The Study On The Mechanism Of Overexpression Of MiR-146a In Evs In Rats With UC And The Effect Of Compound Sophorae Decoction On Intestinal Barrier Function Via Notch Signaling

Part Ⅰ The Study on the Mechanism of Overexpression of miR-146 a in BMSC-EVs in Treating UCObjective: To study the mechanism attenuating UC by enhanced expression of miR-146 a in BMSC-EVs through TRAF6 /NF-κB signaling pathway.Methods: A rat model of acute UC was constructed with a single intrarectal administration of TNBS dissolved in ethanol.Bone-marrow mesenchymal stem cells(BMSCs)were transfected with recombinant lentiviruses overexpressing miR-146 a.The cultured medium was collected from these cells starved by subjecting them to basal media without serum for 48 h and the supernatant was collected.BMSC-EVs were harvested by differential centrifugation.The morphology and protein markers of BMSC-EVs were identified by transmission electron microscope and western blot,respectively.The colitis mice received BMSC-EVs overexpressing miR-146 a suspended in 1 m L PBS via tail vein injection at a dose of 100 μg per rat.Clinical symptoms of rats were monitored daily such as body weight,stool consistency and fecal bleeding.Proteins and m RNAs expression levels of TRAF6/NF-κB signaling pathway and related cytokines downstream were detected through ELISA,western blot and immunohistochemistry.Results: 1.Recombinant lentiviruses overexpressing miR-146 a were successfully constructed and these engineered lentiviruses were favourably transfected BMSCs.BMSCEVs were successfully isolated and served as a stable delivery system of miR-146 a.2.Increased miR-146 a expression packaged in BMSC-EVs can negatively regulate TRAF6 and IRAK1 in colons of rats with TNBS-induced colitis and there was significant difference.3.Elevated miR-146 a in BMSC-EVs can inhibit TNBS-induced inflammatory cytokine production such as TNF-α,IL-6 and IL-1β in rats through suppressing phosphorylation of both IκBα and NF-κB p65 subunit,and the differences are statistically significant.Conclusion: Overexpression of miR-146 a in BMSC-EVs may improve UC by affecting TRAF6 and further down-regulating NF-κB signaling pathway.Part Ⅱ The Effect of Compound Sophorae Decoction on Intestinal Barrier Function via Notch Signaling in UCObjective: To study the mechanism of compound sophorae decoction relieving UC via improving intestinal barrier function.Methods: A mouse model of acute UC was established by administration of 3% DSS in drinking water.The treatment groups were given with mesalazine(0.52 g/kg)or compound sophorae decoction at different doses(3.64 g/kg,7.28 g/kg and 14.56 g/kg)by gavage.During the experiment,the body weight,stool consistency and presence of hemafecia were recorded.DAI and histological score were assessed.Tight junction proteins including ZO-1 and occludin were examined by transmission electron microscope coupled with immunofluorescence.The expression of MMP-9 protein was detected by western blot.Ki67 and cleaved caspase-3 were detected through immunohistochemistry and western blot,respectively.MUC2 and Notch signaling pathway related to intestinal permeability were also studied.The fluorescence intensity of FITC-dextran 4000 in mice plasm was analyzed with a multimode plate reader.Flow cytometer was used to determine the ratio of M1/M2 in spleen and mesenteric lymphoid nodes and m RNA levels of i NOS,Arg1,TNF-α and IL-10 were assayed via q RT-PCR.Results: 1.Compound sophorae decoction can improve body weight and colon length,lower DAI and histological score and mitigate intestinal inflammation.2.Compound sophorae decoction can be beneficial to ZO-1 and occludin,and the potential mechanism may be in connected with suppressing MMP-9.3.Compound sophorae decoction can increase the level of Ki67 and decrease the expression of cleaved caspase-3,and the differences are statistically significant(P<0.05).4.Compound sophorae decoction can inhibit activity of Notch signaling pathway,restrain expression of Hes1,promote level of ATOH1,and thus induce the differentiation direction of IECs into secretory cell like goblet cells,which could produce abundant MUC2,resulting in decreasing gut permeability.5.Compound sophorae decoction can reduce the ratio of M1/M2 and facilitate the M1-to-M2 transition of macrophages,and promote wound healing.Conclusion: Compound sophorae decoction can alleviate DSS-induced experimental colitis via improving intestinal barrier function by regulating Notch signaling,repressing inflammatory response,anti-apoptosis and promoting proliferation of enterocytes,decreasing intestinal permeability and facilitating mucosal healing.