Thus, the present study represents attempted to demonstrate that 1) p66shc expression was significantly increased in NAFLD mice, 2) the p66shc/cytochrome C pathway was a pivotal therapeutic target for preventing NAFLD progression via inhibiting ROS overproduction, cell apoptosis and hepatic steatosis, 3) miR-96-5p could negatively regulate p66shc protein expression via directly binding to p66shc mRNA, 4) the protective effect of catalpol might be associated with upregulating miR-96-5p levels. The gene discussed is CYCS; the disease is metabolic dysfunction-associated steatotic liver disease.