The Study On Antagonism Of EGCG Against Microcystin-LR-induced Oxidative Damage And The Participation Of Phase â…  Enzyme, Phase â…¡ Enzyme And Nrf2

Contamination of natural waters by cyanobacterial blooms is a worldwide environment problem. Most of cyanobacteria produce serious harmful toxin- microcystin(MC) which have been concerned for human health. There are much more researches on the mechanism of MC toxicity, but less on the protective measures against its toxicity. Nowadays there is no effective measure to control microcystis bloom at home and abroad, so to look for effective natural antidote which can prevent and reduce the harmful effect caused by MC has important practical significance.MC-LR,the most toxic of microcystins,is cyclic heptapeptide toxin which have strong hepatotoxicity , cancer-promoting activity and renal toxicity. MC-LR can destroy the body's antioxidant defense system, cause oxidative damage to tissues and organs. Liver is the target organ of MC-LR, which rich in phaseⅠ,Ⅱmetabolic enzymes and antioxidant enzymes. PhaseⅠenzymes mainly responsible for biotransformation of foreign substances. CYP2E1 is a important subtype of cytochrome P450 family which is the most important phaseⅠenzyme. PhaseⅡenzymes mainly responsible for inactivation and detoxification of foreign substances.Among the components of green teapolyphenols(GTP), (-)-epigallocatechin-3-gallate(EGCG) is the most abundant and effective polyphenol with potent antioxidant, anti-tumor effect and chemopreventive activities. Our previous study has found that GTP can reduce the MC-LR-induced liver oxidative damage, but the specific mechanism remains unclear.Research has shown that certain antioxidants including EGCG through Nrf2/ARE pathway induced phaseⅡenzyme and antioxidant enzymes to remove toxins. Other researcher reported that catechins especially EGCG can inhibit the biotransformation of foreign substances via cytochrome P450 enzymes, and thus played its antioxidant, anti-tumor effect.Therefore, this study established animal model of EGCG antagonizes sub-acute exposure to MC-LR, through giving different concentrations of EGCG simultaneously with MC-LR exposure in mice.The first part of our study was to evaluate the changes of antioxidant enzymes caused by MC-LR and the antagonism effect of EGCG against MC-LR-induced oxidative damages. The second part our study was to investigate the different expressions of phaseⅠenzyme, phaseⅡenzyme and Nrf2 in liver cells and their specific role in EGCG antagonizing MC-LR-induced toxicity. Then get ready to have further study on the molecular mechanism of EGCG antagonizes MC-LR-induced oxidative damage.Materials and Methods:1. The establishment of EGCG antagonizes sub-acute exposure to MC-LR model SPF-class male healthy Balb/c mice weighing 18-22g were supplied by Laboratory Animal research Center of the Third Military Medical University (Chongqing,China) and kept for a week using in a well-ventilated room maintained at 75%±5% humidity and 22℃±2℃,12 h light/12 h dark cycles,with free access to water and standard pellet diet. We have done sub-acute experiments respectively for 15 days and 30 days.15 days experiment: twenty-four mice were divided at random into four groups of six animals at each group. control group(0.2 ml 0.9% saline solution i.p and 0.2 ml 0.9% saline solution i.g), MC-LR group(0.01 ml/g 0.9% saline solution i.p and 10μg/kg MC-LR i.g), low dose EGCG group(10mg/kg EGCG i.p and 10μg/kg MC-LR i.g), high dose EGCG group(50mg/kg EGCG i.p and 10μg/kg MC-LR i.g). Once a day, lasting 15 days.30 days experiment: sixty mice were divided at random into six groups of ten animals at each group. control group(0.2 ml 0.9% saline solution i.p and 0.2 ml 0.9% saline solution i.g), MC-LR group(0.2 ml 0.9% saline solution i.p and 10μg/kg MC-LR i.g), EGCG group(10mg/kg EGCG i.p and 10mg/kg EGCG i.g), low dose EGCG group(10mg/kg EGCG i.p and 10μg/kg MC-LR i.g), medium dose EGCG group(25mg/kg EGCG i.p and 10μg/kg MC-LR i.g),high dose EGCG group(50mg/kg EGCG i.p and 10μg/kg MC-LR i.g). Once every other day for 30 days.Recorded the body weight and changes of mice on each experimental day. 24 h after the last exposure, animals were sacrificed by cervical dislocation.Liver were quickly removed,immediately