Research On The Mechanism Of Gypenosides Against Renal Interstitial Fibrosis Based On MicroRNAs

Fibroblasts are the main source of extracellular matrix synthesis and secretion in the process of renal interstitial fibrosis.Renal damage caused by various diseases promotes the accumulation of inflammatory factors in the kidney,activates fibroblasts,and secretes a large amount of extracellular matrix(ECM),then the microenvironment of local cell survival changes.When the extracellular matrix production reaches a certain level,even if the cause of the original kidney damage has been removed,the kidney’s inherent cells will continue to undergo phenotypic transformation under the stimulation of this microenvironment,promoting the development of renal fibrosis in an irreversible direction.MicroRNAs(miRNAs)is a short non-coding RNA that can be widely involved in various life processes by regulating the expression of target mRNA.In the kidney,miRNA can regulate the development of the kidney and participate in the normal function of the kidney.At present,miRNA has also been shown to participate in the regulation of many kidney disease processes including renal fibrosis,they can be used as diagnostic basis and therapeutic targets for diseases.Gypenosides(GPs)are the main components of GPs.It has shown good anti-renal fibrosis efficacy in the high-content screening of more than 300 traditional Chinese medicine compounds based on in vitro models carried out by the research group in the early stage of the research group.The mechanism of action is not yet clear,and there are few reports on the anti-renal interstitial fibrosis of GPs.In addition,studies have shown that GPs can exert anti-tumor,anti-depression,and improve cardiovascular disease effects by regulating miRNA expression.Based on this,this study used TGF-β1 to induce NRK-49F cells to construct an in vitro model to explore the optimal concentration of GPs for anti-fibrosis,and on this basis to further investigate whether its anti-fibrosis effect is related to the regulation of miRNAs expression.Method1.Cultured rat renal interstitial fibroblasts(NRK-49F)in vitro,then stimulated the cells with different concentrations of GPs for 48 h,then CCK-8 method was used to detect the effect on cell proliferation and determine the highest concentration that had no significant effect on cell activity.The concentration to be used in subsequent experiments.2.Stimulated NRK-49F cells with 10 ng/ml TGF-β1 to establish an in vitro model of renal interstitial fibrosis,add 75,50 and 25 μM of GPs to treat the cells,and used immunofluorescence to detect α-SMA,type I collagen(COLI)and type III Collagen(COLIII)expression,preliminary evaluation of the effects of different concentrations of GPs on fibroblast activation and secretion of ECM;3.Construct the renal interstitial fibrosis cell model by the same method as above,add different concentrations of GPs to intervene,RT-PCR and WB to detect the expression of α-SMA mRNA and protein to evaluate the effect of GPs on fibroblasts;4.Construct the renal interstitial fibrosis cell model in the same way as above,add different concentrations of GPs to intervene,RT-PCR and WB to detect the expression of COLI and COLIII mRNA and protein to evaluate the activation of GPs on activation The effect of fibroblast secretion of extracellular matrix;5.Construct the renal interstitial fibrosis cell model as above,add 75 μM GPs to intervene for 48 hours,small RNA sequencing to detect the expression of miRNAs in each group,compare the differential expression of miRNA in the normal group and the model group,use computer analysis methods targetscan,miranda,and Rnahybrid to build an miRNA target gene prediction model,predict downstream target genes for differentially expressed miRNAs,and use GO,KEGG classification analysis and enrichment analysis for prediction results;6.Intersected genes with significant differential expression in the "normal group vs model group" and "model group vs GPs group" to find miRNAs targets that GPs may act on,and analyzed the downstream target genes of the target miRNAs,to find their potential downstream targets,and explore the biological pathway processes related to the targets.Result1.In NRK-49F cells,the IC20 of GPs for 48h is 52.722 μM,and the intervention concentration of GPs