山姜素靶向调控Nrf2抑制H9C2细胞氧化应激反应

Alpinetin targets Nrf2 to inhibit oxidative stress in H9C2 cells

目的 观察山姜素(alpinetin,ALP)对脂多糖(lipopolysaccharide,LPS)诱导大鼠心肌细胞(H9C2)的作用,并探讨其机制。方法 采用不同因素干预H9C2细胞后,使用细胞毒性检测试剂盒(cell counting Kit-8,CCK-8)检测细胞活性;实时荧光定量聚合酶链式反应(real-time fluorescence PCR,RT-PCR)检测基因的表达;Western blot检测蛋白的表达;酶标仪及免疫荧光显微镜检测细胞中活性氧(reactive oxygen species,ROS)的表达;试剂盒检测细胞内乳酸脱氢酶(lactate dehydrogenase,LDH)活性、丙二醛(malondialdehyde,MDA)含量及超氧化物岐化酶(superoxide dismutase,SOD)活力。结果 细胞活性:LPS组细胞活性明显低于对照组(P <0.01),加入山姜素后,可显著提高细胞活性(P <0.01),LPS组LDH活性明显高于对照组(P <0.01);氧化应激分子及基因表达:与对照组相比,LPS组心肌细胞中ROS的表达水平显著上调,细胞内MDA含量显著升高,SOD活力显著下降,抗氧化因子基因表达水平均被显著抑制(P <0.01),加入ALP后可显著抑制ROS的表达水平及细胞内MDA含量,提高细胞内SOD含量,同时上调抗氧化因子基因表达水平(P <0.01);信号通路:LPS组中Keap1蛋白表达水平高于对照组,Nrf2蛋白表达水平低于对照组(P <0.01),加入ALP(10 mg/L)后可逆转以上两种蛋白的表达水平(P <0.01);验证机制:与LPS组相比,LPS+ALP(10 mg/L)可抑制ROS的表达(P <0.01),加入siNrf2后则可显著逆转ALP带来的保护作用,ROS水平显著上调(P <0.01),抗氧化因子基因表达水平同样得到证实。结论 山姜素可靶向调控Nrf2信号通路的活化从而改善LPS诱导的H9C2氧化应激反应。

Objective To observe the effect of alpinetin(ALP)on lipopolysaccharide(LPS)⁃induced rat cardiomyocytes(H9C2),and to explore its mechanism.Methods After intervening H9C2 cells with different factors,cytotoxicity detection kit(CCK⁃8)was used to detect cell activity;real⁃time fluorescence PCR(RT⁃PCR)was used to detect gene expression;Western blot was used to detect the expression of protein;microplate reader and immunofluorescence microscope were used to detect the expression of reactive oxygen species(ROS)in cells;the kits were used to detected the intracellular lactate dehydrogenase(LDH)activity,malondialdehyde(MDA)content and superoxide dismutase(SOD)vitality.Results(1)Cell viability:the cell viability in the LPS group was significantly lower than that in the control group(P<0.01),and the addition of ALP could significantly increase the cell viability(P<0.01),and the LDH activity was significantly higher than that of the control group(P<0.01);(2)oxidative stress molecules and gene expression:compared with the control group,the expression level of ROS in the cardiomyocytes of the LPS group was significantly up⁃regulated,and the intracellular MDA content was significantly increased,the SOD vitality decreased significantly,and the expression levels of antioxidant factor genes were significantly inhibited(all P<0.01).After adding ALP,the expression level of ROS and the content of MDA in cells could be significantly inhibited,and the vitality of SOD in cells could be increased,antiox⁃idant factor genes expression levels are up⁃regulated(all P<0.01);(3)signal pathway:the protein expression level of Keap1 in the LPS group was higher than that in the control group,and the protein expression level of Nrf2 was lower than that in the control group(all P<0.01).After adding ALP(10 mg/L)The expression levels of the above two proteins can be reversed(P<0.01);(4)mechanism confirmation:compared with the LPS group,ALP(10 mg/L)can inhibit the expression of ROS(P<0.01).After adding siNrf2,the protective effect of ALP was significantly reversed,the level of ROS was significantly increased(P<0.01),and the expression level of antioxi⁃dant factor genes was also confirmed.Conclusion Alpinetin can target the activation of Nrf2 signaling pathway to improve LPS⁃induced H9C2 oxidative stress response.