The Protective Effect And Mechanism Of Ginkgetin On Alcoholic Oxidative Injury

As an important global issue on public health, alcohol abuse has been provoked widespread solicitude in the world because of its outcome including functional impairment of multiorgans or multisystems, as well as severe psychiatric disorder and social problem. Alcohol is an intensive oxidative stressor and small hyperosmotic molecule possessing both hydrophilia and lipophilicify. Excessive free radical and reactive oxygen species (ROS) produced during ethanol metabolism result in lipid peroxidation, protein oxidation. DNA damage and adducts formation, which is key mechanism of alcohol induced injury to liver and other organs. However, classic antioxidants medication, i.e. vitamin A, vitamin E, vitamin C, GSH, selenite, zinc and etc, plays little role on preventing ethanol-induced oxidative damage clinically, indicating that endogenous antioxidant system mediated and activated by exogenous antioxidant is crucial for the prophylaxis of alcoholic oxidative damage.Among various genes encoding for proteins in possession of antioxidant characteristics, heme oxygenase-1 (HO-1) has been attracted particular interest as it is finely up-regulated by stress conditions and generates metabolites exhibiting important biological activities and antioxidant capacity after decades quiescence. HO-1 as a potential preferred target gene for preventing oxidative stress, increasing evidences support that its induction represents an adaptive response or enhanced resistance to various oxidative stresses and diseases. However, these research outcomes have not been taken into consideration for practical application owing to potential oxidative toxicity of "classic inductor", i.e. heme or cobalt protoporphyrin, and the safety and efficiency of gene transfection.Of note, some natural flavoniods or polyphenols antioxidant. e.g. curcumin.quercetin, Ginkgo biloba extract (EGB) and ginkgetin, have been found recently to inducethe expression of HO-1 via antioxidant-response element or electrophile response elementin the upstream of gene promoter. These antioxidants might amplify antioxidant effect justby virtue of its induction on HO-1 and exert extensive pharmacological activity. On thebasis of these researches and hypothesis, the effect on serum and hepatic, renal, cardiac.and testicular antioxidant systems and serum lipid level were investigated with ginkgetinas preventer in possession of substantial antioxidant activity and hepatoprotective effect.Additionally, hepatic structure and function, antioxidant system of hepatic mitochondriaand microsomes, and mRNA, protein expression and enzymatic activity of hepatic HO-1were studied after chronic alcohol administration and ginkgetin pretreatment for rats.Enzymatic and biochemical assay, reverse transcriptase-ploymerase chain reaction(RT-PCR), flow cytometry (FCM), Western Blot, ultramicropathology and etc wereemployed in the present study. The details are as following:Part 1 The prophylaxis of ginkgetin on antioxidant system of mice after acute alcohol exposureAlthough the mechanism of alcoholic oxidative stress has been well documented, there is still some dispute on the specific influence or alteration after alcohol administration. Therefore, the dynamic change of serum and hepatic antioxidant systems, i.e. time-response effect, was investigated in mice after acute ethanol intake and ginkgetin pre-administration by enzymatic and biochemical assay in the present part.The results showed that GSH level and enzymatic activity of serum and liver presented U-shape whereas reverse U-shape was found with torpid restoration in MDA level of serum or liver from 1 hour to 15 hours after acute ethanol exposure (4.8 g/kg) for mice intragastrically. Serum GSH level and SOD activity dropped to minimum 2 hours after ethanol administration while GSH-Px activity reduced to the lowest and MDA level climbed to maximum 4 hours after administration. The altering extent of serum GSH, SOD, GSH-Px and MDA was 23.5%, 20.3%, 20.2% and 32.4% respectively. Hepatic GSH level, as well as GSH-Px or SOD activity descended to the minimum 4 hours whereas MDA level increased to the highest 6 hours after ethanol treatment. The altering extent of hepatic GSH, SOD, GSH-Px and MDA was 29.3%, 18.4%, 23.6% and 27.5% respectively. Pretreatment with ginkgetin at a dose of 96 mg/kg, increased the compensation effect at the early stage of ethanol administration and attenuated ethanol-induced GSH depletion, antioxidant enzymes inactivation and lipid peroxidation, by which ginkgetin promoted the restoration of antioxidant systems damaged by ethanol.From the results it could be concluded that serum and hepatic antioxidant capacity had been affected 2-6 hours, especially 4 hours after ethanol administration. Ginkgetin exerted evident protection by alleviating the influence of alcohol on antioxidant system.Part 2 The dynamic variation of serum lipid level and antioxidant system after ethanol administration and the protection of ginkgetin pretreatmentEpidemiological investigations show that ethanol intake with different dose might prognosticate different risk for cardiovascular disease and diabetes milletus, indicating diverse response of drink to health. Therefore, we assayed the serum antioxidant system, as well as serum lipid level of rats treated with different dose of ethanol for different endurance. The effect of ginkgetin on ethanol-fed rats with high dose was also investigated in the present part.The results showed that rats treated with low dose of ethanol (0.8 g/kg) had no obvious change in serum GSH, MDA, TG and TC level for 30, 60. 