Tetramethylpyrazine prevents liver fibrotic injury in mice by targeting hepatocyte-derived and mitochondrial DNA-enriched extracellular vesicles

Liver fibrosis is the common consequence of almost all liver diseases and has become an urgent clinical problem without efficient therapies. Recent evidence has shown that hepatocytes-derived extracellular vesicles (EVs) play important roles in liver pathophysiology, but little is known about the role of damaged hepatocytes-derived EVs in hepatic stellate cell (HSC) activation and following fibrosis. Tetramethylpyrazine (TMP) from Ligusticum wallichii Franchat exhibits a broad spectrum of biological activities including liver protection. In this study, we investigated whether TMP exerted liver-protective action through regulating EV-dependent intercellular communication between hepatocytes and HSCs. Chronic liver injury was induced in mice by CCl₄ (1.6 mg/kg, i.g.) twice a week for 8 weeks. In the last 4 weeks of CCl₄ administration, mice were given TMP (40, 80, 160 mg·kg⁻¹·d⁻¹, i.g.). Acute liver injury was induced in mice by injection of a single dose of CCl₄ (0.8 mg/kg, i.p.). After injection, mice were treated with TMP (80 mg/kg) every 24 h. We showed that TMP treatment dramatically ameliorated CCl₄-induced oxidative stress and hepatic inflammation as well as acute or chronic liver fibrosis. In cultured mouse primary hepatocytes (MPHs), treatment with CCl₄ or acetaminophen resulted in mitochondrial dysfunction, release of mitochondrial DNA (mtDNA) from injured hepatocytes to adjacent hepatocytes and HSCs through EVs, mediating hepatocyte damage and fibrogenic responses in activated HSCs; pretreatment of MPHs with TMP (25 μM) prevented all these pathological effects. Transplanted serum EVs from TMP-treated mice prevented both initiation and progression of liver fibrosis caused by CCl₄. Taken together, this study unravels the complex mechanisms underlying the protective effects of TMP against mtDNA-containing EV-mediated hepatocyte injury and HSC activation during liver injury, and provides critical evidence inspiring the development of TMP-based innovative therapeutic agents for the treatment of liver fibrosis.