| Objective: As aging population, changes in diet and lifestyle, the prevalence of diabetes mellitus is expected to increase to 366 million by the year 2030. Diabetic nephropathy(DN) was one of the most serious chronic microvascular complications of diabetes mellitus, and was the leading cause of kidney failure. DN’s increasing prevalence and incidence had imposed global socio-economic stress on healthcare systems worldwide. Further research of diabetic nephropathy, is an important object in academia.The cause of diabetic renal injury was complex and multi-factorial including hyperglycemia, hypertension, dyslipidemia, production of inflammatory cytokines and oxidative stress. The patients with DN mainly showed edema, proteinuria, and progressive renal function decline. Renal pathology was characterized by mesangial extracellular matrix accumulation, mesangial widening, basement membrane thickening, glomerulosclerosis and tubulointerstitial fibrosis. Podocyte injury plays an important role in the generation and development of proteinuria in DN, but the specific mechanisms of podocyte injury is not clear.Autophagy, a highly regulated lysosomal pathway involved in the recycling of cytosol and the removal of superfluous or damaged organelles, was essential for the survival, differentiation, development and homeostasis of cells. Autophagy worked to maintain cell homeostasis under various stress conditions. The renal cells under high-glucose conditions, nutrient signaling pathways altering impacted cells autophagy. So the cells were abnormal to variety stress, further aggravating the disorder of cells function, which in turn developed into DN. Podocytes had a high level of basic autophagic activity, the mechanism of podocytes autophagy in the progression of DN has become a hot point.Recent studies have shown that renal inflammation was important in promoting the development and progression of DN. TLRs was expressed by podocytes and was up-regulated in inflammatory glomerular disease, where it may mediate glomerular injury by modulating expression of chemokines. These results supported the possibility that TLRs activation in podocytes may play an important role in diabetic nephropathy. There were two major TLR pathways, one was mediated by myeloid differentiation factor 88(My D88) adaptor proteins, and the other was independent of My D88. The My D88-independent pathway was mainly dependent on Toll-Interleukin 1 Receptor(TIR) domain-containing adapter-inducing interferon-β(TRIF). Upon activation TLRs signal via an adaptor molecule My D88 or TRIF, leading to translocation of nuclear factor kappa B(NF-k B) with consequent upregulation of pro-inflammatory cytokines and chemokines. These pro-inflammatory cytokines and chemokines were known to engage in the pathogenesis of DN. But the specific mechanisms of podocyte injury is not clear.Genistein(GEN) is one of the major soy isoflavones isolated from the soybean. Current studies provide good evidence that GEN had an ability to protect kidneys from hyperglycemia-induced oxidative stress, inflammation, and fibrosis in diabetic mice. But there is no clear underlying mechanism by which GEN can boost a protective role in DN progression.In this study, we evaluate the effects of GEN on the autophagy of high glucose-induced podocytes in vitro at physiologically achievable concentration, we also analyzed various inflammatory components of TLRs signaling passways and examined the possible mechanisms involved in it.Methods:1 The influence of GEN on autophagy of high glucose ambient mice podocyteThe conditionally immortalized mouse podocytes were cultured at 33°C with γ-IFN, and then cells were cultured at 37°C without γ-IFN for 10-14 days to induce quiescence and the differentiated phenotype. Synaptopodin was detected by immunofluorescence to validate the differentiation of podocytes. Qualified podocytes were used in experiments. ①Firstly, podocytes were cultivated with the high glucose for 0, 2, 6, 12, 24, 48 and 72 hours. Using western blot method found out the maximal time of LC3Ⅱ. ②Normal glucose(NG) group, mannitol control(MC) group, high glucose control(HG) group, high glucose + GEN(G) group, high glucose + chloroquine(CQ) group and high glucose + CQ + GEN(CG) group were set. According to the results of experiment ①, the podocytes were incubated with 20μM GEN for 6h, then the autophagosome was evaluated by electron microscopy. ③Setting controls as above, LC3 and p-m TOR were detected by immunofluorescence.