Combined therapy with ligustrazine and paeonol mitigates hepatic fibrosis through destroying mitochondrial integrity of stellate cell.

This study investigates the inhibitory effect and potential mechanism of ligustrazine combined with paeonol on hepatic fibrosis, as to provide a new therapeutic strategy for clinical hepatic fibrosis. The degree of liver injury collagen deposition and inflammation was assessed by hematoxylin and eosin staining, Masson, Sirius red staining and biochemically serum analysis. ATP and ROS levels in each group were detected by chemical fluorescence method. The apoptotic rate was measured by Tunel assay. Mito-Tacker fluorescence staining and mitochondrial DNA copy number were measured to observe the effect of ligustrazine or/and paeonol on mitochondrial function of hepatic stellate cell (HSC). The expression of relevant proteins and genes were evaluated by using immunofluorescence RT-PCR and western blot. Ligustrazine or/and paeonol significantly improve the pathological changes in liver tissue induced by CCl₄, however, they reduced the levels of liver and fibrosis markers in tissue and serum. ROS, NOX1 and NOX2 were significantly increased and GSH was decreased in HSC, with the intervention of Ligustrazine or/and paeonol. We further found that Ligustrazine or/and paeonol can effectively inhibit liver inflammation in vivo. The expression of TNF-α, IL-6 and IL-8 was upregulated in HSC. Moreover, Ligustrazine or/and paeonol promotes apoptosis and inhibit proliferation of HSC. Additionally, the inhibiting effects of the drug on collagen deposition was due to the interference with the expression of signaling pathway related proteins and genes such as, MMPS, TGF-β, PDGF and BMP-2 in HSC. Mitochondrial activity of HSC was inhibited by Ligustrazine or/and paeonol. The inhibitory effects of ligustrazine or/and Paeonol on mitochondrial function is partially balanced by mitochondrial protective agent SS-31. Ligustrazine combined with paeonol exerts significant anti-hepatic fibrosis effect in vivo and in vitro. This may due to the disruption of HSC mitochondrial function, thereby induced promoting oxidative stress, apoptosis, inflammation and inhibiting the formation and deposition of extracellular matrix.