Abstract:

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 wassignificantly 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.

Key words:

H9C2 cells, oxidative stress, alpinetin, Nrf2 signaling pathway