Intervention Of Alpha-ketoglutaric Acid On Alcoholic Liver Disease And The Study Of Its Mechanisms Involved

Background and Objective:Alcoholic liver disease (ALD) is a chronic liver disease induced by long-term alcohol abuse, the incidence of which is pretty high in Western Countries. Recently, with development of economy, improvement of living level and modification of life style, the number of alcoholic is increasing, and the incidence of ALD assumes year by year the trend of escalation. Alcohol has been the second leading cause of liver damage following the virus hepatitis in our country. ALD encompasses a broad spectrum of morphological features ranging from steatosis with minimal injury to more advanced liver damage, including steato-hepatitis and fibrosis/cirrhosis. Moreover, approximately15%of the patients with established alcoholic cirrhosis develop hepatocellular carcinoma.Even the pathogenesis of ALD is complex, involving the toxicity of ethanol and its metabolic products, immunological reaction, inflammatory reaction, iron overload, release of cytokine. oxidative stress and lipid peroxidation, the "two hits" centering on oxidative stress and lipid peroxidation can explain the full process of ALD. Acute and chronic alcohol acute have been shown to increase the production of reactive oxygen species (ROS) such as the superoxide amion (O2-) and hydrogen peroxide (H2O2), lower cellular antioxidant levels, and enhance oxidative stress in many tissues, especially in the liver. Recent studies showed that oxidative stress induced each stage of ALD via mitochondria damage, disorder of lipid metabolism, inflammatory reaction, immunological reaction, and so on. The major pathway of oxidative metabolism of ethanol in the liver involves alcohol dehydrogenase (ADH) present in the cytosol and cytochrome P4502E1(CYP2E1) present predominantly in the microsomes, through which ethanol induces oxidative stress. Liver CYP2E1is induced by alcohol consumption, which leads to the injury of mitochondrion, especially the inhibition on mitochondrial respiratory chain. The induction of CYP2E1by ethanol is an important pathway through which ethanol induces oxidative stress, and is a major pathogenesis of ALD.AKG is an intermediate metabolite of Krebs cycle, a common final pathway for the oxidation of fuel molecules. In our common life α-KG is usually used as one component of sports drink; Researches suggested that a-KG could improve the cytoactive and decrease ROS produced in vitro and vivo, which is an antioxidant and plays a key role in the detoxification of ROS; α-KG also antagonizes toxicity of cyanide which induce the oxidative stress and the increase in LPO, depletion in GSH, GPx, SOD. and CAT levels; α-KG mitigates the Al-mediated nuclear accumulation of hypoxia inducible factor-la (HIF-la) and the mitochondrial dysfunction observed during Al stress in cultured human hepatocytes, recovers the activity and expression of a-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and prolyl hydroxylase2(PHD2); AKG treatment up-regulates several stress response proteins, increases plasma levels of several hormones such as insulin, growth hormone and insulin-like growth factor I(IGFI); dietary supplementation with a-KG alleviates oxidative stress and injury in intestinal mucosal cells, while improving intestinal mucosal integrity and absorption of nutrients in endotoxin-challenged pigs. However, few researchers about α-KG are conducted on liver injury induced by ethanol. This research aims to explore the protective effects of α-KG on the treatment of ALD and its possible mechanism in vitro.Methods:(1) Treatment groups:immortalized fetal liver cell line HL7702was cultured and divided into4groups:control group, model group (treated with alcohol for8days and then with normal culture medium for2days), alcohol and a-KG group (cells were treated with alcohol and a-KG at the same time for8days and then with normal culture medium for2days),α-KG group (treated with α-KG only for8days and then with normal culture medium for2days). The above mentioned interfering factors were added after the cells were adherent to the culture dish. The cells were subcultured every3days.(2) Measurement of oxidative stress indicators:After ten days, changes of lipid droplets in HL7702cell were observed using oil red O staining, Reactive oxygen species (ROS) in cells was quantified by optical density measurement and fluorescence microscope and the activity of SOD and CAT in cells were checked with kit. Content of MDA in the cells of each group was detected. Cells apoptosis were analyzed using a flow cytometer. The expression of ADH, CYP2E1in cells was determined by Western Blot.Results:(1) Making models:HL7702ALD cell models were successfully established; results of oil red O staining showed that the content of lipid droplet was higher in alcohol model group than other three groups; (2) Indicators of oxidative stress:Compared with control group, the cells apoptosis rate, content and active fluoresce of ROS, content of MDA was higher, activity of SOD and CAT was lower in alcohol model group; compared with alcohol model group, the cells apoptosis rate, content and active fluoresce of ROS, content of MDA was lower, activity of SOD and CAT was higher in alcohol+α-KG group and α-KG group;(3) Expression of enzyme on ethanol metabolism:Compared with control group, the expression of ADH and CYP2E1increased in alcohol model group; compared with alcohol model group, the expression of ADH and CYP2E1decreased in alcohol+α-KG group and α-KG group.Conclusions:(1) Oxidative stress and lipid peroxidation participated in the occurrence of alcoholic disease;(2) α-KG interfered with the development of ALD and alleviated the hepatocytes damage in ALD modes at the same time of drinking and taking α-KG, and protecting hepatocytes against oxidative stress.(3) α-KG eased the hepatocytes damage in vivo via the role of anti-oxidation.(4) The molecular mechanisms of α-KG’protective effects on ALD involved in down regulating ADH and CYP2E1expression level.