Research On The Protective Effect Of Chlorogenic Acid On The Oxidative Damage Of Turbot Hepatocytes Induced By Metalloprotease Of Vibrio Anguillarum

In order to confirm the antioxidant effect of chlorogenic acid on metalloprotease induced liver injury in turbot(Scophthalmus maximus),we carry out this research.In this study,Nrf2 and β-actin genes were cloned and analyzed for the first time.At the same time,low concentration of metalloproteinase(2μg/ml),medium concentration(10μg/ml)and high concentration(50μg/ml)act on the liver cells of turbotfor 2h,4h,8 h,16 hin vitro.The degree of oxidative damage to liver cells of turbot caused by metalloproteinase was obtained by measuring SOD and CAT activity,MDA content,Nrf2 and HO-1 gene relative expression levels and Reactive oxygen species(ROS)and apoptosis of hepatocytes.We further investigated the antioxidant indices and relative gene expression of chlorogenic acid(9μg/ml,18μg/ml,and 36μg/ml)after metalloproteinase-stressed hepatocytes.In this study,the protective effect of chlorogenic acid on liver cell oxidative damage caused by metalloproteinase of turbot was clarified.The total length of Nrf2 cDNA is 2448 bp.The open reading frame(ORF)is 1983 bp,encoding 660 amino acids.The 5’-untranslated region(UTR)is 44 bp,and the 3’-UTR is 421 bp.The highest homology of Nrf2 gene between turbot and Seriola dumeriis 83.5%,and the lowest is 43.2% with Gallus gallus.Phylogenetic analysis showed that the Nrf2 gene of turbot was the closest relative to H.superba.The results of RT-qPCR showed that Nrf2 was expressed in all 8 tissues.The total length of β-actin cDNA was 1930 bp,ORF was 1128 bp,encoding 375 amino acids,5’-UTR was 104 bp,3’-UTR was 698 bp.The β-actin gene of turbot had the highest homology with Trachidermus fasciatus and Epinephelus coioides,and the lowest homology was 96.9% with mice(Mus musculus)and chicken(G.gallus).Phylogenetic analysis showed that the β-actin gene had the closest relationship with Pagrus auratus and the farthest relationship with chicken(G.gallus)and mammals.The activities of SOD and cat,the content of MDA and the mRNA expression of Nrf2 and HO-1 in hepatocytes were measured by biochemical method and RT-qPCR.The results showed that the activity of SOD in different concentration groups was significantly lower than that in the control group(P<0.05),except that there was no significant difference in SOD activity between the two groups(P>0.05),and the activity of SOD was significantly lower in each concentration group than in the control group at other time points(P<0.05)The activity of cat was significantly lower than that of the control group(P<0.05);the content of MDA was significantly higher than that of the control group(P<0.05),except that the MDA content in the low and high concentration groups at 2h was significantly higher than that in the control group at other time points(P<0.05);compared with the control group,Nrf2 gene expression was significantly higher in each concentration group than that in the control group at other time points(P<0.05),except that there was no significant difference in the levels of Nrf2 in each concentration group at other time points(P<0.05)Compared with the control group,the expression of ROS was significantly higher than that of the control group(P<0.05).The effects of different concentrations of metalloproteases on the ROS and apoptosis rate of hepatocytes were detected by flow cytometry.The results showed that compared with the control group,the content of ROS was significantly increased in the three groups,The apoptotic rate increased significantly.Based on the above experimental results,the antioxidant enzyme activities of SOD and cat,the content of MDA and the mRNA expression levels of antioxidant genes Nrf2 and HO-1 were measured to further clarify the protective effect of chlorogenic acid on the oxidative damage of turbot hepatocytes induced by 50 μg/ml metalloprotease for 4h.The results showed that compared with the control group,the activities of SOD and CAT decreased significantly(P<0.05),the content of MDA increased significantly(P<0.05),and the expression levels of Nrf2 and HO-1 genes were significantly higher in the metalloprotease treated group than in the control group(P<0.05),The activities of SOD and CAT in different concentrations of chlorogenic acid pretreatment group were significantly higher than those of metalloprotease group(P<0.05),and the content of MDA was significantly lower than that of metalloprotease group(P<0.05).The expression of Nrf2 gene in hepatocytes of 36μg/ml chlorogenic acid pretreatment group was significantly higher than that of metalloprotease group(P<0.05),and the expression of HO-1 gene in hepatocytes of 18μg/ml and 36μg/ml chlorogenic acid pretreatment group was significantly higher than that of metalloprotease group(P<0.05).In conclusion,metalloprotease can induce oxidative stress in hepatocytes,which is characterized by the decrease of antioxidant enzymes SOD and CAT activities,the increase of MDA content,the increase of Nrf2 and HO-1 expression,the increase of ROS content and apoptosis rate;the increase of chlorogenic acid can improve the antioxidant capacity of hepatocytes,reduce the content of lipid peroxide MDA,and further up regulate Expression of Nrf2 and HO-1 genes.