Transcriptomics Analysis Revels The Mechanism Of Zishen Qingre Lishi Huayu Recipe In The Treatment Of Granulosa Cell

Objective:In order to explore the possible molecular mechanism of Zishen Qingre Lishi Huayu Recipe(It is referred to as ZSF)meddle the granulosa cells(GC)in patients with PCOS(polycystic ovary syndrome).The differentially expressed genes among the comparable groups(PCOS-ZSF vs PCOS-TSY)were analyzed by transcriptome technology,and the effective targets of ZSF in the treatment of PCOS were screened out,which laid the foundation for further research on the molecular mechanism of ZSF in the treatment of PCOS,it also provided scientific basis for TCM syndrome differentiation of PCOS disease and clinical treatment of PCOS disease.Methods:Experiment 1: Screening the optimal concentration and time of KGN cells1.Preparation of ZSF and intestinal absorption solution: The liquid solution was collected according to clinical decocting Chinese medicine,concentrated and filtered to the drug concentration of about 1 g/m L,then sterilized ang refrigerated for the next experiment;prepare the intestinal absorption solution: after a week of adaptive feeding,these 20 SPF grade SD rats of weighing 180-220 g were randomly divided into two groups(ZSF and TSY groups).A skilled and gentle experimenter sacrificed all rats using cervical dislocation method.Two types of intestinal absorption solutions containing drugs(ZSF-containing or TSY-containing intestinal absorption solutions)were collected,filtered and sub-packed into EP tubes,and stored in the refrigerator at-80℃ for use.2.Using human ovarian granulosa cell line KGN cells for the optimal concentration and time of action: using the cck-8 experimental method,according to the absorbance value at 450 nm at 4 time nodes(0 h,24 h,48 h,72 h);The liquid concentration is designed according to the volume percentage concentration,from higher concentrations(0.5%,1%,2.5%,2.5%,5%,10% and 15%)and then to detect the growth of KGN cells,nararrow the screening after multiple observations of the absorbance OD,more appropriate drug concentration ranges(0.1%,0.2%,0.3%,0.4%,0.5%,0.6% and 0.7%)were selected for three independence experiments,then calculate the cell proliferation rate;In combination with the statistical analysis,the optimal concentration and time of GC growth were selected,lay the experimental foundation for the subsequent sampled co-culture experiments.Experiment 2: Explore the molecular mechanism of GC intervention based on transcriptomics1.Random sampling: according to the diagnostic criteria of hospital PCOS,24 patients with PCOS primary GC,randomly divided into two groups(ZSF group and TSY group).After intervention with the optimal action time of GC in the comparison group(ZSF group and TSY group),GC was collected and stored in liquid nitrogen for sequencing.2.Using RNA-seq(RNA Sequencing,RNA-seq)transcriptomic technology,according to the differential expression gene analysis(differentially expressed genes,DEGs)KEGG enrichment analysis and GO enrichment analysis,find out the difference significant genes and pathways,finally integrate the omics results and domestic and foreign academic literature,screening out the key PCOS target genes and related pathway.3.The selected differentially expressed genes and proteins were verified by realtime PCR(Quantitative Real-time,qPCR)and Western Blot(Western Blot,WB).Results:1.The results of cck-8 showed that the proliferation of KGN was correlated with the concentration and time of the intervention,and compared with the control group,the proliferation at 0.2% volume concentration for 48 h,so it was used as the subsequent experimental condition of the intervention of GC.2.RNA-seq transcriptomics results showed that a total of 260 DEGs were selected between the ZSF group and the TSY group,of which 121 genes were up-regulated and139 genes were downregulated.Combined with domestic and foreign literature studies,we selected 8 relatively representative genes in the academic field as possible key targets for PCOS granule cells,including NGFR,SOCS1,IRS2,SOD2,BRCA1,FOXM1,PLK1 and POLQ.3.GO function enrichment analysis shows that in the comparison group,the biological process mainly involves chromosome separation,mitotic nuclear division,mitotic cell nuclear division regulation,chromosome separation and mitotic cell cycle,its molecular function and movement activity,kinase,protein kinase,protein binding,purine nucleotide binding,ribonucleotide,the cell components mainly located to cell spindle,chromosome and centromere and centromeres,etc.4.Pathway enrichment analysis of KEGG(Kyoto Enc treatment yclopedia of Genes and Genomes,KEGG)pathway identified 7 pathways with potential effects of PCOS: p53,MAPK,FOXO,PI3K-Akt,NF-kappa B,c AMP and JAK-STAT.5.Both qPCR and WB experiments showed that ZSF drug intervention altered the expression of some genes and some proteins in GC.The qPCR experiments showed that BRCA1,FOXM1,PLK1 and POLQ genes were significantly downregulated in the ZSF(P <0.05),NGFR,SOCS1,IRS2 and SOD2(P <0.05),and WB showed IRS2,SOD2,NGFR and protein(P <0.01).Conclusion:(1)The promotion of GC proliferation upon treatment with ZSF may be a manifestation of ZSF in the treatment of PCOS patients.(2)The positive effects of ZSF against PCOS may involve pathways and genes related to autophagy,steroidogenesis,oxidative stress-related longevity,and inflammation in GC.(3)Some components of ZSF applied for the treatment of PCOS may also be effective for the treatment of some complications of PCOS.