该文旨在研究sirtuin家族激活剂非瑟素(fisetin)对乙型肝炎病毒(hepatitis B virus,HBV)复制导致的氧化损伤过程中的保护作用,并初步分析其分子机制。在Huh-7细胞中转染HBV表达质粒p CH9/3091并同时用N-乙酰半胱氨酸(n-acetyl-cysteine,NAC)处理细胞,Mito SOX?Red试剂检测细胞线粒体活性氧类(reactive oxygen species,ROS)水平,DCFH-DA探针法检测细胞内ROS水平,丙二醛(malondialdehyde,MDA)试剂盒检测细胞MDA水平,Western blot检测细胞超氧化物歧化酶1(superoxide dismutase 1,SOD1)和SOD2的蛋白质水平。Huh-7细胞转染p CH9/3091的同时用非瑟素处理细胞,并检测细胞ROS、MDA、SOD1和SOD2蛋白质水平;Huh-7细胞转染p CH9/3091并用非瑟素处理细胞或同时沉默SOD2,用细胞免疫荧光及Western blot检测γ-H2AX(Phosphorylated Histone H2AX)的形成;MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]实验分析氧化应激条件下非瑟素对HBV复制细胞活率的影响;进一步应用Western blot检测丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)家族磷酸化水平及总蛋白水平的变化。结果显示,HBV复制明显增加细胞线粒体和细胞内ROS水平和细胞MDA水平,并且也明显降低SOD1和SOD2水平。NAC处理HBV复制细胞后,线粒体内和细胞内的ROS和细胞MDA水平明显减少。非瑟素处理HBV复制细胞后,HBV复制对细胞内ROS和MDA水平促进作用明显减弱;此外,HBV复制对细胞内SOD1和SOD2水平的抑制作用也明显减弱。非瑟素处理HBV复制细胞后,HBV复制对γ-H2AX形成的促进作用明显减弱,而SOD2沉默则减弱了非瑟素对HBV复制细胞中γ-H2AX形成的抑制作用。在氧化应激条件下,非瑟素明显减弱HBV复制对细胞活力的影响,SOD2沉默则减弱了非瑟素对HBV复制细胞活率的影响。非瑟素拮抗HBV表达对JNK(c-Jun N-terminal kinase)和p38(p38 kinase)磷酸化的促进作用,而SOD2沉默减弱了非瑟素对HBV复制细胞中JNK和p38磷酸化的抑制作用。该研究结果表明,非瑟素可能通过促进SOD2的表达拮抗HBV复制导致的氧化应激反应,并可能通过抑制JNK及p38的激活减少细胞氧化损伤,从而发挥保护细胞的作用。 The aim of this study was to investigate the protective effect of fisetin, a sirtuin family activator, on oxidative damage induced by hepatitis B virus (HBV) replication and its molecular mechanism. In Huh-7 cells transfected with HBV expression plasmid p CH9 / 3091 and simultaneously with N-acetyl-cysteine ​​(NAC) cells, Mito SOX? Red reagent detection of mitochondrial reactive oxygen species (reactive oxygen species oxygen species (ROS) were detected by enzyme-linked immunosorbent assay (ELISA), ROS level was detected by DCFH-DA probe method, MDA level was detected by malondialdehyde (MDA) kit, and superoxide dismutase 1 ) And SOD2 protein levels. Huh-7 cells were transfected with p CH9 / 3091 and treated with non-heptacetin, and the levels of ROS, MDA, SOD1 and SOD2 were detected. Huh-7 cells were transfected with p CH9 / 3091 and treated with non- Silence SOD2, and detect the formation of γ-H2AX (phosphorylated Histone H2AX) by immunofluorescence and Western blotting. MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- -sulfophenyl) -2H-tetrazolium, inner salt] were used to analyze the effect of non-heptacetin on the viability of HBV-infected cells under oxidative stress. The expression of mitogen-activated protein kinase (MAPK) ) Family phosphorylation level and total protein level changes. The results showed that HBV replication significantly increased mitochondrial and intracellular ROS levels and cellular MDA levels, and also significantly reduced the levels of SOD1 and SOD2. NAC treatment of HBV replication cells, mitochondria and intracellular ROS and cell MDA levels were significantly reduced. In addition, the inhibitory effect of HBV replication on the level of intracellular ROS1 and SOD2 was also weakened significantly after the treatment of non-heptacetin HBV replication cells, HBV replication on the role of ROS and MDA levels were significantly reduced. The effect of HBV replication on the formation of γ-H2AX was significantly weakened after the treatment of non-heptacetin HBV replication cells, while the silencing of SOD2 attenuated the inhibitory effect of non-heptacetin on the formation of γ-H2AX in HBV replication cells. Under the conditions of oxidative stress, non-heptacetin significantly attenuated the effect of HBV replication on cell viability, and SOD2 silencing attenuated the effect of non-heptacetin on the viability of HBV-replicated cells. Fetal deprivation inhibited the expression of c-Jun N-terminal kinase (JNK) and p38 (p38 kinase) phosphorylation, whereas silencing of SOD2 attenuated the inhibitory effect of fusidin on JNK and p38 phosphorylation in HBV-infected cells . The results of this study indicate that fusidin may antagonize the oxidative stress response induced by HBV replication by promoting the expression of SOD2, and may play a protective role by inhibiting the activation of JNK and p38 to reduce cell oxidative damage.