The Protective Effect Of OA On Oxidative Injury Induced By Alcohol And The Study Of Mechanism

Background：Alcohol-related disease is metabolic disorders induced by alcoholmetabolism. Alcohol abuse is increasingly becoming a serious public healthproblem in modern society. The role of alcohol on the health involves a varietyof pathological mechanisms. With the deepening research, the role of oxidativedamage caused by alcohol abuse is widely recognized and valued, that theROS-mediated oxidative stress injury is the most important risk factors foralcohol-related diseases. Research progress indicates that the preventment andtreatment for alcohol-related diseases is lack of appropriate drugs and means.Antioxidant is considered to be have a good prospect in prevention ofalcohol-related diseases.Oleanolic acid (OA) is natural triterpenoids derived from the plants, whichhas important biological and pharmacological activity. Many traditional Chinesemedicine plants are rich in OA, such as the Gentianaceae plants mileensis, Melilot plants, Ligustrum, Araliaceae Aralia. Chinese medicine applicationshave a long history, now OA as a prescription drug is used to treat hepatitis-likeillness of adjuvant therapy. Recent study found that OA has a degree ofantioxidant function, but due to its chemical structure, its bioavailability is low,so limit its clinical application. Because OA has good solubility in alcohol, OAdissolved in alcohol may reduce oxidative stress injury induced by the alcoholabuse. It’s important to understand its anti-oxidation mechanism in theprotective effect. It will help to develop alcoholic health products to reduce theincidence of alcohol related disease process and to reduce the health economicburden of alcohol abuse.Objective：(1) Research about the protective effect of OA on alcoholic oxidative injury(2) Research about the mechanism of the protective effect of OA on alcoholicoxidative injury.Methods：(1) Cell model: through the establishment of alcoholic liver cells fromoxidative injury model to evaluate the role of antioxidant activity and protectiveeffect of OA in alcohol injury model. We observe the effects of differentconcentrations OA at different times on QZG cells about Nrf-2, antioxidantenzyme protein expression. Also observed a5,10,15μM of OA intervention foralcohol-induced liver cell proliferative activity of simultaneous detection of cellhomogenate oxidative stress indicators, such as the activity of SOD, MDA, andGSH, GSSG,also T-SH content. Protein expression of Nrf-2, the SOD-1HO-1,Gpx changes measured after OA intervention. In addition, we also observed theinhibition of OA in vitro in the DPPH, O2-, and OH radical chemistry luminescence model.(2) Animal models: through4g/kg/day ethanol feeding rats30days inducedoxidative damage in rats to induce the model of alcoholic, at the same time togive two doses of OA intervention,10mg/kg/day and10mg/kg/day, then takethe serum, liver, muscle, fat homogenate extract of liver mitochondria,determination of the experimental animal body weight and blood glucosechange, observed changes in liver body weight ratio, transaminases ALT andAST and lactate dehydrogenase, markers, serum lipids and mitochondria thecontent of the activity and the swelling degree of change in serum, and otherorganizations within the organ homogenates of advanced glycation end products(AGEs). We also observed oxidative stress related indicators in serum, liver, fatand muscle tissue, such as MDA and GSH, GSSG using and the T-SH content ofCAT, the activities of SOD, Gpx, the T-AOC activity. Extraction of liver totalprotein, determined by Nrf-2, SOD-1, GR, HO-1protein expression levels.Nrf-2protein expression levels were determined in total muscle protein. Nrf-2gene expression levels were determined in liver tissue. We also make slices ofthe liver and stomach for H-E staining. The systematic observation of thereaction of oxidative stress injury, as well as antioxidant enzyme chain changesin alcoholic liver injury model are done, focusing on the protective mechanismof oxidative damage of OA.Results:Successfully constructed and the model of the alcohol intervention in QZGcell oxidative damage, combined with the literature, the200mM EtOH role6has to create QZG cells alcoholic oxidative damage model approach. To verifythe inhibitory effect of OA in vitro three chemical luminescence model such asDPPH, O2and OH, the results show that OA has low ability in directly scavenging free radical. OA affects QZG cells activity for24h, the resultsindicate that below15.625μM OA has no significant effect on cell viability,indicating that OA under the concentration did not produce significantcytotoxicity. We also found that5,10,15μM OA intervention for24hsignificantly improve the Nrf-2protein express, the10μM OA intervention fordifferent time can be significantly induced Nrf-2, HO-1, CAT,, PRX-1proteinexpression levels up regulation within24h.15μM OA can significant inhibitEtOH-induced QZG proliferative activity injury. Oxidative damage-relatedindicators of the EtOH-induced QZG cells results show that200mM of EtOHfor6h can induce significant oxidative stress, manifested as significantly lowerSOD activity, MDA content increased content of GSH reduce GSH/GSSH inratio decreased. Giveing different doses of OA protective intervention, thecellular oxidative stress level was significantly lower performance levels ofMDA, and