摘要
目的探讨补肾养血明目方基于线粒体途径对RPE细胞凋亡的保护作用。方法将ARPE-19细胞分为4组:正常组、模型组、空白肠吸收液组(对照组)、肠吸收液干预组(干预组)。建立氢醌(HQ)诱导的ARPE-19细胞氧化损伤模型。正常组细胞,在常规培养液中培养;模型组细胞,90μM的HQ作用24 h;对照组细胞,空白肠吸收液干预24 h后,加入90μM的HQ作用24 h;干预组细胞,补肾养血明目方含药肠吸收液干预24 h后,加入90μM的HQ作用24 h。分别采用CCK-8法检测细胞活力,荧光分光光度法分析ARPE-19细胞线粒体膜通透转换孔(mPTP)的开放程度,ELISA技术测定细胞色素C(Cyt-c)含量的变化,TUNEL方法观察细胞凋亡情况。结果(1)RPE细胞活力:与模型组比较,干预组(浓度分别为1%,2%,5%)RPE细胞活力提高(t1%=-4.309,P=0.002;t2%=-9.503,P=0.000;t5%=-4.008,P=0.003),其中,浓度为2%的补肾养血明目方含药肠吸收液保护效果最好。(2)RPE细胞mPTP开放:与模型组比较,干预组Calcein平均荧光强度提高(t=-5.521,P=0.001),对照组无明显变化(P>0.05);与对照组比较,干预组Calcein平均荧光强度提高(t=-5.009,P=0.001)。(3)RPE细胞胞浆Cyt-c含量:与模型组比较,干预组ARPE-19细胞胞浆Cyt-c含量降低(t=2.968,P=0.018),对照组无明显变化(P>0.05);与对照组比较,干预组Cyt-c含量降低(t=2.717,P=0.026)。(4)RPE细胞凋亡:与模型组相比,干预组ARPE-19细胞凋亡率下降(t=2.360,P=0.029),对照组凋亡率无明显变化(P>0.05);与对照组相比,干预组细胞凋亡率下降(t=2.932,P=0.008)。结论补肾养血明目方含药肠吸收液可降低氧化损伤ARPE-19细胞mPTP的开放程度,抑制线粒体Cyt-c的释放,减少HQ诱导的ARPE-19细胞凋亡。线粒体保护是补肾养血明目方发挥抗HQ诱导的ARPE-19细胞氧化损伤的重要途径。
OBJECTIVE To observe the protective effect of Bushen Yangxue Mingmu(BSYXMM) formula on apoptosis of retinal pigment epithelium(RPE) cells via mitochondria-dependent pathway. METHODS ARPE-19 cells were divided into four groups: normal group, model group, control group and intervention group. Oxidative injury model of ARPE-19 cells induced by hydroquinone(HQ) was established. Normal group cells were cultured in conventional medium.Model group cells were induced by 90 μM HQ for 24 hours. Cells in the control group were treated with blank intestinal absorption solution for 24 hours and then treated with90 μM HQ for 24 hours. Cells in the intervention group were treated with BSYXMM formula and then treated with 90 μM HQ for24 hours. The CCK-8 method was used to detect cell viability, fluorescence spectrophotometry was used to analyze the opening degree of mitochondrial membrane permeability transition pore(mPTP), ELISA was used to detect the variation of cytochrome c(Cyt-C) content, TUNEL method was used to observe cell apoptosis. RESULTS(1) Activity of RPE cells: compared with the model group, the activity of RPE cells in the intervention group(concentration of 1%, 2%, 5%) was increased(t1%=-4.309,P=0.002;t2%=-9.503,P=0.000;t5%=-4.008,P=0.003)and the best protection effect was at the concentration of 2%.(2) mPTP opening of RPE cells: compared with the model group, the average fluorescence intensity of calcein in the intervention group increased(t=-5.521, P=0.001), but there was no significant change in the control group(P>0.05);compared with the control group, the average fluorescence intensity of calcein in the intervention group increased(t=-5.009, P=0.001).(3) The cytosolic Cyt-C content of RPE cells: compared with the model group, the cytosolic Cyt-C content of ARPE-19 cells in the intervention group decreased(t=2.968, P=0.018), but there was no significant change in the control group(P>0.05);compared with the control group, the cytosolic Cyt-C content in the intervention group decreased(t=2.717, P=0.026).(4)
作者
陈强
梁丽娜
庄曾渊
CHEN Qiang;LIANG Lina;ZHUANG Zengyuan(Eye Hospital,China Academy of Chinese Medical Sciences,Beijing 100040,China)
出处
《中国中医眼科杂志》
2020年第7期461-465,共5页
China Journal of Chinese Ophthalmology
基金
国家自然科学基金青年基金项目(81503621)
中央级公益性科研院所基本科研业务费专项资金(ZZ0908027)
北京市自然科学基金项目(7192236)。
