| Background and ObjectiveIncidence of diabetes mellitus(DM)increases year by year with the change of the contemporary lifestyle,diabetic nephropathy(DN)is one of the most serious chronic microvascular complications of DM,the prevalence of DN also increased year by year.End-stage renal disease(ESRD)caused by diabetic nephropathy also increased year by year.Podocytes are one kind of highly differentiated and non-renewable glomerular epithelial cells,and the injury and loss of podocytes has closely associated with initiation and progression of DN,and involved mechanisms include podocyte apoptosis,detachment,hypertrophy,effacement and loss of foot process proteins,etc.Among them,podocyte apoptosis plays an important role in the pathogenesis and prognosis of DN.The endoplasmic reticulum is a multifunctional organelles,in addition to participate in the synthesis and metabolism of substances and to participate in the regulation of cellular homeostasis.High sugar,oxidative stressand harmful stimulation persistently worked in endoplasmic reticulum,can activate the apoptosis induced by endoplasmic reticulum apoptosis process.Epigallocatechin-3-gallate(EGCG),the most abundant catechin in the green tea,has been shown to exert preventive effect in chronic diseases including cancer,neurodegenerative disease,heart disease.However the effects of EGCG on proliferationand apoptosis of mouse podocytes cultured in high glucose is not entirely clear.This study investigated that EGCG intervention podocytes cultured in high glucose to compare cell proliferation and the expression of Nephrin and WT-1,podocytes apoptosis and endoplasmic reticulum stress iconic apoptotic protein including glucose-regulated protein 78(GRP78),p-PERKand Caspase-12expression.In order to expound that EGCG can ameliorate high glucose-induced podocytes apoptosis through suppressing endoplasmic reticulum stress-induced apoptosis.RESEARCH METHODCells were divided into groups as following:? Normal sugar group(NG:5.6mmol/Lglucose)?Mannitol group(5.6mmol/Lglucose+24.4mmol/L mannitol)?High sugar group(HG:30 mmol/Lglucose)?different concentrations of EGCG group(El:1?mol/L+HG;E10:10?mol/L+HG;E20:20?mol/L+HG;E40:40?mol/L+HG;E60:60?mol/L+HG;E80:80?mol/L+HG;E100:100?mol/L+HG).Cells were exposed to different conditions for 24h,48h,and 72hand detecting the cell proliferation by CCK8 respectively.Cells of four groups(NG,M,HG and E20)in above conditions were used for the following experiments.After 72 hours,the detection of the Nephrin and WT-1 though Western-blotting.Flow cytometry and Hoechst staining were used to examine the cell apoptosis rates.Western-blotting was used to measure the expression of glucose-regulated protein 78(GRP78),p-PERK and caspase-12.RESULTS1.Podocyte proliferation:cell proliferation in HG was significantly decreased compared to those in NG and M groups when cells were incubated for 24h,48h and 72h particularly in 72 h.EGCG with different concentrations was added to high glucose treated cells,EGCG especially at a concentration of 20?mol/L(E20)significantlypromoted cell proliferation after 72h(P<0.05).2.Podocytes injury:WT-1 and Nephrin expression was decreased in podocytes stimulated with HG compared with those with NG andM group(P<0.05).Meantime,their expression was significantly increased intreatment of EGCG(20?mol/L)(P<0.05).3.Podocytes apoptosis:The apoptotic population significantly expanded in HG(22.48%)than NG(0.55%)and M(0.72%)group(P<0.05).Expectedly,EGCG(20?mol/L)treatment in high glucose incubated cells significantly reduced the apoptotic cell population percentage(6.57%)(P<0.05).In the HG,there were much more typical apoptotic cells that nuclear shape became petals or nucleolus pyknosised rather than NG or M group(P<0.05).EGCG(20?mol/L)greatly decreased the number of apoptotic cells treated with high glucose(P<0,05).4.Endoplasmic reticulum apoptosis pathway iconic protein expression:After high glucose stimulation,both GRP78,p-PERK and Caspase-12 were significantly increased than those of NG and Mgroups(P<0.05).However,these proteins were obviously attenuated after EGCG(200?mol/L)treatment(P<0.05)CONCLUSIONEGCG can ameliorate high glucose-induced podocytes apoptosis through suppressing endoplasmic reticulum stress... |
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