## 4 Discussion  
Alcohol abuse can cause severe damage to the liver and ALD due to OS, lipid peroxidation, and inflammation induced by ethanol and its metabolites [26]. Some components of apples have hepatoprotective effects, including antioxidant activity and modulation of lipid metabolism [18; 27; 28]. In addition, apple polyphenols ameliorate alcohol-induced hepatocyte and neuronal injury [29; 30]. Therefore, we hypothesized that apples protected against ALD. ALT and AST are traditional markers of liver injury since they are released into the circulation when xenobiotics impair the structural integrity of hepatocytes [31]. In our study, ethanol treatment for 10 days increased in rats. HE staining showed that ethanol caused significant pathological changes in the liver, including edema and the impairment of hepatocyte morphogenesis, consistent with the previous studies [32; 33; 34], indicating that we successfully established a rat model of ALD. Apple intake improved ALD by decreasing the serum levels of ALT and AST, reducing edema, and improving hepatocyte morphogenesis. Some compounds in apples are known to alleviate ALD [16; 35]. ALD affects the mRNA and protein expression of enzymes involved in alcohol metabolism, and OS and proinflammatory cytokines reduce the mRNA and protein expression of ADH and ALDH [19]. ADH and ALDH play a vital role in alcohol catabolism. The impairment of alcohol metabolism increases alcohol toxicity and stimulates feedback pathways that exacerbate liver injury in vivo [36]. ADH metabolizes ethanol to toxic acetaldehydes [37]. Mammalian ADH has six isoforms (ADH1-ADH6), and ADH1 and ADH4 are involved in ethanol metabolism [38]. Acetaldehydes are metabolized to acetate  
Figure 6: Gene expression levels of involved in lipid metabolism and liver fibrosis. (**A**) Analysis of the gene expression levels of fatty acid-binding protein 2 (_Fabp2_), acetyl-CoA carboxylase alpha (_Acac_), carnitine palmitosyltransferase 1A (_Cpt1a_), collagen type I alpha 1 chain (_Col1a1_), and alpha-2 smooth muscle actin (_Acta2_) in liver tissues by RT-qPCR. (**B**) sPCR to verify differential expression of _Fabp2_, _Acac_, _Cpt1a_, _Col1a1_, and _Acta2_ in the E and EA groups. ns: \(p>0.05\), *: \(p<0.05\), **: \(p<0.01\), ***: \(p<0.001\), ***: \(p<0.001\), ***: \(p<0.001\). by ALDHs, including ALDH1A1[36; 39]. Alcohols are further metabolized by ALDH to carbon dioxide and water [3]. In the present study, we found that apple consumption partially restored the mRNA expression and enzyme activity of ADH and ALDH, suggesting that apples may reduce the effects of ALD by improving the ability to metabolize alcohol. However, the underlying mechanism has not been evaluated. Ethanol abuse disrupts the oxidant-antioxidant balance. Ethanol increases the activity of CYP2E1, which is involved in alcohol metabolism, and the production of superoxide radicals, hydrogen peroxide, nitric oxide, and hydroxyl radicals [40]. Moreover, ethanol reduces the activity of antioxidant enzymes (SOD and GPx) and the expression of antioxidant genes (_Nr_/2, _Ho_-1) in the liver, leading to lipid peroxidation and inflammation [41]. Apple phlorizin exerts antioxidant and hepatotoxicity activity by increasing _Nr_/2 expression and the activity of antioxidant enzymes [42]. Further, apple consumption increases the serum activity of SOD and GPx [43]. Flavonols (quercetin-3-glucose, quercetin-3-D-galactoside, and quercetin-3-rhammoside) from 'Golden Delicious' apples reduced OS in obese rats [12]. These results demonstrate that apples have an excellent antioxidant capacity and protect against ALD by reducing OS. Alcohol abuse induces inflammatory responses in the liver by activating inflammatory genes and pathways, thereby inducing the expression of proinflammatory cytokines. IL-1\(\beta\) and TNF-\(\alpha\) play important roles in ALD [44].