④The expression of LC3 Ⅱand p-m TOR were detected by western blot,the expression of nephrin was detected by western blot and real-time PCR as to evaluate the influence of GEN on autophagy in high glucose ambient mice podocyte.2 The effect of GEN on TLRs/NF-κB signaling passway in high glucose ambient mice podocyte①Firstly, podocytes were cultivated with the high glucose for 0, 2, 6, 12, 24, 48 and 72 hours. Using western blot method found out the maximal time of NF-κB. ②Normal glucose(NG) group, mannitol control(MC) group, high glucose control(HG) group, high glucose + GEN(G) group were set. According to the results of experiment ①, the podocytes were incubated with 20μM GEN for 48 h, then the expression of NF-κB p65 was detected by immunofluorescence. ③Setting controls as above, the expression of My D88, TRIF and NF-κB p65 were detected by western blot, the expression of MCP-1 was detected by ELISA. ④The expression of nephrin was detected by western blot and real-time PCR as to evaluate the effect of genistein on TLRs/NF-κB signaling passway in high glucose ambient mice podocyte.3 After knocking-down the gene expression of My D88/TRIF, the influence of GEN on autophagy of high glucose ambient mice podocyte①Optimizing the transfection conditions: The optimal dose of My D88-si RNA and TRIF-si RNA were confirmed by abviously knockdown the expression of My D88 and TRIF gene.②Differentiated podocytes were divided into three groups: high glucose(HG) group, high glucose+My D88-si RNA(HG+My D88.si RNA,M) group, high glucose+ My D88-si RNA+GEN(HG+My D88.si RNA+GEN,MG) group, high glucose+TRIF-si RNA(HG+TRIF.si RNA,T) group, high glucose+TRIF-si RNA+GEN(HG+TRIF.si RNA+GEN, TG) group, high glucose + My D88-si RNA+TRIF-si RNA(HG + My D88.si RNA+TRIF.si RNA,MT) group and high glucose+ My D88-si RNA+ TRIF-si RNA + GEN( HG+ My D88.si RNA +TRIF.si RNA + GEN,MTG) group. The podocytes were incubated for 6h,then the LC3 and p-m TOR were detected by immunofluorescence. ③Setting controls as above, the expression of LC3 and pⅡ-m TOR were detected by western blot,the expression of nephrin was detected by western blot and real-time PCR as to evaluate the influence of genistein on autophagy in high glucose ambient mice podocyte after knocking- down the gene expression of My D88/TRIF.4 After knocking-down the gene expression of My D88/TRIF, the effect of GEN on TLRs/NF-κB signaling passway in high glucose ambient mice podocyteDifferentiated podocytes were divided into three groups: high glucose(HG) group, high glucose + My D88-si RNA(HG+My D88.si RNA,M) group, high glucose +My D88-si RNA + GEN(HG+My D88.si RNA + GEN,MG) group, high glucose+ TRIF-si RNA(HG + TRIF.si RNA,T) group, high glucose + TRIF-si RNA + GEN(HG + TRIF.si RNA+GEN,TG) group, high glucose + My D88-si RNA + TRIF-si RNA(HG + My D88.si RNA +TRIF.si RNA,MT) group and high glucose + My D88-si RNA +TRIF-si RNA +GEN(HG + My D88.si RNA+ TRIF.si RNA+GEN,MTG) group. ①The podocytes were incubated for 48 h, then the expression of NF-κB p65 was detected by immunofluorescence. ②Setting controls as above, the expression of My D88 and TRIF were detected by western blot, the expression of MCP-1 was detected by ELISA. ③The expression of nephrin was detected by western blot and real-time PCR as to evaluate the effect of genistein on TLRs/NF-κB signaling passway in high glucose ambient mice podocyte after knocking-down the gene expression of My D88/TRIF.Results:1 The influence of GEN on autophagy of high glucose ambient mice podocyte①F-actin immunofluorescence assay showed that at 33 ℃the cells proliferated and maintained cobblestone-like morphology. Immunofluorescence staining showed that there was sparsely expression of synaptopodin in undifferentiated podocytes at 33℃. When cultured at 37℃, F-actin immunofluorescence assay showed that the cells stretched out differently shaped long processes from cell bodies,arised many spindle-like projections from their primary processes. The expression of synaptopodin in differentiated podocytes was enhanced obviously. ②The time-effect relationship experiment disclosed that the protein expression of LC3Ⅱ/ Ⅰsignificantly when cells were treated with HG for 6 h(P < 0.01). ③Electron microscopy showed that the autophagosomes were more in NG than in HG group. ④The results of immunofluorescence showed that LC3 significantly expressed in HG, G, CQ and CG group than in NG group. The results of p-m TOR is increased in CQ group. ⑤The results of western blot showed