美洲大蠊提取物对H2O2诱导的PC12细胞氧化损伤的保护作用

目的:探讨美洲大蠊提取物(PAS840)对大鼠肾上腺嗜铬细胞瘤(PC12)细胞氧化损伤模型的保护作用及机制。方法:采用H₂O₂刺激PC12细胞建立神经细胞氧化损伤模型,实验分为正常组(Con)、模型组(Mod)、PAS840低中高剂量组(20,50,125 μg·mL^-1的PAS840培养基溶液进行处理),采用倒置显微镜观察细胞形态并采用CCK-8法检测各组细胞存活率,生化试剂盒检测各组超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、乳酸脱氢酶(LDH)、谷胱甘肽(GSH)和丙二醛(MDA)的水平;DCFH-DA荧光探针检测各组活性氧簇(ROS)的水平;流式细胞术检测各组细胞凋亡率;JC-1法染色检测各组细胞线粒体膜电位(MMP);RT-qPCR检测各组Nrf2/HO-1通路因子(Nrf2、Keap1、HO-1和NQO1)、凋亡因子(Bcl-2、Bax和Caspase-3)、炎症因子(TNF-α、IL-1β和IL-6)、乙酰胆碱酯酶(AchE)和过氧化氢酶(CAT) mRNA的表达水平;Western blot法检测各组Nrf2、HO-1、Bcl-2、Bax和Caspase-3蛋白的表达水平。结果:PAS840可显著提高氧化损伤细胞的存活率、MMP及SOD、GSH-Px和GSH的水平,降低LDH、MDA和ROS的水平;显著降低Keap1、TNF-α、IL-1β、IL-6和AchE mRNA表达的同时,显著增加CAT和NQO1 mRNA的表达,显著降低Nrf2、Bax和Caspase-3的mRNA及其蛋白表达,显著增加HO-1和Bcl-2的mRNA及其蛋白表达。结论:PAS840可以抑制H₂O₂诱导的PC12细胞凋亡,减轻炎症,其机制可能与降低ROS、调控Nrf2/HO-1通路因子减轻细胞的氧化损伤程度有关。

Protective effect of Periplaneta americana extract on H2O2-induced oxidative damage of PC12 cells

OBJECTIVE To investigate the protective effect and mechanism of Periplaneta americana extract (PAS840) on oxidative damage model of rat pheochromocytoma (PC12) cells.METHODS PC12 cells were stimulated with H₂O₂ to establish the oxidative damage model and divide into control group (Con), model group (Mod) and PAS840 low, medium and high dose groups (the concentrations of 20, 50 and 125 μg·mL^-1 of PAS840 medium solution were used for treatment). Cell morphology was observed under an inverted microscope, and cell survival rate was detected by CCK-8 method. The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), lactate dehydrogenase (LDH), glutathione (GSH) and malondialdehyde (MDA) in each group were detected by biochemical kits. The level of reactive oxygen species (ROS) in each group was detected by DCFH-DA fluorescent probe, cell apoptosis rate in each group was detected by flow cytometry, and mitochondrial membrane potential (MMP) was detected by JC-1 staining. The mRNA expression in each group of Nrf2/HO-1 pathway factors (Nrf2, Keap1, HO-1 and NQO1), apoptosis factors (Bcl-2, Bax and Caspase-3), inflammatory factors (TNF-α, IL-1β and IL-6), acetylcholinesterase (AchE) and catalase (CAT) were detected by RT-qPCR and the protein expression in each group of Nrf2, HO-1, Bcl-2, Bax and Caspase-3 were detected by Western blot.RESULTS PAS840 significantly improved oxidative damaged cells survival rate, MMP and the levels of SOD, GSH-Px and GSH while decreased the levels of LDH, MDA and ROS. The Keap1, TNF-α, IL-1β, IL-6 and AchE mRNA expression were significantly decreased, meanwhile the CAT and NQO1 mRNA expression were significantly increased. The mRNA and protein expression of Nrf2, Bax and Caspase-3 were significantly decreased, and the mRNA and protein expression of HO-1 and Bcl-2 were significantly increased.CONCLUSION PAS840 can inhibit H₂O₂-induced apoptosis of PC12 cells and reduce inflammation. The mechanism may be related to reducing of ROS and regulating Nrf2/HO-1 pathway factors to reduce the degree of oxidative damage cells.