Compound Sophorae Decoction

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Identification of Bone Marrow Mesenchymal Stem Cells derived microvesicles Objective:Obtain and culture bone marrow mesenchymal stem cells(BMSCs)by the method of whole bone marrow adherence.Harvest The BMSCs derived microvesicles(BMSCs-MVs)by serum starvation and ultracentrifugation.Methods:SD rats weighing 80-100 g were used to isolated and cultured BMSCs in aseptic conditions.The cells were identified by flow cytometry.The obtained BMSCs were treated by serum starvation and the supernatants were subjected to ultracentrifugation at low temperature to obtain BMSC-MVs,and then identified them by electron microscopy and flow cytometry.Results:Fusiform BMSCs were arranged in a vortex-like shape and grew well.Flow cytometry identified that the BMSCs were in high expression of CD29 and CD90,and low expression of CD45 and CD11b.BMSC-MVs were in round or oval shape,and their diameter was about 500 nm.Conclusion:BMSCs with high purity could be obtained by the methods of bone marrow adherent.They can be identified by flow cytometiy to meet the characteristics of BMSCs.BMSC-MVs could be obtained by serum starvation treatment combined with low temperature ultracentrifugation.The size of BMSC-MVs was consistent with the characteristics of microvesicles.Part ? Treatment of BMSC-MVs in experimental colitis and Synergistic Action of Compound sophorae decoctionObjective:To investigate the the anti-inflammatory,anti-oxidation and anti-apoptotic effects of BMSC-MVs on 2,4,6-trinitrobenzene sulfonic acid(TNBS)induced experimental colitis rat model and the synergistic effect of compound sophorae decoction.Methods:SD rats weighing 160-180 g were randomly divided into four groups:control group,model group,MVs group,MVs + compound sophorae decoction(TCM)group(combined group).TNBS/ethanol enema was used to establish experimental colitis rat model.Western blot and PCR were used to detect the expression of inflammatory cytokines(NF-?B,TNF-?,iNOS,COX-2,IL-1 ? and IL-10),reactive oxygen species(ROS)(MPO,MDA,GSH and SOD)and apoptosis markers(c-caspase3,c-caspase8 and c-caspase9)in the rats of each group,and the disease activity index was evaluated.Results:In the aspect of inflammatory,compared with the model group,the levels of mRNA and protein of inflammatory mediators such as NF-?B,TNF-?,iNOS,COX-2 were significantly decreased and the level of anti-inflammatory factor IL-10 increased in the MVs group and the combined group.In the aspect of oxidative stress,compared with the model group,the MVs group and the combined group could reduce the expression of oxidative media MPO and MDA,and increase the expression of the antioxidant media GSH and SOD.In the aspect of apoptosis,the expression of c-caspase3,c-caspase8 and c-caspase9 were also decreased in the MVs group and the combined group.Compared with the MVs group,the combined group got a better effect in the treatment of experimental colitis.Conclusion:MVs could relieve experimental colitis induced by TNBS from anti-inflammatory,anti-oxidative and anti-apoptotic affects.Compound sophorae decoction could promote the effect of MVs in the colon.MVs played an important role in the treatment of experimental colitis and compound sophorae decoction had synergistic effect.Part ? miR-200b overexpressed MVs alleviates the deposition of extracellular in experimental colitis related colon fibrosis and Synergistic Action of Compound sophorae decoctionObjective:To investigate the effect of miR-200b overexpressed MVs on the deposition of extracellular in experimental colitis related fibrosis and the synergistic effect of compound sophorae decoction.Methods:The miR-200b-overexpressing lentiviral vectors were transfected into bone BMSCs.miR-200b overexpressed BMSCs were selected by drug sieving.After serum starved,the supernatant was subjected to ultracentrifugation at low temperature.The miR-200b was overexpressed in BMSC-MVs which identified by PCR.The rats were randomly divided into 5 groups:control group,model group,null-MVs group,miR-200b-MVs group,miR-200b-MVs + compound sophorae decoction group(also called the combined group).The experimental colitis related intestinal fibrosis model was induced by multiple TNBS/ethanol enema.The colon tissues were collected for pathological analysis after 6 weeks,and the expressions of fibronectin,collagen ? and collagen? were detected by western blot.Result:MVs were successfully isolated and stably overexpressed miR-200b.The expression of fibronectin,collagen ? and collagen? were significantly increased in the model group.Compared with the model group,in the null-MVs group,the miR-200b-MVs group and the combined group,the expression of fibronectin,collagen ? and collagen?were down-regulated.The effect of the combined group was better than the miR-200b-MVs group.Conclusion:miR-200b overexpressed MVs could alleviate the deposition of extracellular in experimental colitis related colon fibrosis and compound sophorae decoction had a significant synergistic effect.Part ? The mechanism of miR-200b overexpressed MVs in alleviating experimental colitis associated intestinal fibrosis by inhibiting EMT and the synergistic effect of compound sophorae decoctionObjective:To investigate the mechanism of miR-200b overexpressed MVs in alleviating experimental colitis associated intestinal fibrosis and the synergistic effect of compound sophorae decoction.Methods:the experiment was divided into two parts:cell experiment and animal experiment.The animal model of experimental colitis related intestinal fibrosis was established by multiple TNBS/ethanol enema method.The rats were randomly divided into 5 groups:control group,model group,null-MVs group,miR-200b-MVs group,miR-200b-MVs + compound sophorae decoction group(also called the combined group).The expression of E-cad,vimentin,?-SMA,ZEB1 and ZEB2 were detected by Western blot,and the expression of E-cad,FSP-1 and ?-SMA were detected by immunofluorescence.In the cell experiment,IEC-6 cells were stimulated with TGF-P to induce EMT model.The cells were randomly divided into 4 groups:control group,model group,null-MVs group and miR-200b-MVs group.The morphological changes of cells were observed after adding null-MVs and miR-200b-MVs.The expression of E-cad,vimentin,ZEB1 and ZEB2 were detected by western blot.BMSC-MVs dyed by PKH26 were used to observe the location of null-MVs and miR-200b-MVs both in the intestine and in IEC-6 cells.Results:In rat model,we found that the MVs could reach the damaged colon,and compound sophorae decoction could have synergistic effect in improving the homing of MVs.compared with the control group,the expression of vimentin,a-SMA,ZEB1 and ZEB2 in the model group were increased and the expression of E-cad was decreased.After the treatment with miR-200b-MVs and compound sophorae decoction,the expression of vimentin,?-SMA,ZEB1 and ZEB2 decreased and the expression of E-cad increased.In the part of cell experiment,compared with the control group,the expression of vimentin,ZEB1 and ZEB2 increased and E-cad decrease in the model group.In the model of IEC-6 cells,we also got the similar results of miR-200b.After adding miR-200b-MVs,the expression of E-cad increased,and the expression of vimentin,ZEB1 and ZEB2 decreased,compared with the model group.Conclusion:miR-200b-MVs could promote the expression of epithelial cell marker E-cadherin and decrease the expression of vimentin,a-SMA and FSP-1,so as to inhibit the process of EMT.The mechanism may be related to the inhibition of ZEB1 and ZEB2.Compound sophorae decoction could have synergistic effect in this process.