Effects Of Chlorogenic Acid And Aucubin On The Antioxidation Of Grass Carp Liver Cells L8824 Under The Intervention Of Exogenous H₂O₂

Exp.1 Cloning and expression analysis of p38α Mitogen-activated protein Kinase(p38α MAPK)gene c DNA complete sequence from grass carp(Ctenopharyngodon idellus)The p38 mitogen-activated protein kinase(p38 MAPK)pathway is one of the many pathways of MAPK signaling pathway,it is an important signal transduction pathway in animal cells,p38 MAPK genes plays an important role in inflammation,oxidative stress,apoptosis and response to different environmental stimuli.Grass carp(Ctenopharyngodon idellus)p38α MAPK(MAPK 14a)was cloned by using RACE method.The full-length c DNA of MAPK 14 a was 2374 bp containing an open reading frame of 1086 bp,which encoding 361 amino acids with an estimated molecular weight of 41.408 k D and a theoretical p I of 5.15.Amino acid alignment indicated that MAPK14 a possessed a Thr-Gly-Tyr(TGY)dual phosphorylation motif of p38 MAPK family and shared 98% similarity with Cyprinus carpio p38α.The prokaryotic expression vector p ET32-p38α was constructed and then transformed into BL21(DE3),using IPTG to induce expression,SDS-PAGE analysis showed that the molecular weight of recombinant fusion protein was consistent with the predicted value.Real-time quantitative PCR showed that MAPK 14 a gene expressed in a wide range of tissues including the blood,liver,spleen,kidney,heart,brain,foregut,gill,muscle and skin,and the highest expression was in brain,on the contrary,the lowest was in skin and muscle.Exp.2 Cloning,expression analysis of the Nuclear factor erythroid 2-related factor2(Nrf2)gene of grass carp(Ctenopharyngodon idellus)and the effect of dietary Eucommia ulmoides on the gene expressionNuclear factor erythroid 2-related factor 2(Nrf2)is an important transcription factor in the regulation of animal redox homeostasis,which plays a vital role in metabolism,cell cycle and cell protection.In this study,the complete c DNA sequence of Nrf2 gene of grass carp was obtained by RACE(rapid amplification of c DNA ends),and the characteristics of the Nrf2 protein predicted from gene sequence was analyzed by bioinformatics methods.The tissues differential expression pattern was also detected by quantitative real-time PCR.Then,Eucommia ulmoides(EU),a traditional Chinese medicine,was added in diet with a inclusion of 2% to feed grass carp for 3 months to investigate the effects of EU on the expression of Nrf2 gene in different tissues.The results showed that the full-length of Nrf2 gene of grass carp is 2760 bp,encoding 593 amino acids,and the predited Nrf2 protein is a hydrophilic and non-secretory protein.Nrf2 gene highly expressed in kidney,gill,liver,spleen and blood in grass carp.Dietary EU significantly promoted the expression of Nrf2 m RNA in kidney,gill and liver at the end of the feeding trial.This study laid a foundation for a further research on the antioxidant mechanism of EU on fish.Exp.3 Cloning and expression analysis of mitochondrial transcription factor A(TFAM)gene c DNA complete sequence from grass carp(Ctenopharyngodon idellus)Mitochondrial transcription factor A(TFAM)is an important factor encoded by nuclear genes and transferred into mitochondria to participate in mitochondrial DNA transcriptional activation and regulation of mitochondrial DNA copy number.Grass carp(Ctenopharyngodon idellus)TFAM was cloned by using RACE method.The full-length c DNA of TFAM was 1427 bp containing an open reading frame of 837 bp,which encoding 278 amino acids with an estimated molecular weight of 31.777 k D and a theoretical p I of 10.41.The deduced amino acid sequence of the gene contains two high-mobility group(HMG)cassette domains and a low-complexity C-terminal domain,which is highly similar to other species and shared 97% similarity with Megalobrama amblycephala TFAM.Real-time quantitative PCR showed that TFAM gene expressed in a wide range of tissues including the blood,liver,spleen,kidney,heart,brain,foregut,gill,muscle and skin,and the highest expression was in blood,on the contrary,the lowest was in skin.Exp.4 Effect of chlorogenic acid and aucubin on the antioxidation of L8824 cells treated with exogenous H2O2In order to study the effects of chlorogenic acid and aucubin on the antioxidation of grass carp L8824 cells damaged by H2O2 treatment.Established model of cell oxidative damage before trial,and the experiment was divided into blank control group,treatment group,positive control NAC group,different doses of chlorogenic acid(A1,A2,A3,A4 and A5)groups,different doses of aucubins(B1,B2,B3,B4 and B5)groups and chlorogenic acid and aucubin were mixed(A3 + B3)group.The results showed that,the survival rate,mitochondrial membrane potential and SOD activity of NAC group,certain dose group A and B group and(A3+B3)group compared with the treatment group were significantly increased(P<0.05),the level of ROS and the degree of oxidative damage was significantly decreased(P<0.05),the expression of P38 MAPK m RNA was significantly decreased(P<0.05),and the expressions of PGC-1a,Nrf2,and TFAM m RNA were significantly increased(P<0.05),and the differences were most significant in the A3,B3 and(A3+B3)groups.The results showed that chlorogenic acid and aucubin affected the antioxidative capacity of grass carp L8824 hepatocytes.Its possible mechanism is that chlorogenic acid and aucubin initiate the free radical scavenging mechanism by inhibiting the phosphorylation of P38 MAPK and clear excess ROS produced during the mitochondrial biogenesis of L8824 cells,thereby maintaining the normal ROS level of cells,and the appropriate amount of ROS can improve mitochondrial dysfunction and promote mitochondrial biosynthesis by activating the PGC-1a/Nrf2/Tfam signaling pathway.At the same time,the protein that has been phosphorylated by Nrf2 can activate downstream antioxidant enzymes such as SOD and CAT,thus enhancing the antioxidant capacity of grass carp L8824 cells.The recommended drug concentration for chlorogenic acid and aucubin is 40μg/ml.