| H2O2 was used as an inducer to induce oxidative stress in primary hepatocytes of grass carp.The aqueous extract of TCR,RAT,JLT were used as experimental materials,and(VC)was used as the positive control to select the appropriate concentration to add to the primary hepatocyte culture medium.Objective to observe the protective effect of aqueous extracts of natural plants on oxidative damage of grass carp hepatocytes and its possible mechanism.The main research contents and results are as follows:Part one:Establishment of H2O2 damaged grass carp primary hepatocytesThe cells were treated with 0 μmol/L,100μmol/L,200 μmol/L,400 μmol/L and 600μmol/L H2O2 for 0.5 h,1 h,2 h and 4 h respectively.MTT was used to detect cell viability,colorimetric method was used to detect the activity of antioxidant enzymes,cell morphology was observed under microscope,and the changes of apoptosis and intracellular reactive oxygen species(ROS)content were detected by flow cytometry.The results showed that:The cell viability of 200 μmol/L H2O2 group decreased to 50%～70%after 1～4 hours injury,which can be used as the concentration of stress H2O2.the cells were treated with 200 μmol/L H2O2 for 1 h,LDH has(505.67±10.49)U/L increased13.88%(P<0.01).The content of malondialdehyde(MDA)in the cells was increased 5.56%(P<0.05).The activity of superoxide dismutase(SOD)was decreased 24.50%(P<0.01).The cells were treated with 200 μmol/L H2O2 for 1 h,the edge of some cell membrane was blurred,a small number of cells were shrunk and most cells were in a complete state.Flow cytometry was used to analysis the apoptosis rate and ROS content.The results showed that apoptotic rate increased with the increase of H2O2 concentration.The apoptotic rate was(36.78±1.23)%after treated with 200 μmol/L H2O2 for 1 h,which was different significantly from that of the control(P<0.01).The apoptosis rate increased 26.51%,and ROS increased 43.28%content were detected by flow cytometry.Hepatocytes treated with 200 μmol/L H2O2 for 1 h could establish oxidative stress model in vitro.Part two:The effect of natural plant aqueous extract on the repair of H2O2 damaged liver cellsBased on the oxidative stress model established in experiment 1,the repair function of three natural plant aqueous extract(TCR,RAT,JLT)on hepatocytes were studied.The experiment was divided into four groups:control group(Con group),model group(H2O2 group),natural plant aqueous groups(RAT group,JLT group,TCR group)and Vc group.The results of antioxidant test in vitro showed that RAT had the highest activity of scavenging hydroxyl radical and superoxide anion radical,TCR has the highest DPPH radical scavenging activity.Compared with the H2O2 group,the cell viability of the injured hepatocytes treated with natural plant aqueous extract for 24 hours all had trend,and that of the RAT,TCR and Vc group was significantly increased(P<0.05).Compared with the H2O2 group,the CAT activity of RAT,TCR and Vc group all had a significant rising trend(P<0.01).Compared with the H2O2 group,the GSH content of the natural plant aqueous extract groups and Vc group increased significantly(P<0.01),while the content of MDA and the activity of SOD decreased significantly(P<0.05).The apoptosis rate of RAT group and JLT group was significantly decreased(P<0.05),the ROS level of natural plant aqueous extract groups and Vc group was significantly decreased(P<0.01),and the MMP of JLT and TCR group had a significant rising trend.DAPI staining found that the fluorescence intensity of the H2O2 group was much lower,and the number of nuclei was significantly reduced,the nucleus was condensed and irregular.Compared with the H2O2 group,the number of nuclei was increased in the natural plant aqueous extract groups and Vc group,and the phenomenon of nuclear condensation decreased,a small amount of nuclei tended to be normal.Electron microscopic observation showed that mitochondria swelled and cristae disappeared in the H2O2 group,while cristae disappeared slowly in the natural plant aqueous extract groups,and the number of autophagosomes and autophagy lysosomes increased slightly.It may induce autophagy and mediate apoptosis.Western blot results showed that compared with the H2O2 group,Caspase-3 expression in RAT group and TCR group was significantly decreased(P<0.01),and Bax protein expression was significantly decreased(P<0.01).In conclusion,the natural plant aqueous extract of can alleviate the oxidative stress damage of grass carp primary