decrease antioxidant enzyme activity, GSH content increased, whilethe GSH/GSSG ratio was also significantly elevated. Protein expression level ofexperimental results show that after5、10、15μMOA intervention significantlyincreased Nrf-2, the SOD-1HO-1, Gpx protein expression levels.Animal weight loss significantly after the administration of alcohol inalcohol-induced oxidative damage in animal models, no significantimprovement OA administration of alcohol led to weight loss in rats, the animalsin each group glucose no significant change, but the OA after the interventionsignificantly improve the positive control group liver ratio increased, reducingthe pathological changes of liver steatosis. Giving10mg/kg/day and20mg/kg/day of OA, the value of serum ALT and AST activities weresignificantly reduced, show that OA administration significantly impaired liverfunction caused by protection of alcohol. After two doses of OA significantly improved total cholesterol and LDL levels increased trend of low doses of OAalso significantly reduced triglyceride levels, OA also significantly reduced thelevel of high density lipoprotein, the mechanisms to be elucidated. OAadministration in addition to adipose tissue AGEs have a certain degree ofinhibition outside, the rest such as serum, liver and muscle tissue found nosignificant change. OA helps to maintain mitochondrial glutathione redoxbalance, and significant improvements in alcohol-induced mitochondrial activitydecreases, and reduce the degree of swelling of mitochondria. Determination ofoxidative stress injury indicators show that the influence of alcohol can lead todecreased serum and liver SOD and CAT activities, OA can improve the abovetwo kinds of antioxidant enzyme activity in serum and liver homogenate. Gpxactivity and the negative of the positive control group serum control groupshowed no significant changes of OA also failed to change its activity, liverhomogenates Gpx activity of the positive control group no significant change,but to give OA, Gpx activity but significantly reduced. The positive controlgroup, serum T-AOC value increased after administration of the trend to reducethe trend of T-AOC in the liver and serum results contrary, OA after theintervention of liver total antioxidant levels were significantly increased. Reducethe positive control group, serum and liver homogenate GSH content, GSSGusing content increased in OA intervention, GSH significantly higher ratio ofGSH/GSSG Ratio in significantly higher, suggesting that OA by restoring theGSH system play a protective role. The results indicate that the OA cansignificantly protect the alcohol thiol depletion. The positive control group andnegative for the content of MDA in the serum control group showed nosignificant change, MDA decreased in the OA after the intervention. Thepositive control group of liver MDA content was significantly higher OA after the intervention significantly reduced. Positive control group liver Nrf-2, ofHO-1, the GR, of SOD-1and other antioxidant protein expression significantlylowered muscle Nrf-2is also significant with the OA intervention significantlywith increased the expression of the above-mentioned protein of the same timealso increase the liver Nrf-2gene expression. The histopathological resultsshowed that the OA administration is significantly reduced alcohol-inducedstomach and liver pathological damage.Conclusion:Successfully constructed the alcoholic injury QZG cell model and animalmodels, the system of alcoholic liver injury mechanism, and found thatoxidative stress in cellular and animal models of alcoholic liver injury occupiesa prominent and important position, mainly for the free radical inducedmetabolites increase oxidative damage, antioxidant enzyme Nef2as the corechain expression, abnormal activity, reduce the antioxidant defense systemfunctions; chronic liver oxidative damage and lipid metabolism abnormalitiesclosely related; the use of in vitro antioxidant screening technology model,determined hepatoprotective effect of monomer chemical drug-OAanti-oxidation ability to respond to OA in vitro direct antioxidant effect isrelatively weak, may be related to lipid solubility, however, applied to alcoholicliver injury in cell and animal models doses of OA, a significant antioxidanteffect; further studies have shown that activation of Nrf2as the core of theantioxidant enzyme chain the role of the OA and then start the body’s overallantioxidant defense system functions effectively inhibited alcohol-induced liveroxidative stress injury, is an important mechanism to protect the liver againstdamage.These results suggest that: OA directly scavenging free radicals is weak, but in alcohol-induced oxidative damage in vitro model and in vivo animal modelshave played a good role of antioxidant protection. OA may by inducing Nrf-2related antioxidant enzyme expression, protection of the glutathione redoxbalance, improve the body’s antioxidant enzyme activity to protectmitochondrial function and reduce the lipid metabolism disorder to play itsanti-oxidation role. The results of this study indicate that OA has a significantresearch, development in alcoholic liver injury related diseases, applicationprospects. In this study, the deeper study the role of OA in the prevention andtreatment in alcohol-related diseases research base for further development andutilization of effective experimental basis.