Protective Effect Of EGCG On The Oxidative Damage Induced By AFB₁ In HL7702 Liver Cells

ObjectiveEpidemiological investigation indicates that the occurrence of human hepatocellular carcinoma（HCC）is related to dietary AFB₁ exposure.Experimental studies indicate that AFB₁ can induce Hepatocellular Carcinoma in rats through oxidative damage.Some antioxidants have protective effects on AFB₁-induced oxidative stress.Epigallocatechin gallate（EGCG）is one of the main catechins in green tea and has attracted much attention because of its strong antioxidant activity.Our research group showed that EGCG has a certain protective effect on the oxidative damage of rat liver cells caused by AFB₁.The purpose of this study was to investigate the protective effect of EGCG on AFB₁induced normal liver oxidation injury in vitro,and to investigate the role of Nrf2/HO-1 signaling pathway in this process.It provides a theoretical basis for further research on EGCG for the prevention of human and animal liver injury,HCC and other related diseases caused by long-term dietary exposure to AFB₁.MethodsNormal human HL7702 hepatocytes were cultured in 1640 complete medium in vitro.Cells with different densities were seeded in 96-well plates and cell growth status was observed under inverted microscope.Cells were treated with different concentrations AFB₁（40,60,80μg/mL）for 24h and EGCG（5,10,20,40,80,160μg/mL）for 1h,12h,24h and treated for 1h before further culture for 24h which four different treated time groups for EGCG.Cell viability was determined by MTT assay,and appropriate AFB₁ treated concentration and EGCG intervention concentration were selected.In subsequent experiments,the cells of HL7702 hepatocytes were divided into control cells,solvent control cells（0.3%DMSO）,AFB₁ treated-cells（60μg/mL AFB₁）and EGCG-treated cells which include low dose（5μg/mL）,middle dose（10μg/mL）and high dose（20μg/mL）.The cells were treated with 60μg/mL AFB₁ in the AFB₁-treated cells for 24h to establish a oxidative stress model of HL7702hepatocytes.In the EGCG-treated cells,cells were pretreated with the different concentration of EGCG for 1h in advance,and then were treated with a fresh complete culture medium containing 60μg/mL AFB₁ for another 24h.At the end of the experiment,the corresponding components of cells and supernatant were detected.CCK-8 was used to detect cell viability.The reactive oxygen species（ROS）in the cells were measured by 2,7-dichlorodi-hydrofluorescein diacetate（DCFH-DA）fluorescence probe,and biochemical analysis was used to measure the contents of glutamic-pyruvic transaminase（GPT）,glutamic oxalacetic transaminase（GOT）,lactate dehydrogenase（LDH）in the supernatant of cells and the total intracellular superoxide dismutase（T-SOD）,malondialdehyde（MDA）in cells.Enzyme-linked immunosorbent assay was used to determine the concentration of 8-hydroxy-2 deoxyguanosine in cell supernatant.The expression of transcription factor nuclear factor-erythroid-2-related factor 2（Nrf2）,Heme oxygenase-1（HO-1）and quinone oxidoreductase-1（NQO1）mRNA were detected by real-time fluorescence quantitative PCR.The protein expressions of HO-1 and NQO1 were detected by western blot.Results1.They havesignificantly inhibited the cell proliferation of HL7702hepatocytes when cells were treated with 40,60 and 80μg/mL AFB₁ compared with the control cells（P<0.05）.When cells were treated with 60μg/mL of AFB₁for 24 hours,the relative survival rate of normal HL7702 hepatocytes was70.27%,which was selected as the AFB₁ treated concentration.EGCG concentration is higher than 80μg/mL will significantly damagedthe cells when5,10 and 20μg/mL EGCG have obvious promoting effect on cells proliferation（P<0.05）.So 5,10 and 20μg/mL EGCG were selected as low,middle and high intervention doses of EGCG respectively.2.The survival rate of cells in the EGCG-treated cells was improved to different degrees compared with the AFB₁-treated cells.In addition,compared with the AFB₁-treated cells,the cell survival rate of the high-dose EGCG-treated cells was significantly improved（P<0.05）.3.Compared with the control cells,GPT,GOT and LDH enzyme level in cells supernatant were significantly increased in AFB₁-treated cells（P<0.05）.The level of GPT,GOT were significantly decreased after treatment with different doses EGCG and LDH level of medium and high dose EGCG-treated cells were significantly lower compared with the AFB₁-treated cells（P<0.05）.In addition,compared with the low dose EGCG-treated cells,the activity of GPT and GOT decreased significantly in the high dose EGCG-treated cells（P<0.05）.4.Compared with the control cells,the levels of T-SOD in cells were significantly decreased when MDA and ROS in cells and 8-OHdG in cells supernatantwere significantly increased in the AFB₁-treated cells（P<0.05）.Compared with AFB₁-treated cells,the level of T-SOD was significantly increased and ROS,8-OHdGwere significantly decreasedafter treatment with different doses EGCG（P<0.05）.In medium and high dose EGCG-treated cells,MDA levels were significantly decreased compared with the AFB₁-treated cells（P<0.05）.In addition,compared with the low dose EGCG-treated cells,the level ofROS and MDA decreased significantly in the high dose EGCG-treated cells（P<0.05）.5.Compared with the control cells,the expression of Nrf2 mRNA in hepatocyte was significantly up-regulated and the HO-1,NQO1 mRNA were significantly down-regulated in AFB₁-treated cells（P<0.05）.Compared with the AFB₁-treated cells,the expressions of HO-1 and NQO1 mRNA in hepatocytes were significantly increased in EGCG-treated cells（P<0.05）.In low and high dose EGCG-treated cells,the expression of Nrf2 mRNA in hepatocyte was significantly up-regulated compared with the AFB₁-treated cells（P<0.05）.6.Compared with the control cells,the expressions ofHO-1and NQO1proteins in hepatocyte were significantly down-regulated in AFB₁-treated cells（P<0.05）.Compared with the AFB₁-treated cells,the expressions of HO-1 and NQO1 protein in hepatocytes were significantly up-regulated in EGCG-treated cells（P<0.05）.In addition,compared with the low dose EGCG-treated cells,the expression of NQO1 protein in hepatocytes were significantly up-regulated in the high dose EGCG-treated cells（P<0.05）.ConclusionEGCG can promote the growth of normal HL7702 hepatocytes in a certain range.When over 80μg/mL has significantly damage to hepatocytes.EGCG can inhibit the oxidative damage of hepatocytes caused by AFB₁ at low concentration（5-20μg/mL）.The mechanism may be mainly through activatingthe Nrf2/HO-1 signaling pathway,increasing the expressions of antioxidant enzymes,scavenging free radicals and reducing oxidative stress.