| In order to investigate whether soybean meal and soybean have damage effect on hepatocytes and its mechanism of action.Soybean aqueous extract(SAE)and soybean meal aqueous extract(SMAE)were used as the experimental material,the primary hepatocytes cultured in vitro of grass carp were used as the experimental objects.The final concentrations of SMAE and SAE were 0,0.5,5.0 and 10.0 mg/mL that respectively added to the cell culture medium for 24 hours,and then the samples were collected for analysis to study the oxidative damage,regulation and metabolism mechanism of the hepatocytes.The main contents of this paper are as follows:Part 1:Effect of SAE and SMAE on oxidative damage of grass carp primary hepatocytesCCK-8 method was used to detect cell viability,and using electron microscope to observe cell ultrastructure.Hoechst 33285 fluorescent staining was used to observe cell nuclear morphology and detect the activity of antioxidant related enzyme.Flow cytometry is used to detect cell apoptosis rate.The results show that with the increase of the concentration of aqueous extract,the activity of hepatocytes decreased gradually,with a significant dose-response relationship(P<0.05).No significant difference of the cell relative activity has been observed among the low SAE and SMAE concentration(0.5 and 2.5 mg/mL)groups and the control group.In the 5.0 and 10.0 mg/mL SAE dose groups,the relative cell viability was negatively correlated with the concentration.The cell viability was 78.10±8.57%and 65.97±7.35%respectively,which was significantly different from the control group(P<0.01).The relative cell viability in the SMAE groups(5.0 and 10.0 mg/mL)was 86.35±7.17%and 80.26±7.08%,respectively.which was significantly lower than the control group(P<0.01).At the same concentration,the relative activity of cells in SAE group was less than that in SMAE group.The ultrastructure of hepatocytes showed that the annular condensation of chromatin along the nuclear membrane,sparse light density,swelling of mitochondria,decrease of number and accumulation of lipid droplets.Hoechst 33285 staining showed that the fluorescence intensity of SAE and SMAE groups(10.0 mg/mL)was significantly weakened,the staining was uneven(chromatin agglutination),the nucleus was broken,and a few apoptotic bodies appeared.Compared with the control group,the contents of lactate dehydrogenase(LDH)and malondialdehyde(MDA)in the cell culture medium were significantly increased,and the activities of superoxide dismutase(SOD)and glutathione(GSH)were significantly different(P<0.05).Flow cytometry was used to detect mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)levels.SAE and SMAE could decrease MMP and increase ROS.With the increase of water extract concentration,the early apoptosis rate of cells increased.There was no significant difference in the apoptotic ratio between the low dose group(0.5,2.5 mg/mL)and the control group.The apoptotic ratio of the SAE group with 5.0 and 10.0 mg/mL concentrations was 22.55± 4.35%,55.03± 2.76%,and that of the SMAE group was 32.67±5.79%,37.11±8.57%,respectively,which was significantly different from that of the control group(P<0.01).The above results show that the damage degree of SAE and SMAE to hepatocytes was positively correlated with the amount of added,and SAE and SMAE showed the same damage effect.When the amount of added was more than 5.0 mg/mL,there was a significant difference compared with the control group.SAE and SMAE can lead to the changes of cell ultrastructure,the increase of ROS level,the disorder of cell antioxidant system and the anomalies of mitochondrial structure and function,which ultimately affect cell viability and trigger the pathway of cell death.Part 2:Transcriptome analysis of primary hepatocyte damage in grass carps caused by SAE and SMAEIn order to clarify how the aqueous extract affects the normal life activities of hepatocytes at the overall level,the transcripts of hepatocytes in the normal group,SAE(5.0 mg/mL)group and SMAE(5.0 mg/mL)group were analyzed at the overall level by using RNA-seq transcriptomics.According to the statistical difference standard,the default parameters:p-adjust<0.05 and |log2fc|>=1,to screen differentially expressed genes,go function annotation,enrichment analysis and KEGG signal pathway enrichment analysis were used to study them systematically.In this study,869 differentially expressed genes were obtained in SAE group,of which 337 genes were up regulated and 532 genes were down regulated.The results showed that the most significant differentially enriched gene group in SAE group were mainly involved in the biological processes such as "regulation of protein hydrolysis","negative regulation of hydrolase activity","cellular lipid metabolic process","isoprenoid metabolic process".Molecular function includes "enzyme inhibitor activity","peptidase inhibitor activity","endopeptidase inhibitor activity","oxidoreductase activity",etc.Located in"extracellular space","extracellular region part" and other cell components.Most of the significantly enriched KEGG pathways are focused on steroid biosynthesis,complement coagulation cascade,PPAR signal pathway,cytokine receptor interaction and metabolic pathway of nutrients.In SMAE group,1451 differentially expressed genes were obtained,440 of which were up-regulated and 1011 down regulated.The annotated genes were further analyzed for differential expression analysis.In biological processes,the most common entries for differentially expressed genes include "oxidation-reduction process","negative regulation of catalytic activity","small molecule metabolism process","negative regulation of proteolysis",etc.Molecular functions such as"oxidoreductase activity","peptidase regulation" and "peptidase inhibitor activity".Cell components located in the "extracellular space" and "extracellular region part".Most of the KEGG pathways are concentrated in "complement and coagulation cascade","cytokine receptor interaction","glycine,serine and threonine metabolism","triglyceride metabolism" and NF-?B signaling pathway.The above results showed that the enriched pathway was mostly related to the inflammatory reaction,protein metabolism,amino acid metabolism and lipid metabolism,which indicated that the nutritional metabolism of hepatocytes was disordered,the signal transduction function mediated by cell membrane receptor was regulated,and the energy metabolism of the body was changed.In addition,hepatocytes of SAE group were enriched in several fatty acid metabolism pathways,such as steroid biosynthesis,PPAR signaling pathway,arachidonic acid metabolism,fatty acid metabolism,etc.,which regulated the fatty deposition of hepatocytes through steroid biosynthesis pathway.Part 3:Metabolomics study on the regulation of SAE and SMAE on primary hepatocyte metabolism in grass carpsUsing UPLC/MS technology to obtain the profile information of hepatocyte metabolites in the control group,5.0 mg/ml concentration of SAE group and SMAE group,including intracellular metabolites and extracellular metabolites.Multivariate statistical analysis methods such as principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OLPS-DA)were used to select potential biomarkers by means of variable importance projection(VIP>1),and the structure of potential biomarkers was identified by combining mass spectrometry fragment information and database retrieval.The results showed that most of the samples were located in the ellipse of 95%confidence interval without outliers.Moreover,the clustering distribution of hepatocyte metabolite phenotypes in both SAE group and SMAE group deviated from the control group.a total of 11 endogenous small-molecule metabolites were screened from the control group,SAE group and SMAE group respectively,which were regarded as potential biomarkers for the disturbance of endogenous metabolism of liver cells caused by aqueous extract.The are UDP-D-galactose,1-hydroxy-3-nonanone,7-hydroxymethotrexate and members of sulfonyltransferase family({[(3 E)-4-(5-hydroxy-1-oxo-1 H-isochromen-3-yl)but-3-en-1-yl]oxy}sulfonic acid)and arginyl-proline were up-regulated.Glutathione,1-arginine,indole,phosphatidylserine(PS(14:0/16:1(9Z))),amylose and indoleacetaldehyde showed a downtrend.The identified differential metabolites were input into metabo Analyst's metpa platform to construct related metabolic pathways.It was found that the differential metabolites were mainly concentrated in glutathione metabolism,arginine metabolism,arginine and proline metabolism pathways.In the extracellular fluid metabolizing group,30 different metabolites were screened,which were n-acyl-l-phenyl,alanine,n-acyl-d-phenyl,alanine,n-acetylomithine,choline,L-histidine,5,6-p-toluidine,vanillin,amylose,guanosine guanine,pyridine derivatives,and concentrated in two pathways of histidine metabolism and purine metabolism.The above results showed that endogenous products produced strong disturbance in the whole metabolic track and closely related with hepatocytes damage.Histidine metabolism and purine metabolism pathway led to the disorder of TCA cycle,nucleotide metabolism and amino acid metabolism.It then causes inflammation in the liver,the accumulation of lipid and the influence of cell membrane integrity. |
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