基于Nrf2/ARE信号通路探讨当归-川芎含药血清对H_(2)O_(2)致PC12细胞氧化损伤的保护作用

Protective Effect of Angelicae Sinensis Radix-Chuanxiong Rhizoma Medicated Serum Against HO-induced Oxidative Damage of PC12 Cells Based on Nrf2/ARE Signaling Pathway

目的:研究当归-川芎含药血清(Angelicae Sinensis Radix-Chuanxiong Rhizoma medicated serum,ASRCRS)对过氧化氢(H_(2)O_(2))诱导高分化大鼠肾上腺嗜铬瘤细胞(PC12细胞)氧化损伤的保护作用及其分子机制。方法:采用H_(2)O_(2)体外诱导PC12细胞氧化损伤,并以终体积分数为15%的ASRCRS低、中、高剂量组进行干预。采用噻唑蓝(MTT)比色法检测细胞存活率;荧光倒置显微镜观察细胞形态;试剂盒检测细胞上清液乳酸脱氢酶(LDH),丙二醛(MDA)含量和细胞内超氧化物歧化酶(SOD)活力及活性氧(ROS)分布水平;Annexin V-FITC/PI双染法检测细胞凋亡情况;蛋白免疫印迹法(Western blot)检测核转录因子E2相关因子2(Nrf2),Kelch样环氧氯丙烷相关蛋白-1(Keap1),血红素加氧酶-1(HO-1),SOD1蛋白的表达水平。结果:选择300μmol·L^(-1)H_(2)O_(2)刺激24 h作为氧化损伤造模的条件。与正常组比较,H_(2)O_(2)模型组细胞形态异常,细胞存活率显著降低(P<0.01);LDH,MDA含量升高(P<0.01),SOD活力降低,ROS在细胞内分布增加;Nrf2,HO-1,SOD1蛋白表达水平均明显下调(P<0.05,P<0.01),Keap1蛋白表达水平明显上调(P<0.01)。与模型组比较,ASRCRS不同剂量组均能改善细胞形态,提高细胞存活率,抑制细胞凋亡;显著提高SOD酶活力(P<0.01),抑制LDH的释放(P<0.01)和ROS的生成,降低MDA含量(P<0.01);明显上调Nrf2,HO-1,SOD1蛋白表达水平(P<0.05,P<0.01),显著下调Keap1蛋白表达水平(P<0.01)。结论:当归-川芎含药血清可能是通过上调Nrf2的蛋白表达,激活Nrf2/抗氧化反应元件(ARE)信号通路,增强细胞抗氧化损伤能力,抑制细胞凋亡,从而发挥保护H_(2)O_(2)损伤的PC12细胞的作用。

Objective: To investigate the protective effect and molecular mechanism of Angelicae Sinensis Radix-Chuanxiong Rhizoma medicated serum(ASRCRS)against oxidative damage of PC12 cells induced by H_(2)O_(2). Method: Oxidative damage of PC12 cells was induced by H_(2)O_(2) in vitro,and intervention was performed in the low-,medium-,and high-dose ASRCRS groups with a final volume fraction of 15%. The cell viability was determined by methyl thiazolyl tetrazolium(MTT)assay. Cell morphology was observed by an inverted fluorescence microscope. The content of lactate dehydrogenase(LDH)and malondialdehyde(MDA),the activity of superoxide dismutase(SOD),and the distribution of reactive oxygen species(ROS)in the cell supernatant were detected by the kits. Cell apoptosis was detected by Annexin V-FITC/PI double staining. The protein expression levels of nuclear factor E2-related factor 2(Nrf2),Kelch-like epichlorohydrin associated protein-1(Keap1),heme oxygenase-1(HO-1),and SOD1 were detected by Western blot. Result: Oxidative damage was induced by 300 μmol·L^(-1) H_(2)O_(2) for 24 hours. Compared with the normal group,the model group showed abnormal cell morphology,reduced cell viability(P<0.01),increased LDH and MDA(P<0.01),blunted SOD activity,elevated intracellular distribution of ROS,down-regulated protein expression of Nrf2,HO-1,and SOD1(P<0.05,P<0.05),and up-regulated protein expression of Keap1(P<0.01). Compared with the model group,ASRCRS groups displayed improved cell morphology,increased cell viability,inhibited cell apoptosis,potentiated SOD activity(P<0.01),suppressed release of LDH(P<0.01)and generation of ROS,decreased content of MDA(P<0.01),up-regulated protein expression of Nrf2,HO-1 and SOD1(P<0.05,P<0.01),and down-regulated protein expression of Keap1(P<0.01). Conclusion: ASRCRS could protect PC12 cells from oxidative damage induced by H_(2)O_(2) by up-regulating the expression of Nrf2 to activate the Nrf2/antioxidant response element(ARE)signaling pathway,enhancing the ability to resist oxidative damage,and inhibiting cell apoptosis.