Study On Liver Protection Of Quercetin And Its Mechanism In High Fat Diet-induced NAFLD In Rats

With improved living standards and changed dietary habits, people have more chances to intake high-fat diets, and thus, the incidence of non-alcoholic fatty liver disease (NAFLD) is gradually increased, as a main chronic liver disease threatens human health, and yet there are no optimal treatments. In recent years, more studies have focused on using active natural plant ingredients as potential treatments for chronic liver diseases. Quercetin is a natural polyphenolic flavonoid, which is widely distributed in fruits, vegetables, and Chinese herbs. As quercetin has a polyphenol hydroxyl structure, so it has a variety of biological and pharmacological activities, such as anti-oxidation, anti-inflammation, anti-cancer, anti-virus and anti-cardiovascular disease. More studies have confirmed that oxidative stress injury and inflammation constitute key pathogenic mechanisms of NAFLD. Our previous study showed that quercetin had antioxidant activity that could alleviate fructose or streptozotocin-induced NAFLD and liver inflammatory response in rats. Based on the above these results, this study used a rat model of high fat-induced NAFLD to investigate the protective effect and possible mechanism of quercetin.In this thesis, male Sprague Dawley (SD) rats were fed with high fat diet to develop a animal model of NAFLD. Model group and respective treatment groups were continuously given high fat diet for 8 weeks. After first 4 weeks, quercetin (50 and 100 mg/kg/d) and allopurinol (5 mg/kg/d) were treated on these rats by gavage for the next 4 weeks. Normal control group received normal water and diet for 8 weeks. Compared with normal control group, significantly increased body weight, as well as triglyceride (TG) and total cholesterol (TC) levels in serum and liver were observed in model control group. Hematoxylin and eosin (HE) staining results showed the infiltration of inflammatory cells in model control group, indicating liver inflammation. Oil red staining showed red lipid droplets in the liver of model group, suggesting lipid accumulation phenomenon. These observations demonstrated that the animal model of NAFLD was successful by high fat diet. Compared with the model group, quercetin (50 and 100 mg/kg/d) could significantly reduce body weight, serum and liver TG and TG levels, indicating that quercetin could effectively relieve high fat diet-induced hepatic lipid accumulation and lipid disorders. Positive drug allopurinol (5 mg/kg/d) significantly reduced body weight, serum and liver TG and TC levels, as well as alleviated liver inflammation and lipid accumulation in this animal model.Compared with normal group, the model group presented oxidative stress, evidenced by significant increase of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and superoxide anion (O2·-) levels. Correspondingly, liver lipid peroxidation product malondialdehyde (MDA) levels were also significantly increased in this animal model. The further tests showed that inducible nitric oxide synthase (iNOS) and xanthine oxidase (XO) activity were significantly enhanced in the liver of this animal model. Compared with the model group, quercetin could inhibit iNOS and XO activity to reduce ROS, H2O2, O2·- and MDA levels in high fat diet-fed rats. In addition, allopurinol as a XO specific inhibitor could also significantly inhibit oxidative stress in this animal model. These results suggested that quercetin may have antioxidant effect to reduce endogenous ROS generation by inhibiting XO activity in the liver of high fat diet-fed rats.In addition, compared with the normal group, inflammatory factors interleukin-1β (IL-1β) and IL-18 levels were both increased in high fat diet-fed rats, which were consistent with the HE staining of liver inflammatory cell infiltration, further suggesting liver inflammatory response. Quercetin (50 and 100 mg/kg/d) and allopurinol could significantly reduce the inflammatory response, and decrease IL-1β and IL-18 levels, indicating that quercetin alleviated hepatic inflammation in animal model of NAFLD.Compared with normal control group, thioredoxin-interacting protein (TXNIP) protein levels were significantly increased, and nucleotide binding oligonucleotide poly domain receptor family of pattern recognition receptors protein 3 (NLRP3) inflammasome were also remarkably activated, accompanying with high IL-1β and IL-18 protein levels in the liver of this animal model. Quercetin (50 and 100 mg/kg/d) could significantly reduce hepatic TXNIP protein levels, and then suppress NLRP3 inflammasome activation in animal model of NAFLD. Allopurinol (5 mg/kg/d) also showed the down-regulation trends of TXNIP protein levels to inhibit NLRP3 inflammasome activation in this animal model. These data indicate that TXNIP may be the key protein to mediate anti-oxidant and anti-inflammatory effects of quercetin and allopurinol in high fat diet-fed rats.Generally, oxidative stress and inflammatory response further aggravate abnormal lipid metabolism. Compared with normal control group, sterol regulatory element binding protein 1c (SREBP-1c) and stearoyl-CoA desaturase 1 (SCD1) protein levels were markedly up-regulated, and acetyl coenzyme A carboxylase phosphorylation (p-ACC) protein levels were down-regulated in high fat diet-fed rats. Quercetin (50 and 100 mg/kg/d) could reduce SREBP-1c and SCD1 protein levels, and up-regulated p-ACC protein levels in the liver of high fat diet-fed rats. These results showed that quercetin reduced oxidative stress and inflammatory response, and regulated lipid metabolic disorder with the decrease of lipid accumulation, resulting in alleviate hepatic injury in high fat diet-induced NAFLD in rats.In summary, this thesis demonstrated that high fat diet induced NAFLD with liver oxidative stress and inflammatory damage in rats. Oxidative stress-related protein TXNIP-mediated NLRP3 inflammasome activation caused hepatic inflammatory response, which may be the key linking to pathological injury in this animal model of NAFLD. Quercetin and allopurinol could effectively reduce oxidative stress, down-regulate TNXIP protein levels to inhibit the activation of NLRP3 inflammasome, and then reduce IL-1β和 IL-18 levels to relieve inflammatory injury in the liver of high fat diet-fed rats. Furthermore, they down-regulated SREBP-1c and SCD1, and up-regulated p-ACC and then reduce lipid accumulation in the liver of high fat diet-fed rats. These results suggested that the inhibition of TXNIP and NLRP3 inflammasome activation may be one of the key molecular mechanisms for improving liver injury in high fat diet-induced NAFLD of rats. This thesis may also provide a scientific basis for the treatment of chronic liver diseases such as NAFLD and other chronic liver diseases.