槟榔碱对H_(2)O_(2)诱导SH-SY5Y神经细胞氧化应激损伤的保护作用及机制研究

Neuroprotective Effect and Mechanism of Arecoline on H_(2)O_(2)-induced Damage in SH-SY5Y Cell

本研究通过建立过氧化氢(H_(2)O_(2))诱导的SH-SY5Y神经细胞氧化应激模型,旨在探究槟榔碱对氧化应激诱导神经细胞损伤的保护作用及其作用机制。采用CCK-8法检测细胞活力,采用分光光度法检测乳酸脱氢酶(LDH)释放、丙二醛(MDA)含量、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,采用流式细胞术检测细胞凋亡、线粒体膜电位,采用Western blot技术检测Nrf2、HO-1、Keap1、Bcl-2、Bax、Caspase-3的表达。结果表明:槟榔碱干预均能有效降低细胞凋亡并显著上调线粒体膜电位;在提高SOD、CAT水平的同时降低MDA水平;140μmol/L槟榔碱处理能够显著上调Nrf2、HO-1、Bcl-2蛋白的表达(P<0.001,P<0.0001),并下调Keap1、Bax、Caspase-3蛋白的表达(P<0.001,P<0.0001)。表明槟榔碱能够有效改善H_(2)O_(2)诱导的氧化应激损伤,其作用机制与激活Nrf2/HO-1信号通路、提升细胞抗氧化活力、调控Bcl-2/Bax/Caspase-3信号通路和抑制细胞凋亡有关。

The aim of this study was to explore the protective effect of arecoline on the cell model of H_(2)O_(2)-induced oxidative stress damaged model in SH-SY5Y cells and elucidate its mechanism.Cell viability was detected by CCK-8 assay.Spectrophotometry was used to detect LDH release,MDA,SOD,and CAT contents.Flow cytometry was used to detect cell apoptosis and mitochondrial membrane potential.Western blot was used to detect the expressions of Nrf2,HO-1,Keap1,Bcl-2,Bax and Caspase-3.Arecoline could reduce cell apoptosis and significantly increase mitochondrial membrane potential.The level of SOD elucidateand CAT increased while the level of MDA decreased.Arecoline 140μmol/L group could significantly up-regulate the protein expression of Nrf2,HO-1 and Bcl-2(P<0.001,P<0.0001),and down-regulate the protein expression of Keap1,Bax and Caspase-3(P<0.001,P<0.0001).Conclusions:arecoline can effectively improve H_(2)O_(2)-induced oxidative stress injury in SH-SY5Y cell by activating the Nrf2/HO-1 signaling pathway,enhancing the activities of antioxidant enzymes and regulating the oxidation reduction system of cells,as well as regulating the Bcl-2/Bax/Caspase-3 signaling pathway and inhibiting cell apoptosis.