决明子总蒽醌调控Sirt1/PPARγ通路对H_(2)O_(2)诱导人晶状体上皮细胞氧化损伤的影响

Effects of Cassia seed total anthraquinone regulating Sirt1/PPARγpathway on oxidative damage induced by H_(2)O_(2) in human lens epithelial cells

目的探讨决明子总蒽醌(CSTA)对过氧化氢(H_(2)O_(2))诱导人晶状体上皮细胞(LEC)氧化损伤的影响及其相关作用机制。方法体外培养人LEC细胞系HLE-B3细胞,利用不同浓度梯度CSTA作用于HLE-B3细胞以筛选其最适处理浓度。实验分为对照组(CG组)、H_(2)O_(2)组(H_(2)O_(2)组)、CSTA组和沉默信息调节因子1(Sirt1)抑制剂组(EX527组)。四甲基偶氮唑盐比色法(MTT)检测HLE-B3细胞活力;流式细胞仪检测HLE-B3细胞凋亡情况;酶联免疫(ELISA)检测HLE-B3细胞内丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)含量;实时荧光定量PCR(qRT-PCR)法检测HLE-B3细胞内Sirt1、PPARγmRNA表达;Western Blot检测HLE-B3细胞内Sirt1、过氧化物酶增殖物活化受体γ(PPARγ)、p-PPARγ蛋白水平。结果通过设置浓度梯度选择20 mg/L作为CSTA的处理浓度。(1)细胞活力:H_(2)O_(2)组与CG组比较降低(t=23.048,P=0.000),CSTA组与H_(2)O_(2)组比较升高(t=5.669,P=0.000),EX527组与CSTA组比较降低(t=4.151,P=0.002),差异均有统计学意义。(2)细胞凋亡率:H_(2)O_(2)组与CG组比较升高(t=18.480,P=0.000),CSTA组与H_(2)O_(2)组比较降低(t=9.670,P=0.000),EX527组与CSTA组比较升高(t=7.982,P=0.000),差异均有统计学意义。(3)氧化损伤指标:①MDA含量,H_(2)O_(2)组与CG组比较升高(t=13.040,P=0.000),CSTA组与H_(2)O_(2)组比较降低(t=9.602,P=0.000),EX527组与CSTA组比较升高(t=7.575,P=0.000),差异均有统计学意义。②GSH-Px含量,H_(2)O_(2)组与CG组比较降低(t=15.220,P=0.000),CSTA组与H_(2)O_(2)组比较升高(t=11.810,P=0.000),EX527组与CSTA组比较降低(t=9.090,P=0.000),差异均有统计学意义。③SOD含量,H_(2)O_(2)组与CG组比较降低(t=14.930,P=0.000),CSTA组与H_(2)O_(2)组比较升高(t=11.150,P=0.000),EX527组与CSTA组比较降低(t=8.783,P=0.000),差异均有统计学意义。(4)Sirt1和PPARγmRNA:①Sirt1 mRNA,H_(2)O_(2)组与CG组比较降低(t=33.803,P=0.000),CSTA组与H_(2)O_(2)组比较升高(t=13.561,P=0.000),EX527组与CSTA组比较降低(t=10.312,P=0.000),差异均有统计学意义。②PPARγmRNA,四组间比较差异无统计学意义(P>0.05)。(5)Sirt1及p-PPARγ/PPARγ蛋白:①Sirt1蛋白,H_(2)O_(2)组与CG组比较降低(t=11.000,P=0.000),CSTA组与H_(2)O_(2)组比较升高(t=7.667,P=0.000),EX527组与CSTA组比较降低(t=6.333,P=0.000),差异均有统计学意义。②p-PPARγ/PPARγ蛋白,H_(2)O_(2)组与CG组比较升高(t=16.410,P=0.000),CSTA组与H_(2)O_(2)组比较降低(t=13.060,P=0.000),EX527组与CSTA组比较升高(t=10.630,P=0.000),差异均有统计学意义。结论CSTA可能通过调节Sirt1/PPARγ通路,减轻H_(2)O_(2)诱导HLE-B3细胞氧化损伤。