90 days. Serum SOD, GSH-Px and CAT activity gradually increased during experimental peroid and HDL-C significantly increased on the 90th day. Ethanol treatment with middle dose (1.6 g/kg) resulted in evident accumulation in serum MDA, TG and TC level, and gradual depletion in GSH. Serum HDL-C content, as well as the activity of SOD and GSH-Px declined after temporal and slight increase. High dose of ethanol (2.4 g/kg) led to conspicuous decrease in GSH and HDL-C level, as well as the activity of SOD and GSH-Px. but evident augument in MDA, TG and TC content. CAT activity declined rapidly after slight increase. Pretreatment of ginkgetin, especially high dose of ginkgetin (96 ing/kg), alleviated or counteracted the alteration of serum antioxidant system and lipid level induced by ethanol with high dose.From the results we could find that low dose of ethanol might activate antioxidant system and increase HDL-C level by which alcohol accordingly exerted protective effect. Serum lipid and lipid peroxidation aggravated, but antioxidant level evidently declined after trivial increase at the first stage overall after ethanol exposure with middle dose, especially for a long endurance. High dose of ethanol resulted in exacerbation in the disorder of lipid metabolism and impairing antioxidant system. Ginkgetin medication, especially high gingetin ptetreatment, alleviated the influences induced by ethanol, indicating evident lipid regulating and prophylaxis on alcoholic oxidative damage.Part 3 The effect of chronic ethanol administration on antioxidant system in rat tissues and prophylaxis of ginkgetinChronic alcohol binge not only impacted on liver function severely, but also induced pathological changes and dysfunction of multiorgans, including myocardium damage, renal inadequacy, spermatiferous disorder and etc. As a small molecule with high diffusibility, ethanol induced massive free radical and ROS during metabolism, by which ethanol impacts intensive oxidative stress on multiorgan and accordingly leads to alcohol-related diseases. Therefore, in the present part we investigated the effect of chronic ethanol administration on antioxidant system of liver, kidney, heart and testes, and explored the prophylactic effect of ginkgetin on ethanol-induced injury in the tissues.The results showed that MDA and ROS accumulated in all measured tissues. whereas GSH level, total antioxidant capacity (T-AOC) and the activity of SOD. GSH-Px and CAT in measured tissues significantly decreased except renal SOD and cardiac CAT in rats treated with ethanol at a dose of 2.4 g/kg for 90 days. Pretreatment with ginkgetin. especially high dose of ginkgetin (96 mg/kg), alleviated or counteracted the alteration of antioxidant system in tissues induced by high dose of ethanol.The results indicated that chronic ethanol intake might result in evident redox disbalance in liver, heart, kidney and testis. Ginkgetin medication, especially at high dose, alleviated the influences on antioxidant system of multiorgans induced by ethanol, exerting excellent prophylaxis on alcoholic oxidative damage.Part 4 The study on preventive mechanism of ginkgetin on alcoholic liver damageAs main organ for ethanol metabolism, liver becomes critical target of alcohol intoxication. Alcoholic liver disease (ALD) is the second biggest category ranking after infectious hepatopathy. In the present part we further explored prophylactic effect and related mechanism of ginkgetin on alcohol-induced liver damage. Hence, enzymatic or biochemical assay, pathological and ultra-micropathological method were employed to investigate the changes of liver structure and function, and of antioxidant system in hepatic mitochondria and microsomes of ethanol-fed and ginkgetin-intervention rats. Additionally, RT-PCR, FCM, Western Blot and enzymatic assay were deployed to determined the activity of CYP 2E1 and HO-1, and the expression of rat liver after ethanol administration and ginkgetin pretreatment.The results showed that chronic ethanol treatment (2.4 g/kg for 90 days) led to prevalent hepatocytes steatosis, watery and parenchymatous degeneration, and liver disorder. Mitochondria and rough endoplasmic reticulum (RER) in liver showed swollen, deformative and degenerative pathological changes, and redox disequilibrium. HO-1 level decreased by 55.0% in mRNA, 49.3% in protein, and 45.5% in activity, whereas CYP 2E1 activity increased by nearly 4 fold following chronic ethanol administration. Rat hepatic pathological change and oxidative damage in mitochondria and microsomes were attenuated in the presence of ginkgetin with different dose (48 and 96 mg/kg). especially with high dose (96 mg/kg). Of note, ginkgetin induced mRNA, protein expression and activity of HO-1 in ethanol plus ginkgetin group compared with ethanol-fed group, and in ginkgetin control versus normal control. No evident activation was found in other antioxidant enzymes after ginkgetin supplement.From above we could find that chronic ethanol administration impaired the antioxidant capacity of mitochondria and microsomes and inhibited the expression and inactivated the activity of HO-1. Ginkgetin induced HO-1 and exerted synergetic and enhanced antioxidant effect to scavenge ethanol-derived ROS, by which ginkgetin protected the antioxidant system from ethanol-induced oxidative stress.In summary, low ethanol might exhibit synergetic protection by promoting HDL-C synthesis and activating antioxidant system. However, excessive ethanol administration could result in disorder of lipid metabolism and oxidative stress in muhiorgans. Ginkgetin pretreatment attenuated or counteracted the adverse effects of ethanol and alleviated ethanol-induced oxidative damage. The induction of HO-1 by ginkgetin might mediate the prophylacitc effect. However, it is needed to elucidate the protection mediated by HO-1 via introduction of specific inhibitor or revulsivum on cellular level. In addition, it is still remained unclear about the signal pathways of ginkgetin induces HO-1 and whether the protective mechanism of HO-1 is related to the substrate eliminating, contribution of metabolism product, or their combination effect in the present study.