Effects Of Human Umbilical Cord Mesenchymal Stem Cells On Renal Fibrosis In Vivo And In Vitro And Mechanism Involved

Objective:To evaluate the effect and its mechanism of human umbilical cord mesenchymal stem cell(HucMSC)on aristolochic acid(AA)-induced renal fibrosis in mice.Methods:Experiments in vivo:Adult male C57BL/6 mice were randomly divided into 5 groups:normal control group(intraperitoneally injected with PBS),MSC control group(intraperitoneally injected with PBS,tail intravenously injected with MSC),AA group(intraperitoneally injected with AA),MSC+AA group(tail intravenously injected with ucMSCs and intraperitoneally injected with AA subsequently)and AA+MSC group(intraperitoneally injected with AA and intravenously injected with MSC through the tail subsequently).AA was intraperitoneally injected at a dosage of 5 mg/kg every other day for 2 weeks.The amount of MSC injections was 1×10~6.At 4 weeks post injection completion,mice were euthanized and their serum and kidneys were collected.The renal function and morphological changes of kidney tissue were detected.Western blot and immunohistochemical were used to detect the expression of E-cadherin,N-cadherin,TGF-β1 and p-Smad2/3.Experiments in vitro:Human renal tubular epithelial cells HK-2 were randomly divided into 4 groups:normal control group,MSC control group,TGF-β1 group and TGF+MSC group.The dose of TGF-β1 cytokine was 5ng/ml.MSC and HK-2 were co-cultured in transwell chambers with an intervention time of 48 h.After the intervention,Western blot and immunofluorescence were used to detect the expression of epithelial-mesenchymal transition indicators and TGF-β/Smad signaling pathway indicators.Results:In vivo experiments,there was no significant difference in renal function and kidney morphology between the MSC control group and the normal control group.The levels of Scr and BUN were found significantly increased in AA group,while decreased in two groups of MSCs-treated mice(p<0.05).HE,PAS and Masson staining showed that the glomerular and tubular structures of the control and MSC groups were structurally intact,with no obvious abnormalities.In the AA group,renal tubular dilatation or atrophy,and a large number of inflammatory cells infiltrated and collagen fibers deposited in the interstitial space were observed.Among the two groups of mice intervened with MSCs,it was shown that the number of sites and severity of renal tubular damage were less than that in AA group,and collagen fibers deposition was reduced.The results of Western blot and immunohistochemistry showed that the expression of E-cadherin in the AA group was lower than that in the control group.The expression of N-cadherin,TGF-β1 and p-Smad2/3 was significantly higher than that in the control group.The expression of E-cadherin was higher in the two groups with MSC intervention than that in the AA group,while the expression of N-cadherin,TGF-β1 and p-Smad2/3 was decreased.In the vitro experiment,in the TGF-β1 cytokine intervention group,the expression of E-cadherin was decreased than that in the control group,and the expression N-cadherin and TGF-β/Smad signaling pathway related indicators was significantly higher than that of the control group;the expression of E-cadherin was higher than that of the TGF group after co-culture with MSC,and the expressions of N-cadherin,TGF-β1 and p-Smad2/3 were lower than those in the TGF group(p<0.05).Conclusion:It was shown that aristolochic acid can induced renal fibrosis in C57BL/6 mice,and epithelial-mesenchymal transition may play an important role in the process.The intervention of human umbilical cord mesenchymal stem cells can effectively alleviate the renal damage induced by aristolochic acid.It suggested that MSCs play as a nephron-protective role in anti-fibrosis through inhibiting the activation of TGF-β/Smad signaling pathway.