Hepatoprotective Mechanism Of Phenolics From Lycopus Lucidus Turcz. Through Activating The Nrf2/ARE Signal Pathway In Vitro

ObjectiveTo discuss protective activity of phenolic extracts（free phenolics and bound phenolics）from Lycopus lucidus Turcz.on CCl₄-induced BRL injury through activating the Nrf2/ARE signal pathway.And to reveal the antioxidant mechanism of the phenolic extracts from Lycopus lucidus Turcz.on the hepatoprotective effect in vitro.MethodsBRL were separated into normal group,CCl₄injury group,PCL-treated groups（treated by FPL and BPL）and PCL-intervention groups（first pretreated with FPL and BPL,respectively,than used CCl₄to injury）.The oxidative damage indexs of reactive ROS,CytC and 8-OHd G in the BRL was determined based on the method of ELISA.The mRNA expression levels of Nrf2 and Keap1,biphasic detoxifying enzymes including HO-1 and NQO1,and antioxidant enzymes including GSR and SOD were measured through the method of fluorescence immunoassay.Meanwhile,the protein expression levels of Nrf2 and Keap1,biphasic detoxifying enzymes including HO-1,NQO1 and GCLC were tested using the method of Western blot.In addition,protein levels of Nrf2 and Keap1 in nucleus and cytoplasm,and ERK,JNK and p38accompanied by theirs phosphorylated form were also tested through the method of Western blot.ResultsThe increase of CytC,ROS and 8-OHd G in CCl₄injury BRL was reduced by the FPL and BPL extract.While,comparasion to the normal group,the mRNA expression of Nrf2 and Keap1,phase II detoxifying enzymes including NQO1 and HO-1,and antioxidant enzymes including GSR and SOD were not increased by CCl₄injury,but the expression of Nrf2 protein was increased and the expression of downstream protein was decreased.Additionally,FPL and BPL treatment did not change the mRNA expression of Nrf2 gene,but increased the mRNA expression of Keap1 gene,of which the 1.6mg/m L FPL and BPL treatment groups increased by 352%and 23.5%,respectively,comparasion to the normal group.It also increased the mRNA expression of phaseⅡdetoxifying enzymes including NQO1 and HO-1 and antioxidant enzymes including GSR and SOD.The mRNA expression of NQO1 and HO-1 genes in 1.6mg/m L FPL and BPL treated groups increased by 10.03 and 67.38 times and 9.0 and2.35 times of the normal group,respectively,and the mRNA expression of GSR and SOD increased by 8.69,6.1 times and 6.02,5.95 times of the normal group,respectively.A divergence was observed of the effects among different concentrations of FPL and BPL on the expression of Nrf2 protein.As far as the FPL and BPL intervention groups were concerned,the expression of Nrf2 and Keap1 genes was up-regulated,and the mRNA expression of Nrf2 and Keap1 genes at the dosage of 1.6 mg/m L was 5.85 and4.18,and 146.48 and 110.84 times higher than that in the CCl₄injury group,respectively.At the same time,an increase in the mRNA expression of phase II detoxifying enzymes including NQO1 and HO-1 and antioxidant enzymes including GSR and SOD was observed.At the dosage of 0.4 and 0.8 mg/m L FPL and BPL intervention groups,the expression of Nrf2 protein was increased.Meanwhile,a divergence was observed in Keap1,GCLC,HO-1 and NQO1 protein expression among different FPL and BPL intervention groups.Comparasion to the injury group,a decrease was observed in the phosphorylation degree of p38 and JNK protein in PCL intervention group,while an increase was observed in the phosphorylation degree of ERK protein.ConclusionIn the range of tested concentration,FPL treatment did not have the ability of activating Nrf2 signal pathway,but it had the ability of increasing the gene and protein expression of downstream phase II detoxification enzymes（HO-1 and NQO1）,as well as the gene level of antioxidant enzymes（SOD and GSR）,while BPL treatment had the ability of activating Nrf2 signal pathway and increasing the gene and protein expression of downstream phase II detoxification enzymes（HO-1 and NQO1）,as well as the gene level of antioxidant enzymes（SOD and GSR）.Additionally,a effectively reduce in FPL and BPL intervention on the CCl₄injury BRL was observed.FPL and BPL intervention increased the expression of downstream phase II detoxifying enzymes and antioxidant enzymes by activating Nrf2/ARE signal transduction pathway,so as to protect hepatocytes from oxidative stress and protect BRL.