is set to 75 μM,50 μM,and 25 μM,respectively;2.Immunofluorescence results showed that the expression levels of α-SMA,COLI,and COLⅢ in NRK-49F cells increased after TGF-β1 induction.After intervention with different concentrations of Gypenosides,the expression levels of α-SMA,COL Ⅰ,and COL Ⅲ were higher than those of the model,with 75 μM GPs showed the best effect;3.The results of RT-PCR and WB showed that the intervention of GPs of different concentrations can inhibit the expression of α-SMA mRNA and protein,and the inhibitory effect was concentration-dependent.,with 75 μM GPs showed the best effect.Among them,25μM GPs were Inhibitory effect of α-SMA mRNA expression was not significantly different from the model group;4.The results of RT-PCR and WB showed that GPs of different concentrations could inhibit the expression of COLⅠ and COLⅢ mRNA and protein,and the inhibitory effect was concentration-dependent,with 75 μM GPs showed the best effect.Among them,the inhibitory effect of 25 μM and 50 μM GPs on the expression of COLI protein was not significantly different from that of the model group;5.Small RNA sequencing results show that miRNAs are abundant in NRK-49F cells and have a regulatory effect on multiple biological processes and signaling pathways;after TGF-β1 induces fibroblast activation,the expression of 151 miRNAs changes significantly,among which 77 expressions were up-regulated and 74 expressions were down-regulated;after GPs intervention,the expression of 18 miRNAs could be significantly improved,of which 10 expressions were up-regulated and 8 expressions were down-regulated;the results of target gene classification analysis and enrichment analysis showed that after fibroblast activation,The cytoplasm,nucleus,cell components,cytosol,cell junctions,cytoskeleton and other parts of the cell composition changes,phosphorylation,RNA polymerase Ⅱ transcriptional positive regulation,protein phosphorylation,ion migration,transcriptional positive regulation A variety of molecular functions have been changed;proteins involved in biological processes such as metal ion binding,molecular function,protein binding,ATP binding,and nucleotide binding have changed.The differentially expressed 151 miRNAs downstream regulate targets and metabolic pathways,PI3K-Akt signaling pathway,Ras signaling pathway,phospholipase D signaling pathway,Ras signaling pathway,Rap1 signaling pathway,cAMP signaling pathway,agonist protein cytoskeleton regulation,axon guidance and other signal transduction processes have significant correlation;6.Intersection analysis showed that TGF-β1 induced down-regulation of miR-3588 and miR-378a-5p expressions,and up-regulated miR-135b-5p and miR-3068-5p expressions.The intervention of GPs can reverse this effect.These 4 miRNAs It may be a potential key upstream regulator of GPs regulating renal interstitial fibrosis;its downstream target genes are predicted and analyzed.The results show that these four miRNAs regulate target genes in the MAPK signaling pathway,PI3K-Akt signaling pathway,and phospholipase D signaling There are significant enrichments in the pathway,Ras signaling pathway,Rap1 signaling pathway,cAMP signaling pathway,mTOR signaling pathway,calcium signaling pathway,and Wnt signaling pathway,among which the PI3K/Akt signaling pathway enriches the most target genes,a total of 19,among which 16 target genes for miR-378a-5p,including Tp53,Tgfa,Ifnarl,Lpar1,Ccnel,Ngfr,G6pc,Lpar4,Tsc1,Pik3ca,Col6a2,Rela,Brcal,Phlpp2,Igf2,Rptor,and mainly concentrated in PI3K And its upstream targets.Based on this analysis,miR-378a-5p/PI3K-Akt may be one of the important signaling pathways for GPs to exert anti-renal interstitial fibrosis.ConclusionGPs can significantly inhibit the activation of NRK-49F cells induced by TGF-β1,reduce the abnormal deposition of ECM and play a role in anti-renal interstitial fibrosis.Its inhibitory effect is concentration-dependent,and the effect of 75 μM is the most significant.The mechanism of action may be related to the up-regulation of miR-3588 and miR-378a-5p expressions,and down-regulation of miR-135b-5p and miR-3068-5p expressions,which may lead to changes in the expression of related target genes in downstream pathways,of which miR-378a-5p/PI3K-Akt may be One of the important pathways of renal interstitial fibrosis.