Hepatoprotective Effects of Astragaloside IV and Formononetin Against Oxidative Injury

The mitochondrial SIRT3 is a major deacetylase protein that regulates important mitochondrial biology, including metabolic pathways, reactive oxygen species detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response. We found that astragalus aqueous extract increased the activity of SIRT3 in vitro. We further explore the potential antioxidative effects of Astragaloside IV (AST-IV) and Formononetin (FMR) on induced oxidative stressed AML12 (alpha mouse liver 12) cells. The AML12 cell line was established from healthy hepatocytes from a mouse (CD1 Strain, Line MT42) transgenic for human TFG alpha. We first tested whether AST-IV or formononetin increases SIRT3 expression in cell model. We cultured AML12 and treated with various concentrations of AST-IV or formononetin in parallel with untreated control, negative control, and positive control. We found that AST-IV and formononetin induced SIRT3 expression through Western Blotting (WB) analysis. In addition, we assessed rescue effects of AST-IV and formononetin on t-BHP-induced oxidative injury in AML12 hepatocytes under three separate conditions: pre-treatment with AST-IV or formononetin, post-treatment with AST-IV or formononetin, and both pre-treatment and post-treatment with AST-IV and formononetin. Through CCK-8 analysis, we identified that both pre-treatment and post-treatment significantly increased cell proliferation on induced-oxidative stress AML12 cells compared to no treatment and control. Pre-treatment of AML12 cells before induction of oxidative stress with AST-IV and formononetin also showed cell proliferation but was less significant compared to AML12 cells that received both pre-treatment and post-treatment with AST-IV or formononetin. Post-treatment AST-IV or formononetin after induction of oxidative stress on AML12 cells did not significantly show any difference in cell proliferation compared to non-treatment and control. Significantly AST-IV and formononetin showed induction of the SIRT3 protein on tert-butyl hydroperoxide (t-BHP) induced oxidative stressed AML12 hepatocytes by WB. Taken together, the increase in cell proliferation is indicative of the antioxidative effects of AST-IV and formononetin through induction of SIRT3 protein. Further investigation is currently conducted to provide evidence of AST-IV and formononetin's mechanistic effect on inducing the expression of the SIRT3 protein-linked downstream pathways for their antioxidative benefit to hepatocytes. The ultimate goal is to utilize these SIRT3 modulators as pharmacologic agents in treating oxidative injury hepatocytes. © FASEB.