Multi-omics Analysis Of The Mechanism Of Cholesterol Regulated By Gypenosides In Liver

Objective:In this study,clinical data were extensively used to investigate the effect of Thunb on intrahepatic cholesterol levels,and at the same time,combined with animal experiments to further study the regulation of gypenosides on genes and proteins in endogenous cholesterol synthesis pathway and some cholesterol metabolism pathways except bile acid pathway,to further reveal the important target and mechanism of gypenosides to reduce liver cholesterol.Methods:1.A collection of recent clinical trial articles on the treatment of hypercholesterolemia by Thunb and a comprehensive evaluation of the cholesterol-lowering efficacy of Thunb in humans using Meta analysis.Using "Gynostemma pentaphyllum" as the subject word,databases such as Pubmed,CNKI and Web of science were systematically searched for eligible literature.Sample sizes,cholesterol levels and SD values were extracted from the literature for treatment and control populations.Standardized mean difference(SMD)and 95% confidence interval(95% CI)were calculated by STATA14.0 to evaluate treatment effect,and sensitivity analysis and publication bias was performed on the collected literature.2.The effect of gypenosides on the gene level of the steroid pathway was investigated by transcriptomics.Liver RNAs were extracted from mice at the endpoint of administration in the stage of previous study,respectively,from the control group(Con),the hypercholesterolemia group(HC),and gypenosides group(GP).Transcriptome analysis was performed using the BGISEQ-500 sequencing platform.KEGG pathway and NCBI databases were used to find steroid pathway-related genes,and the expression levels of steroid pathway genes were analyzed using volcanogram,heat map,and PCA.3.Real-time PCR validation of transcriptome data: selected genes that are significantly regulated by gypenosides in the transcriptome were validated using real-time PCR.Primer specificity was tested after primer design by Primer 3.0 software.RNA were extracted from mouse liver tissue and reverse transcribed to cDNA for real-time PCR,and Ct values were measured to express gene transcript levels.4.The effect of gypenosides on the expression of steroid pathway proteins was investigated by high-resolution mass spectrometry.Five mice were selected from each of the three groups of Con,HC and GP mice at the endpoints of administration,liver proteins were extracted and hydrolyzed by trypsin,peptides were separated by NanoElute ultra-efficient liquid-phase system,and peptides were identified and assembled proteins were reduced using high-resolution mass spectrometry data.The proteomic data were mined in depth using volcanograms,volcanogram,heat map,PCA,etc,and correlation analysis was performed with the transcriptome.Results:1.Meta-analysis results indicated that gypenosides was significantly negatively correlated with total cholesterol(TC)levels(SMD=-1.45849,95%CI:-1.78791~-1.12908,P=0.000)and low-density protein cholesterol(LDL-C)levels(SMD=-1.93328,95%CI:-2.74991~-1.11664,P=0.000),which was consistent with the pharmacodynamic results of previous animal experiments.It further proved the clinical efficacy of cholesterol levels in blood or liver were reduced by Gynostemma pentaphyllum.2.Transcriptome results indicated that 138 steroid pathway-related genes were obtained by literature search and database query,of which 128 genes were detected in the transcriptome.Further analysis showed that 38% of the 128 steroid pathway genes were significantly up-regulated by a high-fat diet,while 33% of these genes were significantly down-regulated.PCA and heatmap analyses revealed that the overall expression profile of the steroid pathway at the transcriptional level could be significantly up-regulated by gypenosides.The expression of key genes for cholesterol synthesis such as Hmgcs1,Fdps,and Fdft1 was significantly repressed by high-fat diet and significantly up-regulated by gypenosides.The expression of cholesterol efflux transporter and steroid hormone metabolism-related genes such as ApoB,Abca1,Cyp3a11,Cyp3a25 were significantly up-regulated by gypenosides.3.RT-PCR results showed that the expression levels of 21 hepatic steroid pathway-related genes such as Hmgcs1,Hmgcr,Tm7sf2,Fdps,Fdft1,Cyp3a25,Cyp3a11,and Cyp7a1 were highly consistent with the transcriptome results,further confirming the accuracy of the transcriptome results.4.80 steroid pathway proteins were detected in liver tissues by LC-MS,44% of which were significantly regulated by a high-fat diet,while 18% of the steroid pathway proteins were significantly altered after treatment with gypenosides.Gypenosides could inhibit the expression of cholesterol synthesis-related proteins such as HMGCS1,FDPS,FDFT1,and promote the expression of cholesterol efflux transporter proteins such as SCARB1,CYP3A25 and steroid hormone metabolizing enzymes.Transcriptome-proteome association analysis showed that CYP3A25,FDFT1,TM7SF2,HMGCS1,FDPS,MVD1 and PMVK were significantly associated with the therapeutic effects of gypenosides.The expression of all seven proteins except CYP3A25 was positively correlated with serum cholesterol levels in mice.Conclusion:Inhibiting the synthesis of endogenous cholesterol,accelerating further metabolism of cholesterol downstream products,and accelerating cholesterol efflux may play a indispensable part in the mechanisms by which gypenosides reduce hepatic high cholesterol levels.Among them,gypenosides may exert desirable effects on the regulation of the transcriptional or protein expression levels of cholesterol synthesis key enzymes HMGCS1,FDPS,FDFT1,cholesterol efflux key transporter and vector protein ABCA1,APOB,cholesterol downstream products key metabolites CYP3A25,CYP3A11,etc.