Study On The Protective Effect Of Phenolics From Lycopus Lucidus Turcz. On EtOH-induced Hepatocyte Injury

ObjectiveTo establish a model of Ethyl Alcohol(Et OH)-induced Buffalo Rat Liver(BRL)cell injury in vitro,the protective effect of phenolics from Lycopus lucidus Turcz.(FP and BP)on Et OH-induced BRL cell injury was clearly determined,and the protective mechanism was revealed from the perspective of alcohol metabolism,antioxidant,inflammatory response and iron metabolism.MethodsThe phenolics from Lycopus lucidus Turcz.were extracted with the aid of ultrasound,FP and BP powder were obtained by X-5 macroporous resin purification and freeze-drying.The vitro Et OH-induced cell damage model of BRL cells was established using cell viability as an index,which was divided into Normal group,Model group,FP and BP dose group.The cell viability was detected by Cell Counting Kit-8(CCK-8)kit,Aspartate Transaminase(AST)and Lactate Dehydrogenase(LDH)were determined by automatic biochemical analyzer,and intervention dose was screened.The activity of Alcohol Dehydrogenase(ADH)and Acetaldehyde Dehydrogenase(ALDH),the content of Triglyceride(TG)and Total Cholesterol(TC),the content of Reactive Oxygen Species(ROS),8-Hydroxydeoxyguanosine(8-OHd G)and Malondialdehyde(MDA),and the activity of Glutathione(GSH),Superoxide Dismutase(SOD)and Catalase(CAT)in each cell group were detected by the kit.The protein expression levels of Nuclear Factor erythroid 2-Related Factor 2(Nrf2),Recombinant Kelch Like ECH Associated Protein 1(Keap1),Heme Oxygenase 1(HO-1),Recombinant Glutamate Cysteine Ligase,Catalytic(GCLC)and NADPH: Quinone Oxidoreductase 1(NQO1)were detected by Western blot.The expression levels of Tumor Necrosis Factor Alpha(TNF-α),Interleukin 6(IL-6)and Interleukin 10(IL-10)were detected by immunohistochemistry,and the expression levels of Ferrum(Fe),Ferritin(Fer)and Transferrin(TRF)were detected by the kit.Results1.The Et OH-injury model was established with BRL cells as the research object,and the CCK-8 results showed that the cell viability was less than 70% at 1.0 mmol/L Et OH,which was the optimal concentration for the establishment of the injury model.The results of CCK-8 and hepatocyte injury indexes showed that pretreatment of FP and BP(0.4～1.6 mg/m L)could significantly improve cell viability,and significantly reduce Et OH-induced AST and LDH leakage levels in BRL cells.2.Compared with the Normal group,the activity of ADH and ALDH in the Model group showed an increasing trend,and the content of TG and TC showed an increasing trend;Compared with Model group,the activity of ADH and ALDH in FP and BP dose groups showed a decreasing trend,while the content of TG and TC showed a decreasing trend.3.Compared with the Normal group,the contents of ROS,8-OHd G and MDA were increased in the Model group,while the activities of GSH,CAT and SOD were decreased;Compared with Model group,the contents of ROS,8-OHd G and MDA were decreased in FP and BP dose groups,while the activities of GSH,CAT and SOD were increased.4.Compared with Normal group,Keap1 protein levels in model group were increased,while Nrf2,HO-1,GCLC and NQO1 protein levels were decreased;Compared with model group,Keap1 protein levels decreased in FP and BP dose groups,while Nrf2,HO-1,GCLC and NQO1 protein levels increased.5.Compared with the Normal group,the expression levels of TNF-α and IL-6 in the Model group were increased,while the expression levels of IL-10 were decreased;Compared with Model group,the expression levels of TNF-α and IL-6 in FP and BP dose groups were decreased,while the expression levels of IL-10 were increased.6.Compared with the Normal group,the contents of Fe and Fer in the Model group increased,while the contents of TRF decreased;Compared with model group,Fe and Fer in FP and BP dose groups showed a decreasing trend,while TRF content showed an increasing trend.Conclusion1.Phenolics from Lycopus lucidus Turcz.inhibited the decrease of BRL cell activity induced by Et OH,and prevented AST and LDH extravasation in BRL cells.2.Phenolics from Lycopus lucidus Turcz.reduced the levels of ADH,ALDH,TG,TC,Fe,Fer and increased the levels of TRF,affecting the abnormal metabolism of alcohol,lipids and iron.3.Phenolics from Lycopus lucidus Turcz.reduced the levels of ROS,8-OHd G,MDA,TNF-α,IL-6,and increased the levels of GSH,CAT,SOD,IL-10,which enhanced the antioxidant and anti-inflammatory ability of cells.4.Phenolics from Lycopus lucidus Turcz.increased Nrf2 and its downstream target enzymes HO-1,GCLC and NQO1 protein expression levels,and reduced Keap1 protein expression levels,which activated the Nrf2-Keap1 signaling pathway to resist oxidative stress.