OBJECTIVE To investigate the effect of Cassia seed total anthraquinone on the oxidative damage of human lens epithelial cells(LEC)induced by hydrogen peroxide(H_(2)O_(2))and its related mechanism.METHODS Human LEC cell line HLE-B3 cells were cultured in vitro,and Cassia seed total anthraquinone were used to act on HLE-B3 cells with different concentration gradients to screen the optimal treatment concentration.The experiment was divided into control group(CG group),H_(2)O_(2)group,Cassia seed total anthraquinone group(CSTA group)and silent information regulator 1(Sirt1)inhibitor group(EX527 group).Methyl thiazolyl tetrazolium assay(MTT)method was used to detect the viability of HLE-B3 cells;Flow cytometry was used to detect the apoptosis of HLE-B3 cells;Enzyme-linked immunoassay(ELISA)was used to detect malondialdehyde(MDA),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)levels in HLE-B3 cells;Quantitative real-time PCR(qRT-PCR)method was used to detect the Sirt1 and PPARγmRNA expression in HLE-B3 cells;Western Blot was used to detect the Sirt1,peroxidase proliferator activated receptorγ(PPARγ)and p-PPARγprotein levels in HLE-B3 cells.RESULTS By setting the concentration gradient,20 mg/L was selected as the treatment concentration of the Cassia seed total anthraquinone.(1)Cell viability:H_(2)O_(2)group was lower than CG group(t=23.048,P=0.000),CSTA group was higher than H_(2)O_(2)group(t=5.669,P=0.000),EX527 group was lower than CSTA group(t=4.151,P=0.002),the differences were all statistically significant.(2)Apoptosis rate:H_(2)O_(2)group was higher than CG group(t=18.480,P=0.000),CSTA group was lower than H_(2)O_(2)group(t=9.670,P=0.000),EX527 group was higher than CSTA group(t=7.982,P=0.000),the differences were all statistically significant.(3)Oxidative damage:①MDA content,H_(2)O_(2)group was higher than CG group(t=13.040,P=0.000),CSTA group was lower than H_(2)O_(2)group(t=9.602,P=0.000),EX527 group was higher than CSTA group(t=7.575,P=0.000),the differences were all statistically significant.②GSH-Px content,H_(2)O_(2)group was lower than CG group(t=15.220,P=0.000),CSTA group was higher than H_(2)O_(2)group(t=11.810,P=0.000),EX527 group was lower than CSTA group(t=9.090,P=0.000),the differences were all statistically significant.③SOD content,H_(2)O_(2)group was lower than CG group(t=14.930,P=0.000),CSTA group was higher than H_(2)O_(2)group(t=11.150,P=0.000),EX527 group was lower than CSTA group(t=8.783,P=0.000),the differences were all statistically significant.(4)Sirt1 and PPARγmRNA:①Sirt1 mRNA,H_(2)O_(2)group was lower than CG group(t=33.803,P=0.000),CSTA group was higher than H_(2)O_(2)group(t=13.561,P=0.000),EX527 group was lower than CSTA group(t=10.312,P=0.000),the differences were all statistically significant.②PPARγmRNA,there was no significant difference in the comparison between the four groups(P>0.05).(5)Sirt1 and p-PPARγ/PPARγSirt1 protein:①Sirt1 protein,H_(2)O_(2)group was lower than CG group(t=11.000,P=0.000),CSTA group was higher than H_(2)O_(2)group(t=7.667,P=0.000),EX527 group was lower than CSTA group(t=6.333,P=0.000),the differences were all statistically significant.②p-PPARγ/PPARγprotein,H_(2)O_(2)group was higher than CG group(t=16.410,P=0.000),CSTA group was lower than H_(2)O_(2)group(t=13.060,P=0.000),EX527 group was higher than CSTA group(t=10.630,P=0.000),the differences were all statistically significant.CONCLUSIONS Cassia seed total anthraquinone may reduce H_(2)O_(2)-induced oxidative damage in HLE-B3 cells by regulating the Sirt1/PPARγpathway.