青蒿琥酯通过Akt/HO-1信号通路抑制诱导H2O2的晶状体上皮细胞氧化应激和凋亡

Artesunate inhibits HO-induced oxidative stress and apoptosis of lens epithelial cells via Akt/HO-1 signaling pathway

目的研究青蒿琥酯(Art)对H2O2诱导的晶状体上皮细胞(HLEB3)氧化应激和凋亡的影响及其机制。方法补充分组及各组处理情况采用CCK8法检测细胞活力;通过测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GSH)和丙二醛(MDA)水平来评估氧化应激损伤;流式细胞仪检测细胞凋亡;实时荧光定量聚合酶链反应(qRT-PCR)检测血红素氧合酶-1(HO-1)mRNA水平;western blot分析Bax、Bcl-2、磷酸化蛋白激酶B(p-Akt)及HO-1蛋白表达。结果(1)细胞活力:A组、B组和C组之间比较,差异有统计学意义(F=26.794,P=0.000);B组与A比较细胞活力降低,有统计学意义(t=34.364,P=0.000),C组与B组比较细胞活力升高,有统计学意义(t=17.358,P=0.000)。(2)氧化应激损伤:(1)SOD活性:A组、B组和C组之间比较,差异有统计学意义(F=21.634,P=0.000)。B组与A组比较SOD活性降低,有统计学意义(t=31.024,P=0.000),C组与B组比较SOD活性升高,有统计学意义(t=21.526,P=0.000);(2)CAT活性:A组、B组和C组之间比较,差异有统计学意义(F=15.607,P=0.000)。B组与A组比较CAT活性降低,有统计学意义(t=43.629,P=0.000),C组与B组比较CAT活性升高,有统计学意义(t=25.564,P=0.000);(3)GSH活性:A组、B组和C组之间比较,差异有统计学意义(F=38.347,P=0.000)。B组与A组比较GSH活性降低,有统计学意义(t=31.416,P=0.000),C组与B组比较GSH活性升高,有统计学意义(t=11.503,P=0.000);(4)MDA水平:A组、B组和C组之间比较,差异有统计学意义(F=23.614,P=0.000)。B组与A组比较MDA水平升高,有统计学意义(t=26.342,P=0.000),C组与B组比较MDA水平降低,有统计学意义(t=6.215,P=0.000)。(3)细胞凋亡:(1)细胞凋亡率:A组、B组和C组之间比较,差异有统计学意义(F=49.356,P=0.000)。B组与A组比较细胞凋亡率升高,有统计学意义(t=53.264,P=0.000),C组与B组比较细胞凋亡率降低,有统计学意义(t=10.629,P=0.000);(2)Bax表达水平:A组、B组和C组之间比较,差异有统计学意义(F=34.751,P=0.000)。B组与A组比较Bax表达升高,有统计学意义(t=32.169,P=0.000),C组与B组比较Bax表达降低,有统计学意义(t=14.317,P=0.000);(3)Bcl-2表达水平:A组、B组和C组之间比较,差异有统计学意义(F=29.648,P=0.000)。B组与A组比较Bcl-2表达降低,有统计学意义(t=49.634,P=0.000),C组与B组比较Bcl-2表达升高,有统计学意义(t=22.258,P=0.000)。(4)Akt/HO-1信号通路:(1)p-Akt表达水平:A组、B组和C组之间比较,差异有统计学意义(F=46.391,P=0.000)。B组与A组比较p-Akt表达降低,有统计学意义(t=38.561,P=0.000),C组与B组比较p-Akt表达升高,有统计学意义(t=26.146,P=0.000)。(2)HO-1表达水平:A组、B组和C组之间比较,差异有统计学意义(F=34.268,P=0.000)。B组与A组比较HO-1表达降低,有统计学意义(t=41.028,P=0.000),C组与B组比较HO-1表达升高,有统计学意义(t=26.551,P=0.000)。结论Art通过激活Akt/HO-1信号通路保护H2O2诱导的氧化应激和细胞凋亡,提示Art对氧化应激损伤诱导的白内障具有保护作用。

OBJECTIVE To investigated the effect of artesunate(Art)on H2O2-induced oxidative stress and apoptosis in HLEB3 cells and the associated mechanisms.METHODS Cell viability was detected by CCK8 assay.The oxidative damage was assessed by measuring the activities of superoxide dismutases(SOD),catalase(CAT),glutathione reductase(GSH),and malondialdehyde(MDA).Apoptosis was analyzed by flow cytometry analysis.The changed expressions of heme oxygenase-1(HO-1)and protein kinase B(Akt)pathways were evaluated by qRT-PCR and western blot.RESULTS(1)Cell viability:The difference of cell viability among A group,B group,and C group was statistically significant(F=26.794,P=0.000);the cell viability reduced in B group compared with A group(t=34.364,P=0.000),and cell viability increased in C group compared with A group(t=17.358,P=0.000).(2)Oxidative stress injury:(1)SOD activity:The difference of SOD activity among A group,B group,and C group was statistically significant(F=21.634,P=0.000);the SOD activity reduced in B group compared with A group(t=31.024,P=0.000),and SOD activity increased in C group compared with A group(t=21.526,P=0.000).(2)CAT activity:The difference of CAT activity among A group,B group,and C group was statistically significant(F=15.607,P=0.000);the CAT activity reduced in B group compared with A group(t=43.629,P=0.000),and CAT activity increased in C group compared with A group(t=25.564,P=0.000).(3)GSH activity:The difference of GSH activity among A group,B group,and C group was statistically significant(F=38.347,P=0.000);the GSH activity reduced in B group compared with A group(t=31.416,P=0.000),and GSH activity increased in C group compared with A group(t=11.503,P=0.000).(4)MDA level:The difference of MDA level among A group,B group,and C group was statistically significant(F=23.614,P=0.000);the MDA level increased in B group compared with A group(t=26.342,P=0.000),and MDA level reduced in C group compared with A group(t=6.215,P=0.000).(3)Cell apoptosis:(1)Cell apoptotic rate:The difference of cell apoptotic rate among A group,B group,and C group was statistically significant(F=49.356,P=0.000);the cell apoptotic rate increased in B group compared with A group(t=53.264,P=0.000),and cell apoptotic rate reduced in C group compared with A group(t=10.629,P=0.000).(2)Bax expression:The difference of Bax expression among A group,B group,and C group was statistically significant(F=34.751,P=0.000);the Bax expression increased in B group compared with A group(t=32.169,P=0.000),and Bax expression reduced in C group compared with A group(t=14.317,P=0.000).(3)Bcl-2 expression:The difference of Bcl-2 expression among A group,B group,and C group was statistically significant(F=29.648,P=0.000);the Bcl-2 expression reduced in B group compared with A group(t=49.634,P=0.000),and Bcl-2 expression increased in C group compared with A group(t=22.258,P=0.000).(4)Akt/HO-1 pathway:(1)p-Akt expression:The difference of p-Akt expression among A group,B group,and C group was statistically significant(F=46.391,P=0.000);the p-Akt expression reduced in B group compared with A group(t=38.561,P=0.000),and p-Akt expression increased in C group compared with B group(t=26.146,P=0.000).(2)HO-1 expression:The difference of HO-1 expression among A group,B group,and C group was statistically significant(F=34.268,P=0.000);the HO-1 expression reduced in B group compared with A group(t=41.028,P=0.000),and HO-1 expression increased in C group compared with B group(t=26.551,P=0.000).CONCLUSIONS Art protected H2O2-induced oxidative stress and apoptosis through activating the Akt/HO-1 pathway,suggesting the protective effect of Art against H2O2-induced cataract.