Regulation Of Hepatic Immune-related Genes In Hyperlipidemia Mice Treated With Gypenosides

Objective:To study the regulation of hepatic immune-related genes during the treatment of gypenosides by using hyperlipoidemia mouse fed with high-fat diet and providing a basics of mechanism of gypenosides.Methods:1.The samples were from the previous animal experiments.The male C57BL/6J mice were divided into five groups,including group 1(Normal diet(ND)16W),group 2(High fat diet(HFD)16W),group 3(ND 38W),group 4(HFD 38W)and group 5(gypenosides(GP)).Group 1 and group 2 were fed respectively with normal diet(ND)and high fat diet(HFD)for 16 weeks to judge whether the model was successful.Group 3 and group 4 were continuously fed with normal diet and high fat diet,and treated with equal 0.1%CMC-Na from 16 weeks to 38 weeks.Group 5 were continuously fed with high fat diet,and treated with gypenosides(250 mg/kg)from 16 weeks to 38 weeks.At week 16 or week 38,liver tissue were taken.2.Hepatic RNA was extracted and were sequenced by a Beijing Genomics Institute(BGI)BGISEQ-500 Sequencing Platform.Original sequence data were aligned and annotated referring to UCSC GRCm38/mm10 mouse genome.795 immune-related genes were filtrated through OMIM and NCBI database.Genes expression data were extracted from the expression matrix,and 6 differentially expressed immune-related genes were randomly screened.Real-time fluorescence quantitative PCR was used to verify the mRNA expression of hepatic immune-related genes.The mRNA data matrix were analyzed by heat map,PCA and volcano map to find out the differentially expressed immune-related genes.After the genes with significant expression were screened out,the fold change of GP/HFD and HFD/ND were used to make scatter plot.The differentially expressed genes were found out the immune-related genes significantly regulated by gypenosides.3.KEGG pathway of differential genes were analyzed by DAVID database.4.The differential genes of HFD group and ND group,and GP group and HFD group were filtrated.A total of 277 were filtrated,and their expression values(FPKM),body weight,blood glucose,serum total cholesterol(TC),and low density lipoprotein(LDL-C)were used to correlation analysis.5.Real-time PCR was used to verify the expression level of transcriptome genes.Results:1.Overall profile of hepatic immune-related genes at week 16 of high-fat diet:At week 16 of high-fat diet,795 immune-related genes were analyzed by heat map and PCA,and HFD group was completely separated from ND group.According to the volcano map analysis,the expression level of 151 immune-related genes was significantly changed(P<0.05).2.Overall profiling of hepatic immune-related genes by gypenosidesGP group,HFD group and ND group were distinguished signficantly from the heat map and PAC map.A comparative analysis of hepatic immuine-related genes fed normal and high-fat diets were carried out using volcano maps,and 242 genes were significantly expressed(P<0.05).And the most genes showed an up-regulation trend.After treatment with gypenosides,90 genes were significantly changed(P<0.05).31 immune-related genes were significantly regulated by high fat diet and gypenosides,including Il18,Ngf,Cd4,Vnn1,Ephx1,Mtor,etc.3.KEGG pathway analysis of immune-related genes regulated significantly by gypenosides.At week 16 of high-fat diet,compared with ND group,differentially expressed genes generated by high fat diet mainly enriched in herpes simplex infection,pathways in cancer,hepatitis C,RIG-I-like receptor signal pathway and Toll-like receptor signaling pathway,etc.RIG-I receptor signal pathway were most enriched.Compared with the high fat diet group,the differential genes in mice treated with gypenosides was significantly enriched in Herpes simplex infection,Influenza A,Toll-like receptor signaling pathway,Measles,etc.The number of genes enriched in the herpes simplex infection pathway was the highest and the Toll-like receptor signaling pathway was most enriched.The results showed that gypenosides could regulate the immune system through multiple pathways.4.Correlation analysisCorrelation analysis showed that LDL-C was positively correlated with TC.Il18 bp and body weight,Cnpy3 and TC,H2-Q10 and LDL-C,Il18 and LDL-C,Il18 and blood glucose were positively correlated;H2-Eb1 and Cfp,Fpr1 and Cfp,Cd74 and H2-Eb1,H2-Aa and H2-Eb1 were highly positively correlated;Ikbkg and H2-Q10,Ikbkg and Irf5,Prkar1 a and Msrb1 were highly negatively correlated.Some highly correlated genes were enriched in the same pathway.5.Validation of the reliability of transcriptome dataUnder the influence of high-fat diet and gypenosides,Il18,Ngf and Cd4 were significantly up-regulated after significant down-regulation(P<0.05);Vnn1,Ephx1 and Mtor were significantly down-regulated after significant up-regulation(P<0.05).The real-time PCR data were generally in line with the trend of transcriptomic data.PCR results of Il18,Ngf,Cd4,Vnn1,Ephx1 and Mtor were positively correlated with RNA-seq data,indicating that transcriptome data were reliable,indicating that transcriptome data are reliable.Conclusion:Gypenosides significantly regulated 31 immune-related genes(including Vnn1,Ephx1,Mtor,Il18,Ngf and Cd4,etc.)during hyperlipidemia mice by treated with gypenosides,which may be one of its effects in the treatment of hyperlipidemia.