## 4 Discussion  
In 2012, the Korea Ministry of Food and Drug Safety approved the use of _Rhus verniciflua_ stokes (RV) extracts devoid of urushiol, which causes allergic contact dermatitis known as urushiol-induced contact dermatitis. RV extracts are commercially available in various forms as food and medicine in Korea. Although RV contains urushiol, it is useful as food and medicine due to its wide range of biological activities. We have previously reported the presence of many bioactive compounds, including gallic acid, fustin, fiesetin, 1-hexacosanol, and quercetin in fermented _Rhus verniciflua_ stokes (FRVE). These compounds act on nonalcoholic fatty liver disease (NAFLD) by upregulating AMP-activated protein kinase (AMPK) [5]. Here, we performed a preclinical study to further confirm the anti-hepatic lipogenic effect of FRVE and observed results consistent with our previous in vitro study. Outcomes of our present study showed anti-nonalcoholic fatty liver effect and safety of FRVE usage in an HFD-induced NAFLD animal model. As demonstrated in Figure 1, urushiol was not detected in the FRVE used in the animal study by HPLC. We confirmed the safety of FRVE and the presence of the main active ingredients (fiesetin and fustin). Obesity is associated with the development of NASH, NAFLD, liver fibrosis, cirrhosis, and hepatocellular carcinoma [26; 27]. In a HFD, 30-75% of the total calories are derived from saturated fatty acids, and HFD mouse model is a useful system to study metabolic alterations and NAFLD. HFD-fed rodents develop metabolic diseases such as obesity, dyslipidemia, and diabetes and exhibit changes in lipogenic regulators such as sterol regulatory element-binding protein 1 ( SREBP-1), liver X receptor, and inflammatory cytokines, depending on the duration of the HFD intake; these pathologies  
Figure 6: Effect of FRVE on SPREBP-1, LDLR, and AMPK protein expression in HFD-induced nonalcoholic fatty liver model. Representative pictures of SREBP-1, LDLR, and AMPK expression in the liver tissue section via immunohistochemical staining. Staining intensity was quantified using Image pro plus program. Values were expressed as mean \(\pm\) SD in each group (\(n=6\)). \({}^{*}p<0.05\), \({}^{**}p<0.01\), and \({}^{***}p<0.001\) (compared to HFD control group). \(\#p<0.05\), \(\#p<0.01\) (compared to NCD control group). closely resembles the pathological (liver inflammation and fibrosis) and molecular alterations observed in human NAFLD [28; 29]. Interestingly, FRVE decreased the body weight, intra-abdominal fat weight, and liver weight in the HFD-induced NAFLD model, without inducing toxicity; status of liver damage was confirmed by testing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (Figure 2). Serum AST and ALT levels are widely used as serum biomarkers of liver injury like NASH. Increased ALT and AST are hallmark features of NAFLD, and their levels also increase in obesity in humans [30] as well as rodents [31]. Our results showed that FRVE protected against liver injury by decreasing the levels of ALT and AST, which were induced by HFD (Figure 3A,B). The reduction of body weight by FRVE treatment in HFD-fed mice is associated with the inhibition of lipid synthesis. FRVE decreased the levels of TG and TC in both the serum and liver, and decreased hepatic lipid accumulation as observed by oil-red O staining (Figures 3C,D and 4). These results are in concordance with a previous study, such as a hyperlipidemia study of western HFD for two weeks with the use of allergen-free RVS obtained by boiling, demonstrated a decrease in hepatic lipid accumulation, body weight, and total cholesterol in the RVS-treated group [32].