黄芩苷对高糖诱导的人晶状体上皮细胞凋亡和氧化应激的影响及其可能机制

目的　探讨黄芩苷对高糖诱导的人晶状体上皮细胞凋亡和氧化应激的影响，并探讨其可能机制。方法　以人晶状体上皮细胞系HLE-B3为研究对象，用不同浓度黄芩苷处理后，采用CCK-8法检测细胞活力以筛选黄芩苷最佳作用浓度用于后续实验。将细胞随机分为正常组、高糖组、黄芩苷组，采用CCK-8法检测各组细胞活力，流式细胞术检测各组细胞凋亡率，RT-PCR检测Kelch样ECH相关蛋白1 (Keap1)、NQO1、核因子红细胞2相关因子2 (Nrf2)、HO-1、Bax、Bcl-2的mRNA相对表达量，Western blot检测Keap1、Nrf2、NQO1、HO-1、Bax、Bcl-2的蛋白表达水平，酶标法检测各组细胞超氧化物歧化酶（SOD）活力和丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）含量。结果　CCK-8法检测结果显示，5 μmol·L^-1黄芩苷可以显著增强HLE-B3细胞活力，随着黄芩苷浓度升高，细胞活力下降，故以5 μmol·L^-1黄芩苷进行后续实验。与正常组HLE-B3细胞活力［（100.00±0.00)%］相比，高糖组细胞活力［（73.52±1.71)%］显著降低（P<0.01)；与高糖组相比，黄芩苷组细胞活力［（92.36±3.61)%］显著升高（P<0.01）。流式细胞术检测结果显示，与正常组HLE-B3细胞凋亡率［（7.93±0.22）%］相比，高糖组细胞凋亡率［（57.12±2.63）%］显著升高（P<0.01）；与高糖组相比，黄芩苷组细胞凋亡率［（42.09±1.04）%］显著降低（P<0.01）。RT-PCR检测结果显示，与正常组相比，高糖组HLE-B3细胞中Nrf2、HO-1、NQO1、Bcl-2 mRNA相对表达量均显著降低，Keap1、Bax mRNA相对表达量均显著升高（均为P<0.05）；与高糖组相比，黄芩苷组HLE-B3细胞中Nrf2、NQO1、HO-1、Bcl-2 mRNA相对表达量均显著升高，Keap1、Bax mRNA相对表达量均显著降低（均为P<0.05）。Western blot检测结果显示，高糖组HLE-B3细胞中Keap1、Bax蛋白相对表达量均较正常组显著升高，黄芩苷组Keap1、Bax蛋白相对表达量均较高糖组显著降低（均为P<0.05）；高糖组HLE-B3细胞中Nrf2、HO-1、NQO1、Bcl-2蛋白相对表达量均较正常组显著降低，而黄芩苷组Nrf2、HO-1、NQO1、Bcl-2蛋白相对表达量均较高糖组显著升高（均为P<0.05）。酶标法检测结果显示，高糖组HLE-B3细胞中SOD活力、GSH-Px含量均较正常组降低，MDA含量较正常组升高（均为P<0.05）；黄芩苷组HLE-B3细胞中SOD活力、GSH-Px含量均较高糖组明显升高，MDA含量较高糖组降低（均为P<0.05）。结论　低浓度黄芩苷可抑制高糖诱导的人晶状体上皮细胞凋亡和氧化应激，其作用机制可能与Keap1-Nrf2-ARE信号通路有关。

Effects of baicalin on apoptosis and oxidative stress of human lens epithelial cells induced by high glucose and its possible mechanisms

Objective　To explore effect of baicalin to the apoptosis and oxidative stress of human lens epithelial cells (HLE-B3) induced by high glucose and its possible mechanisms.Methods　CCK-8 was used to detect the proliferation rate of HLE-B3 which was cultured in vitro with a certain concentration of baicalin. According to the proliferation rate of HLE-B3 detected by CCK-8, the optimal concentration of baicalin was selected for follow-up experiments. HLE-B3 cells were divided into three groups randomly including control group, high glucose group and baicalin group. CCK-8 and flow cytometry were used to detect the proliferation and apoptosis rate of every group, respectively. RT-PCR was used to detect the expression of mRNAs of Keap1, NQO1, Nrf2, HO-1, Bax and Bcl-2. Western blot was used to detect the expression levels of proteins of Keap1, NQO1, Nrf2, HO-1, Bax and Bcl-2. Enzyme labeling method was used to detect the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in each group.Results　CCK-8 results showed the proliferation of HLE-B3 cultured in 5 μmol·L^-1 baicalin was higher than other group, so we chosed it for follow-up experiments. The cell activity of HLE-B3 in the high glucose group was (73.52±1.71)%, which was significantly lower than that in the control group ［(100.00±0.00)%］, and the baicalin group ［（92.36±3.61）%］ was higher than the high glucose group (both P<0.01). Flow cytometry showed that the apoptosis rate of high glucose group［（57.12±2.63）%］ was higher than that in control group ［（7.93±0.22）%］, and the baicalin group ［（42.09±1.04）%］ was lower than the high glucose group (both P<0.01). RT-PCR showed that, when compared with the control group, the expression levels of mRNAs of Nrf2, HO-1, NQO1 and Bcl-2 of high glucose group decreased, while the expression levels of Keap1 and Bax increased (all P<0.05). The expression levels of mRNAs of Nrf2, NQO1, HO-1 and Bcl-2 in baicalin group was higher than those in the high glucose group, while the expression levels of Keap1 and Bax mRNAs decreased (all P<0.05). Western blot showed the expression levels of proteins of Keap1 and Bax in the high glucose group were higher than those in the control group, while their expression levels in the baicalin group were lower than those in the high glucose group (all P<0.05). The expression levels of Nrf2, HO-1, NQO1 and Bcl-2 proteins in the high glucose group were lower than those in the control group, while their expression levels in the baicalin group were higher than those in the high glucose group (all P<0.05). The activity of SOD and the content of GSH-Px in the high glucose group were lower while the content of MDA was higher compared to the control group (all P<0.05). The activity of SOD and the content of GSH-Px in the baicalin group were higher while the content of MDA was lower compared to the high glucose group (all P<0.05).Conclusion　Low concentration of baicalin can inhibit the apoptosis and oxidative stress of HLE-B3 cells induced by high glucose, which may related to the signal pathways Keap1-Nrf2-ARE.