## 4 Discussion  
Incidence of MASH, a chronic liver disease characterized by hepatic steatosis, inflammation, and fibrosis, steadily increases with obesity and metabolic syndrome [14]. Untreated MASH progresses to cirrhosis and liver cancer, necessitating the development of effective measures for its prevention and treatment [15]. _D. racemosum_, belonging to the _Hanamelidaceae_ family, is an evergreen plant species native to East Asia, including Jeju Island in Korea and Japan. Traditionally used for its anti-inflammatory and antioxidant properties, DRE also exerts anti-obesity effects on 3T3-L1 adipocytes in vitro and in high-fat diet-induced obese mouse models in vivo by inhibiting lipid accumulation and body weight gain [10]. As oxidative stress and chronic inflammation caused by excessive lipid accumulation are key pathological features of MASH, natural compounds with antioxidant and anti-inflammatory properties are potential candidates for its prevention and treatment [16]. Based on the previously reported anti-obesity activity of DRE, we hypothesized that its extract protects against MASH. However, specific effects of the \(D\). _racemosum_ extract on MASH remain unclear. Therefore, in this study, we investigated the protective effects of DRE in a cellular model of MASH to elucidate its action mechanisms. Our findings provide valuable insights for the development of new therapeutic agents against MASH. In this study, we demonstrated the hepatotoxicity effects of DRE using in vitro and in vivo MASH models. We showed that DRE significantly attenuated lipid accumulation, inflammation, and fibrosis in PA-induced AML12 cells and HFMCD diet-induced MASH model mice. In AML12 hepatocytes, DRE reduced intracellular TG levels and lipid droplet accumulation, as evidenced by oil red O staining. Moreover, DRE downregulated the levels of key lipogenic (FAS, SREBP1c, and ACC) and pro-inflammatory (CD68, IL-6, and monocyte chemoattractant protein-1) genes. These results suggest that DRE exerts anti-lipogenic and anti-inflammatory effects on hepatocytes under lipotoxic conditions. DRE decreased the fibrotic marker (_COL1A1_, _COL1A2_, and _TIMP1_) levels in PA-treated cells. Additionally, it significantly reduced liver weight and the liver-to-body weight ratio and improved the histological features by decreasing steatosis and hepatocellular ballooning in mice. Metabolic and fibrogenic gene expression levels in liver tissues were determined for the mechanistic analysis of these effects. Indeed, DRE increased the _CPT-1_ levels and decreased the _CD36_ and transforming growth factor-\(\beta\) levels, exerting potent antifibrotic effects. Although changes in _PPARa_ and _IL-6_ levels were not statistically significant, the overall expression patterns were consistent with a hepatotoxicity profile. Among the well-studied plant-derived candidates for MASH treatment, QUE and its plant sources exhibit comparable biological activities. For instance, QUE-enrichedextracts of onion peel, Morus alba, and Sophora japonica reduce hepatic steatosis and downregulate the pro-inflammatory cytokine and fibrogenic marker levels in high-fat- and methionine- and choline-deficient-fed rodent models [17; 18; 19]. In this study, rutin, a glycosylated derivative of QUE, was identified as the major compound in DRE via HPLC analysis. Rutin shares structural and functional properties with QUE, suggesting potentially equivalent modes of action [20]. This similarity suggests that the hepatoprotective efficacy of DRE is partly attributed to its rutin content. Silymarin extracted from _Silybum marianum_ (milk thistle) is a well-known hepatocyte agent with antioxidant and antifibrotic activities. Although silymarin strongly suppresses oxidative stress and collagen deposition, it shows limited efficacy in reversing established steatohepatitis [21].