艾塞那肽对高糖诱导的小鼠足细胞损伤的保护作用及机制研究

目的探讨艾塞那肽对高糖诱导的小鼠足细胞损伤的作用及相关机制。方法将足细胞系MPC5细胞按照不同葡萄糖培养浓度和干预因素分为以下5组:正糖对照组(5.5 mmol/L葡萄糖,n=3)、甘露醇高渗对照组(5.5 mmol/L葡萄糖+19.5 mmol/L甘露醇,n=3)、高糖组(25.0 mmol/L葡萄糖,n=3)、高糖+艾塞那肽组(25.0 mmol/L葡萄糖+100 nmol/L艾塞那肽,n=3)和高糖+艾塞那肽+LY294002组(25.0 mmol/L葡萄糖+100 nmol/L艾塞那肽+50μmol/L磷脂酰肌醇-3-激酶抑制剂LY294002,n=3)。采用原位缺口末端标记法荧光染色观察细胞凋亡,膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶双染法检测细胞凋亡率,Western blot检测细胞内裂孔膜肾病蛋白(nephrin)、磷酸化蛋白激酶B(p-AKT)以及磷酸化Bcl-xL/Bcl-2相关死亡启动子(p-BAD)水平。多组间比较采用单因素方差分析,组间多重比较采用最小显著性差异t检验。结果与正糖对照组和甘露醇高渗对照组比较,高糖组足细胞凋亡率显著增加分别为(28.60±2.65)%、(4.50±0.75)%和(4.55±0.65)%,t=-19.19、-19.15,均P<0.01],细胞内nephrin蛋白表达降低(分别为0.22±0.03、0.72±0.06和0.73±0.08,t=11.43、11.52,均P<0.01),p-AKT和p-BAD蛋白表达下调(分别为0.14±0.03、0.83±0.06和0.86±0.04,0.16±0.03、0.66±0.06和0.68±0.04,均P<0.01)。与高糖组比较,高糖+艾塞那肽组足细胞凋亡率显著下降,细胞内nephrin表达升高,p-AKT和p-BAD蛋白表达上调(均P<0.01)。而高糖+艾塞那肽+LY294002组较高糖+艾塞那肽组足细胞凋亡率增加,细胞内nephrin蛋白表达降低,p-AKT和p-BAD蛋白表达下调(均P<0.05)。结论艾塞那肽通过上调足细胞中nephrin蛋白表达和减少足细胞凋亡从而保护高糖诱导的足细胞,其机制可能与激活AKT/BAD信号途径有关。

Protective effect and possible mechanisms of exenatide in podocyte injury induced by high glucose in mice

Objective To investigate the effects of exenatide on podocyte injury induced by high glucose(HG)in mice and its possible mechanisms.Methods According to different glucose concentrations and interventions,podocytes MPC5 cells were assigned to the following 5 groups:normal glucose(NG)group(5.5 mmol/L glucose,n=3),mannitol(MA)group(5.5 mmol/L glucose+19.5 mmol/L mannitol,n=3),HG group(25.0 mmol/L glucose,n=3),HG+exenatide group(25.0 mmol/L glucose+100 nmol/L exenatide,n=3),and HG+exenatide+LY294002 group(25.0 mmol/L glucose+100 nmol/L exenatide+50μmol/L phosphatidylinositol-3-kinase inhibitor LY294002,n=3).Apoptosis of podocytes were stained with DeadEndTM fluorometric terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling system,AnnexinⅤ-flourescein isothiocyanate and propidium iodide(PI),and assessed by flow cytometry.The expression levels of slit diaphragm protein(nephrin),phosphorylated protein kinase B(p-AKT)and phosphorylated Bcl-xL/Bcl-2 related death promoter(p-BAD)were measured by western blotting.Data among multiple groups were compared by using one-way analysis of variance(ANOVA)and the least significant difference t test.Results Compared with the NG group and MA group,the percentage of apoptotic podocytes significantly increased(28.60±2.65)%vs.(4.50±0.75)%,(4.55±0.65)%,t=-19.19,-19.15,both P<0.01],the protein levels of nephrin decreased(0.22±0.03 vs.0.72±0.06,0.73±0.08,t=11.43,11.52,both P<0.01),the protein expressions of p-AKT and p-BAD were down-regulated(0.14±0.03 vs.0.83±0.06,0.86±0.04;0.16±0.03 vs.0.66±0.06,0.68±0.04,all P<0.01)in the group cultured with HG.Compared with the HG group,the percentage of apoptotic podocytes significantly decreased and the protein levels of nephrin increased,and the protein expressions of p-AKT and p-BAD were also up-regulated in the HG+exenatide group(all P<0.01).However,compared with the HG+exenatide group,the percentage of apoptotic podocytes increased and the protein levels of nephrin decreased,and the protein expressions of p-AKT and p-BAD were also down-regulated in the HG+exenatide+LY294002 group(all P<0.05).Conclusions Exenatide prevents high glucose-induced podocyte injury by preserving slit diaphragm protein expression and preventing podocyte apoptosis.The possible mechanisms may involve the AKT/BAD signaling pathway.