Effect Of Human Umbilical Cord Mesenchymal Stem Cells On The Innate Immunity Of Podocytes Under High Glucose

BackgroundDiabetic nephropathy(DN)is one of the major causes of end-stage renal disease(ESRD),which specific pathogenesis has not been fully illustrated yet.Recently studies have suggested that innate immunity and inflammation is very important in the development of DN.Innate immunity as the first defense in the immune system can recognize the Pathogen-associated molecular pattern(PAMP)which is highly expressed on the pathogens as well as Damage-associated molecular pattern(DAMP)which is released after the damage of tissues and cells such as Heat shock proteins(HSP)and High-mobility groupbox 1(HMGB1).Toll?like receptors(TLR)is one of the major components of innate immunity.In DN,high glucose,disorder of lipid metabolism and anoxia can induce many endogenous ligand,Which can be recognized by TLR and activated the innate immunity and induce inflammation.Mesenchymal stem cells(MSCs)have multipotent differentiation ability and can alleviate the progress of DN.In this experiment we explored the effects of human umbilical cord mesenchymal stem cells(HUC-MSCs)on the innate immunity and the inflammation of podocytes under high glucose(HG)condition.ObjectiveTo explore the effects of HUC-MSCs on the innate immunity of podocytes mediated by TLR signaling pathway under high glucose(HG)condition.Methods1.Extraction of HUC-MSCs from umbilical cord of full-term newborns.2.The phenotype of HUC-MSCs were identified by flow cytometry and the differentiation of HUC-MSCs were induced under osteoinductive and adipogenic differentiation medium.3.CCK8 was used to detect the cell viability under normal and high glucose.4.Podocytes were divided into four groups according to the treatment:normal glucose group(NG),mannitol control group(NG+MA),high glucose group(HG)and HUC-MSCs co-culture group(HG+MSC).After 72 hours treatment,the protein levels of IL-6,IL-?,TNF-?,MCP-1 and HSP70,HMGB1 in culture medium were measured by ELISA.Real-time PCR was used to detect the mRNA expressions of TLR2 and TLR4.Western blotting was used to detect the protein expressions of TLR2,TLR4,MyD-88 and p-P65.Immunofluorescence staining was used to study the localization of p-P65 in podocytes.Results1.The identification of HUC-MSCs:1.1 Flow cytometric analysis showed that HUC-MSCs had a high expression of CD73,CD90 and low expression of CD34,CD45.1.2 HUC-MSCs were differentiate into osteogenic while they were cultured in osteoinductive differentiation medium.1.3 HUC-MSCs were differentiate into adipogenic cells while they were cultured in adipogenic differentiation medium.2.The viability of podocytes were decreased under high glucose.3.High glucose induced the inflammation of podocytes by activating the TLR signaling,which increased the secretion of IL-6,IL-?,TNF-?,MCP-1,HSP70,HMGB1,the mRNA level of TLR2,TLR4 and the protein level of TLR2,TLR4,MyD-88 and p-P65(all P<0.05).High glucose also activated NF-kB and induced its nuclear translocation.HUC-MSCs co-culture decreased the inflammation and restrained the TLR signaling.Conclusions1.High glucose can enhance the innate immune response of podocytes via TLR.2.High glucose can decrease the viability of podocytes.3.HUC-MSCs co-culture can decrease the inflammation and innate immunity of podocytes induced by HG via TLR.