washing out the blood with ice-cold 0.9% saline solution ,weighted and stored at -70℃.2. Antagonism of EGCG on the MC-LR induced oxidative damage The liver of mice has been treated accordingly, then used to detect liver/body weight index, liver histopathological changes, lipid peroxidation, enzyme activity and mRNA and protein expression of antioxidant enzymes (SOD, GSH, GPx).3. The study on the effect of phaseⅠenzyme, phaseⅡenzyme and Nrf2 involved in antagonism of EGCG on microcystin-LR-induced oxidative damageTo detect the activity of phaseⅠenzyme (CYP2E1), phaseⅡenzyme (GST) in liver cells.Used RT-PCR and immunohistochemistry to detect the mRNA and protein expression changes of CYP2E1,GST and Nrf2 in liver cells of each experimental group.Results:1. The growth and development of mice with MC-LR treatment were obviously inhibited compared to control(P<0.05). The body weight was increased significantly in medium,high dose EGCG group compared to MC-LR group(P<0.05).It suggested that EGCG could antagonize the MC-LR-induced growth and weight inhibition to some extent .2. MC-LR group has the highest liver /body weirht index(LBI) among all groups and there were significant differences compared to the control (P<0.05).The LBI in three doses EGCG groups significantly decreased compared to MC-LR group(P<0.05).The result showed that EGCG could inhibit the increase in LBI which caused by MC-LR.3. Under light microscope, the structure of liver cells is unclear,disordered and fusion, cell necrosis,infiltration was founded around lobular central veins, Kupffer cell hypertrophy locally in MC-LR group.When under electron microscopy,we observed mitochondria swelled greatly, rough endoplasmic reticulum expansed, a large number of lysosomes and fat droplets appeared and nuclear morphology was abnormal.But those pathological damages in liver ,to a certain extent,lessened in medium,high dose EGCG group,showed EGCG could antagonize MC-LR caused pathological damages.4. Liver MDA level in MC-L R group significantly elevated in response to MC-LR treatment compared with control (P<0.05). MDA levels in three doses EGCG groups obviously diminished compared with MC-LR group(P<0.05).It showed EGCG could inhibit the LPO caused by MC-LR.5. The activity and mRNA and protein expression of SOD,GPx,GSH in liver cells increased significantly in medium,high dose EGCG group compared to MC-LR group (P<0.05)and dose-response relationship appeared. It showed MC-LR caused obviously oxidative damages on animals. EGCG could help elevate antioxidant enzymes activities and expression,therefore lessen MC-LR-induced oxidative damages.6. The activity of phaseⅠenzyme CYP2E1 in MC-LR group increased significantly compared to contro(lP<0.05)while decreased in three EGCG groups compared to MC-LR group(P<0.05). The activity of phaseⅡenzyme GST increased significantly in medium,high dose EGCG group compared to MC-LR group(P<0.05)and dose-response relationship appeared. The same changes were happened on mRNA and protein levels of CYP2E1 and GST.7. The mRNA and protein expression of Nrf2 upregulated in MC-LR group and all EGCG groups compared to control(P<0.05).AS compared with MC-LR group,the expression of Nrf2 significantly upregulated in medium,high dose EGCG group(P<0.05).It was shown that EGCG and MC-LR could increase the expression of Nrf2.Conclusion1. EGCG antagonized MC-LR sub-acute exposure induced liver oxidative damage through alleviated liver pathological damages,reduced lipid peroxides and incresaed antioxidant enzymes activity.2. CYP2E1 might be a potential source responsible for ROS generation by MC-LR. EGCG could inhibit CYP2E1 activity and expression ,reduce ROS generation,involved in antagonism to MC-LR toxicity.3. EGCG could induce upregulation of phaseⅡdetoxifying and antioxidant enzymes via activating Nrf2 and increasing its expression, thereby enhanced the activity and expression of enzymes,scavenged excessive free radicals and ROS, to play its antagonistic effect to MC-LR to a certain extent.In brief,our study suggests EGCG can antagonize MC-LR induced liver oxidative damage through inhibit phaseⅠenzyme CYP2E1 and upregulate phaseⅡdetoxifying and antioxidant enzymes via Nrf2/ARE signaling.The same reports have not been found ,which involved in our study.