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  • br Materials and methods br Results br Discussion Oxidative


    Materials and methods
    Discussion Oxidative stress is a disturbance in the balance of pro- and antioxidant species, resulting in oxidative damage to membrane proteins, lipids, and DNA. Oxidative stress is a predisposing factor for the progression of metabolic diseases and liver injury (Rani et al., 2016, Yu et al., 2016). Possible sources of oxidative stress include mitochondrial dysfunction, peroxisomal β-oxidation, the release of ROS by pro-inflammatory cells, the activity of hypoxia-induced enzymes such as NADPH oxidase, and the activities of hepatic CYP450 enzymes (Rani et al., 2016, Shaik and Mehvar, 2010). A multitude of different high-fat diets have been used with relative fat fractions ranging between 20 and 60% of energy as fat, and the basic fat source varying between animal‐derived fats, e.g., lard or beef tallow, and plant oils, e.g., corn or safflower oil (Buettner, Schölmerich, & Bollheimer, 2012). A previous study in an impaired glucose tolerance model showed that C57BL/6 mice fed a high-fat diet (58% energy by fat) for 12 weeks exhibited signs of diabetes mellitus type 2 (Winzell & Ahrén, 2004). In this study, the ICR mice were given a daily HFFD diet consisting of hydrogenated soybean oil and a 20% fructose solution for 8 weeks (35.99% energy from fat), which increased the glucose AUC value and the mean glucose tolerance of the mice. In a high-fat diet rodent model, hepatic gluconeogenesis was observed to be reduced by inhibition of hepatic fatty pexidartinib oxidation (Lee et al., 2017, Oakes et al., 1997). Thus, a prolonged high-fat diet may induce fatty acid oxidation, leading to high gluconeogenesis and glucose tolerance. The antioxidant properties of VitE have been reported to prevent diabetes mellitus type 2 by protecting pancreatic β-cells against glucose toxicity, reducing MDA and TBARS and elevating GPx and SOD (Balbi et al., 2018, Kaneto et al., 1999). The results of this study revealed that a significant restoration of the glucose AUC value was an additional benefit of the antioxidant activity of THC toward glucose toxicity in mice with HFFD-induced oxidative stress. Correspondingly, the MDA level was elevated in liver, indicating the oxidative stress occurred during the HFFD period. In addition, effects on the antioxidative system were measureable through expression of Sod, Cat, and Gpx mRNA (Kumar et al., 2013, Zitka et al., 2012). Dhuley (1999) reported the elevation of antioxidative enzymes, namely SOD, CAT, and GPx, in the livers of rats receiving 20% coconut oil mixed into their normal diet for 90 days, along with as an increase in MDA and hydroperoxide levels (Dhuley, 1999). Moreover, GPx and SOD activities, and MDA level have been shown to be elevated in the plasma of NAFLD patients (Kumar et al., 2013). In the present study, expressions of hepatic Sod and Cat mRNA were increased in HFFD mice. The SOD enzymes, cytosolic (CuZn-Sod) and mitochondrial (Mn-Sod), catalyze dismutation of superoxide (O2–) into oxygen (O2) and hydrogen peroxide (H2O2) (Polavarapu et al., 1998). Mn-SOD is a more inducible enzyme and is relatively resistant to chemical inhibitors of CuZn-SOD, such as cyanide. CuZn-SOD has also been shown to be sensitive to H2O2-induced inactivation (Hodgson & Fridovich, 1975). In the cell, these antioxidant enzymes work together; SOD lowers the steady state intracellular concentration of superoxide while CAT and GPx remove H2O2 generated by SOD (Chance, Sies, & Boveris, 1979). Thus, our findings indicate that the HFFD caused oxidative stress via production of both superoxide and hydrogen peroxide. The consumption of a high-fat and/or high-fructose diet has been implicated in the progression of many diseases, including metabolic disorders, NAFLD, and NASH (Ouyang et al., 2008, Yang et al., 2012). In the present study, oxidative stress and liver injury were induced in the HFFD mice as demonstrated by increases in lipid peroxidation, the serum AST level, and the AST/ALT ratio. These findings confirmed our previous data in which a HFFD induced oxidative stress and depleted GSH stores in the livers and brains of ICR mice (Jarukamjorn et al., 2016). The increases in serum AST and ALT levels are indicative of hepatocellular damage based on the release of AST and ALT from hepatocytes into the bloodstream (Lin et al., 2015). Dramatic increases in AST and ALT levels were observed in mice fed a trans-fatty acid in a severe NAFLD model (Obara et al., 2010). Regarding NAFLD, an AST/ALT ratio of less than one is considered to be typical, while an AST/ALT ratio of more than one indicates hepatic fibrosis and progression of the disease (Cortez-Pinto & Ermelinda Camilo, 2004). A ratio greater than two is generally observed in alcoholic fatty liver disease (AFLD) and indicates an advanced stage of the disease, such as alcoholic cirrhosis, especially in patients with higher AST activities. In the present study, HFFD mice with an AST/ALT ratio of 1.14 ± 0.20 (Table 2) were considered as being consistent with progressive NAFLD with hepatic fibrosis. As antioxidants, both VitE and THC improved the signs of oxidative damage in the liver. VitE is widely accepted as one of the most potent antioxidants in nature and a therapeutic agent against human diseases (El Hadi et al., 2018, Peh et al., 2016). VitE is used as a medicinal treatment of NAFLD, the outcomes of which include improved hepatic fibrosis scores and liver stiffness, and reduced AST and ALT levels (Fukui et al., 2015, Sato et al., 2015). THC was previously noted to protect against oxidative damage to the liver, kidneys, and heart, promoting significant decreases in AST and ALT levels associated with myocardial infarction in rats (Ali et al., n.d., Muthumani and Miltonprabu, 2015, Naito et al., 2002, Okada et al., 2001). Moreover, THC has been shown to normalize AST and ALT levels in rats following arsenic-induced oxidative damage and to improve metabolic condition through the reduction of cholesterol, free fatty acid, and triglyceride levels (Muthumani and Miltonprabu, 2015, Pari and Murugan, 2007), consistent with our results.