that LC3Ⅱsignificantly expressed in HG, G, CQ and CG group(P<0.01). The results of p-m TOR is significantly increased in CQ group(P < 0.01). ⑥The results of western blot and real-time PCR showed that the expression of nephrin is lower in HG and CQ group than in NG and G group, is higher in CG group than in CQ group(P<0.01). Collectively, these results disclosed that GEN could promote the autophagy of high glucose ambient mice podocyte.2 The effect of GEN on TLRs/NF-κB signaling passway in high glucose ambient mice podocyte①The time-effect relationship experiment disclosed that the protein expression of NF-κB p65 nuclear translocation significantly when cells were treated for 48 h(P < 0.05). ②The results of immunofluorescence showed that HG group showed a strong nuclear staining for NF-κB p65 when compared to cytoplasmic distribution in NG group and MC group. Nuclear translocation of NF-κB p65 in G group was blocked by GEN as demonstrated by decreased nuclear staining. ③The results of western blot showed that the expression of My D88 and TRIF were increased in HG group compared to NG, the nuclear translocation of NF-κB p65 was obvious in HG compared to NG(P < 0.01). This observation suggests that GEN administration to high-glucose cultured murine podocytes inhibited the translocation of NF-κB p65. ④The results of western blot and real-time PCR showed that the expression of nephrin is higher in NG and G group than in HG group. This observation suggests that GEN administration could relieve the HG induced imflammation injury in podocytes.⑤The results of ELISA showed that the expression of MCP-1 was increased in HG group compared to NG and G group(P < 0.01).3 After knocking-down the gene expression of My D88/TRIF, the influence of GEN on autophagy of high glucose ambient mice podocyte①The verification experiment results of My D88-si RNA and TRIF-si RNA showed that at 70 pmol/well My D88/ TRIF-si RNA knocked down the protein and m RNA expression of My D88/ TRIF significantly in podocytes cultured in 6 well plate. ②The results of immunofluorescence showed that LC3 significantly decreased in MT group. The results of p-Mtor is opposite to LC3. ③The results of western blot showed that LC3Ⅱdecreased in MT group(P < 0.01). The results of p-m TOR is opposite to LC3Ⅱ. ④The results of western blot and real-time PCR showed that the expression of nephrin is lower in HG group than in other groups(P < 0.01). These results disclosed that knocking-down the gene expression of My D88/TRIF and using GEN could protect the high glucose ambient mice podocyte.4 After knocking-down the gene expression of My D88/TRIF, the effect of GEN on TLRs/NF-κB signaling passway in high glucose ambient mice podocyte①The results of immunofluorescence showed that HG group and HC group showed a strong nuclear staining for NF-κB p65 when compared to cytoplasmic distribution in M, T group and MT group. Nuclear translocation of NF-κB p65 in G group was blocked by GEN as demonstrated by decreased nuclear staining. ②The results of western blot showed that the expression of My D88 and TRIF were decreased in M/T,MT and GEN using group(P < 0.01). The results of western blot about the expression tendency of NF-κB p65 was similarly to the results of immunofluorescence. ③The results of western blot and real-time PCR showed that the expression of nephrin is lower in HG and HC group than in other groups(P < 0.01). ④The results of ELISA showed that the expression of MCP-1 was decreased in M group, T group and MT group compared to HG group and HC group, especially in G group(P < 0.01).Conclusion:1 GEN could enhance the autophagy of podocyte, and it would relieve the inhibition of autophagy by CQ and the reduction of nephrin.2 GEN could inhibit the nuclear translocation of NF-κB p65 and decrease the expression of MCP-1, and relieve the reduction of neprhin.3 GEN could relieve the reduction of autophagy. Knocking-down the gene expression of My D88/TRIF and using GEN could relieve the HG induced imflammation injury in podocytes.4 When high glucose stimulate mice podocyte and knocking-down the gene expression of My D88/TRIF and using GEN could inhibit the nuclear translocation of NF-κB p65, decrease the expression of MCP-1, and relieve the reduction of neprhin.In summary, GEN plays a crucial role in the pathogenesis of podocyte injury in DN, perhaps by increasing autophagy and inhibiting the action of TLRs/NF-κB signaling passway. |
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