hepatocytes,reduce the degree of cell damage,and have a certain effect on liver protection.Part 3:Transcriptome analysis of primary hepatocytes of grass carp induced by hydrogen peroxide in natural phytoremediationTranscriptome technique was used to detect gene expression in con group,H2O2 group,natural plant aqueous extract group and VC group.GO annotation and analysis of KEGG pathway were used to explore the protective mechanism of natural plant aqueous extract on H2O2 induced oxidative damage of grass carp primary hepatocytes.The results showed that.There were 1073 differentially expressed genes,363 of which were up regulated and 710 down regulated.Oxidative stress may be caused by abnormal expression of redox related genes that damaged Mitochondrial and cellular oxidative and may be related to the interference of intracellular Ca2+homeostasis or the P13K/AKT/FOXO pathway mediated by LAR.Compared with H2O2 group,there were 3637 differential expression genes in TCR group,1565 of which were up regulated and 2072 down regulated.The differential expression genes mainly involved "iron drooping" related to apoptosis pathway,and TCR might alleviate oxidative damage by upregulation of GPX4.There were 482 differentially expressed genes,156 of which were up regulated and 326 down regulated in JLT group,mainly involving autophagy related pathways.JLT promoted normal apoptosis by up regulating Beclin 1 by affecting HMGB1 and DAPK.There were 200 differential expression genes in RAT group,89 of which were up regulated and 111 down regulated genes,which mainly involved the regulation of calcium resorption by endocrine and other factors and autophagy related pathways,RAT regulated autophagy and the role of intracellular Ca2+balance;There were 359 differential expression genes,103 of which were up regulated genes and 256 down regulated genes in Vc group.Vc might alleviate apoptosis by promoting thyroxine synthesis.Part 4:RAT has effect on repaired liver injury cells of cultured grass carpTo verify whether natural plants can repair liver damage in animals,It was found that the hepatopancreas of grass carp and allogynogenetic crucian carp was damaged in the process of fish culture.RAT was added to the dietary fed with 0.5 g/kg RAT in pellets for 1d,3 d and 5 d respectively.The results showed that the hepatosomatic index(HSI)of RAT group were induced trend,and the difference was significant except for the 3d group(P<0.05).Compared with the 0 d group,the viscerasomatic index(VSI)of 3 d and 5 d groups decreased significantly(P<0.05).The contents of total bilirubin(TB),direct bilirubin(DB)and indirect bilirubin(IBIL)were reduced significantly compared with the 0 d group(P<0.05);The TB acid content of RAT group has downward trend,but it was not significant(P>0.05).Compared with 0 d group,the contents of cholesterol(CHOL),total protein(TP)and globulin(GLB)in RAT group decreased significantly,but the difference was not significant(P>0.05),and the content of albumin(ALB)showed downward trend,in which there was significant difference between 1 d and 5 d groups(P>0.05);Compared with the 0 d group,AST in RAT group were significantly lower.AST in 0 d group were significantly higher than that in the 0 d group(P<0.05),Compared with the 0 d group,AST/ALT was significantly lower(P<0.05),and the trend was the same as that of aspartate aminotransferase.The results of H&E staining of liver tissue sections of grass carp showed that the nuclei of 3 d and 5 d groups were stained deeply.Most of the nuclei were located in the center of the cell,with more nuclei and clear cell structure.0.5 g/kg RAT was added to the feed of allogynogenetic crucian carp,T-AOC,SOD,CAT,GST and GSH-PX of RAT groups were increased significantly compared with 0 d group(P<0.05)After 70 days of feeding,while the GSH and MDA content were decreased significantly,but there was no significant difference between RAT groups and the 0 d group(P>0.05).In conclusion,the hepatopancreas of grass carp was damaged,adding 0.5 g/kg RAT for 1 d,3 d and 5 d might repaired the liver of grass carp,which was mainly manifested in maintaining the normal morphology and healthy tissue structure of grass carp liver.The hepatopancreas of allogynogenetic crucian carp was damaged,adding 0.5 g/kg RAT for 70 days,the activity of antioxidant enzymes increased significantly,which might alleviate the antioxidant stress of allogynogenetic crucian carp. |
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