The TLR4/NF-κB Signaling Pathway Mediates The Effects Of The Extract Of Polygala Fallax Hemsl.on Human Glomerular Mesangial Cells Cultured With High Glucose

Objective: To research the effects of the extract of Polygala fallax Hemsl.(EPF)on the injury of human glomerular mesangial cells(HMCs)induced by high glucose(HG),and explore the protective mechanism of the kidney through TLR4/NF-κB inflammatory pathway.Methods: HMCs induced by HG were used to establish the cell damage model.The experiment was divided into the control group(normal glucose,NG)and model(high glucose,HG)group,EPF(0.05,0.1 and 0.5μg/m L)group,and Gliquidone(GLQ)positive group.EPF was obtained by concentration ethanol extraction.MTT method was used to determine the optimal dose concentration and time of HMCs cell injury induced by HG and to analyze the effect of EPF on HMCs cell proliferation induced by HG.Hoechst 33342 staining experiment was performed to observe the morphological changes and apoptosis of HMCs induced by EPF on HG.The effect of EPF on the mitochondrial membrane potential of HMCs induced by HG was observed by JC-1 staining.Flow cytometry was used to detect the effect of EPF on HMCs apoptosis induced by HG.The effects of EPF on the contents of IL-1β,IL-6,IL-18,MCP-1,and TNF-α secreted by HMCs cells induced by HG were analyzed by ELISA.RT-q PCR or western blotting was used to detect the effect of EPF on the m RNA or protein expression levels of TLR4、My D88、NF-κBp65、p-NF-κBp65、MMP-9、MMP-2、Col Ⅳ、FN、IL-1β、IL-6、IL-18、MCP-1、TNF-α、caspase-3、Bcl-2 and Bax of HG-induced HMCs.The effect of EPF on NF-κBp65 nuclear transport in HMCs cells induced by HG was observed.TLR4 inhibitors were further used for intervention,and the effects of EPF and TAK-242 on the m RNA or protein expression levels of TLR4/NF-κB pathway and downstream inflammatory factors in HMCs cells were detected by RT-q PCR or Western blotting.The effects of EPF and TAK-242 on NF-κBp65nuclear transport in HMCs cells induced by HG were observed.Results:(1)MTT results showed that compared with the NG group,EPF had no obvious cytotoxicity to normal HMCs at 0.01 to 1μg/m L,but showed cytotoxicity when it was greater than 1μg/m L(P<0.01).The abnormal proliferation of HMCs was obvious at 48 h after 30 m M HG treatment(P<0.01),so as the best conditions for modeling.Compared with the HG group,EPF or GLQ significantly inhibited HMCs cell proliferation induced by HG(P<0.01).(2)Hoechst 33342 staining results showed that EPF could significantly reduce fluorescence intensity,significantly improve the morphology of cell damage,reverse the effect of HG on HMCs injury,and reduce the apoptosis rate(P<0.01).JC-1 staining results showed that EPF could reverse cell damage caused by HG,showing enhanced green fluorescence intensity and increased mitochondrial depolarization cell rate(P<0.01).Flow cytometry results showed that compared with NG group,the apoptosis rate of HMCs cells in the HG group was significantly increased(P<0.01),and EPF administration could significantly reduce the apoptosis rate of HMCs cells(P<0.01).(3)ELISA results showed that compared with the NG group,the contents of inflammatory factors IL-1β,IL-6,IL-18,MCP-1,and TNF-α in HMCs cells in the HG group were increased(P<0.01),and the expression level of inflammatory factors in HMCs cells was significantly decreased after EPF administration(P<0.05 or P<0.01).(4)According to RT-q PCR and Western blotting results,EPF or TAK-242 could significantly reverse the expression of HG-induced HMCs target and signal pathway-related proteins or m RNA.The expression levels of TLR4/NF-κB signaling pathway and downstream inflammatory factors,fibrosis and apoptosis factor caspase-3related protein or m RNA were significantly down-regulated,and the expression levels of Bcl-2/Bax protein or m RNA were up-regulated(P<0.05 or P<0.01).After combined treatment with EPF and TAK-242,the protein or m RNA expression levels of TLR4,My D88,p-NF-κBp65/NF-κBp65,and downstream inflammatory factors were decreased(P<0.01),and had a synergistic inhibitory effect.(5)According to the nuclear transport staining results of NF-κBp65,the red fluorescence of NF-κBp65 in the EPF or TAK-242 group was weaker than that in the HG group,which could inhibit the nuclear transport of NF-κBp65induced by HG in HMCs.Conclusion: EPF can reduce HMCs cell injury induced by HG,inflammatory response to cell injury,and accumulation of extracellular matrix mediated by MMP-2/9,and its anti-inflammatory effect may be regulated by targeting TLR4 and TLR4/NF-κB inflammatory pathways,and has the effect of alleviating apoptosis.