周期蛋白依赖性激酶-5抑制肽通过上调神经生长因子缓解高糖环境对人肾足细胞凋亡的影响

Cyclin dependent kinase-5 inhibitory peptide alleviates high glucosee induced apoptosis of human renal podocytes by upregulating nerve growth factor

目的探究截短型周期蛋白依赖性激酶5(Cdk5)抑制肽(TFP5)对高糖(HG)条件下人肾足细胞损伤和凋亡的影响,及其作用机制。方法将人肾足细胞分为低糖组(LG,1.0 g·L^(-1))、HG组(4.5 g·L^(-1))、HG+Scramble组(4.5 g·L^(-1)+100.0 nmol·L^(-1))、HG+TFP5组(4.5 g·L^(-1)+100.0 nmol·L^(-1))和HG+联合组(4.5 g·L^(-1)+50.0 ng·mL^(-1)K252a+100.0 nmol·L^(-1)TFP5)。用实时荧光定量聚合酶链反应检测细胞凋亡基因和神经生长因子(NGF)及Cdk5的表达水平,用荧光红染料法和DCFH探针法分别检测H_(2)O_(2)和活性氧(ROS)的水平,用流式细胞术检测细胞的凋亡情况。结果LG组和HG组的B淋巴细胞瘤-2(Bcl-2)mRNA分别为(0.04±0.02)×10^(-2)和(0.09±0.01)×10^(-3),Cdk5 mRNA分别为(0.04±0.01)×10^(-1)和(0.07±0.01)×10^(-1),细胞凋亡比率分别为(1.61±0.25)%和(3.77±0.17)%,H_(2)O_(2)分别为0.56±0.27和2.94±1.15,ROS分别为0.51±0.28和1.04±0.07,Cdk5酶活力分别为0.14±0.06和2.11±0.02,差异均有统计学意义(均P<0.01)。LG组、HG+Scramble组和HG+TFP5组的H_(2)O_(2)分别为1.13±0.20、2.49±0.38和1.08±0.18,ROS分别为0.87±0.18、2.61±0.71和0.83±0.22,Bcl-2 mRNA分别为(0.07±0.02)×10^(-2)、(0.01±0.03)×10^(-2)和(0.04±0.06)×10^(-2),NGF mRNA分别为(0.15±0.53)×10^(-1)、0.04±0.06×10^(-1)和0.22±0.08×10^(-2)。HG+Scramble组的上述指标和HG+TFP5组比较,差异均有统计学意义(均P<0.01)。HG+Scramble组、HG+TFP5组和联合组的NGF mRNA分别为0.04±0.01×10^(-1)、0.05±0.01×10^(-1)和0.01±0.03×10^(-1),Bcl-2 mRNA分别为(0.05±0.03)×10^(-2)、(0.01±0.00)×10^(-1)和(0.03±0.02)×10^(-2),NGF蛋白相对表达水平分别为1.14±0.07、1.69±0.44和0.48±0.06,HG+TFP5组的上述指标和联合组比较,差异均有统计学意义(均P<0.01)。结论TFP5可通过Cdk5-NGF调控轴显著降低HG培养条件下人肾足细胞内的氧化应激压力和细胞凋亡。

Objective To investigate the effect of truncated cyclin dependent kinase 5(Cdk5)inhibitory peptide(TFP5)on the injury and apoptosis of human renal podocytes under high glucose(HG)and its mechanism.Methods Human renal podocytes were divided into low glucose group(LG,1.0 g·L^(-1)),HG group(4.5 g·L^(-1)),HG+Scramble group(4.5 g·L^(-1)+100.0 nmol·L^(-1)),HG+TFP5 group(4.5 g·L^(-1)+100.0 nmol·L^(-1))and combination group(4.5 g·L^(-1)+50.0 ng·mL^(-1)K252a+100.0 nmol·L^(-1)TFP5).The expression levels of apoptosis gene,nerve growth factor(NGF)and Cdk5 were detected by real-time fluorescence quantitative polymerase chain reaction,and the levels of H_(2)O_(2) and reactive oxygen species(ROS)were detected by Amplex Red method and DCFH probe method,and the cell apoptosis was detected by flow cytometry.Results The expression levels of Bcl-2 mRNA in LG and HG groups were(0.04±0.02)×10^(-1)and(0.09±0.01)×10^(-3);Cdk5 mRNA were(0.04±0.01)×10^(-1) and(0.07±0.01)×10^(-1);the apoptotic rates were(1.61±0.03)%and(3.77±1.17)%;H,0,levels were 0.56±0.27 and 2.94±1.15;R0S levels were 0.51±0.28 and 1.04±0.07;Cdk5 enzyme activities were 0.14±0.06 and 2.11±0.02;the differences were statistically significant(all P<0.01).In LG,HG+Scramble and HG+TFP5 groups,H,0,levels were 1.13±0.20,2.49±0.38 and 1.08±0.18;R0S levels were 0.87±0.18,2.61±0.71 and 0.83±0.22;Bcl-2 mRNA were(0.07±0.02)×10^(-2),(0.01±0.03)×10^(-2) and(0.04±0.06)×10^(-2);NGF mRNA were(0.15±0.53)×10^(-1),0.04±0.06×10^(-1) and 0.22±0.08×10^(-2);the differences were statistically significant between HG+Scramble group and HG+TFP5 group(all P<0.01).In HG+Scramble,HG+TFP5 and combination groups,NGF mRNA were 0.04±0.01×10^(-1),0.05±0.01×10^(-1) and 0.01±0.03×10^(-1);Bcl-2 mRNA were(0.05±0.03)×10^(-2),(0.01±0.00)×10^(-1) and(0.03±0.02)×10^(-2);NGF protein relative expression levels were 1.14±0.07,1.69±0.44 and 0.48±0.06;the differences were statistically significant between HG+TFP5 group and combination group(all P<0.01).Conclusion TFP5 can significantly reduce oxidative stress and apoptosis in human renal podocytes cultured in HG through the Cdk5-NGF regulatory axis.