MiRNA Sequencing Of Changbai Mountain Ginseng Decoction And Its Effect On Partial Target Genes In Rats With Qi Deficiency

Objective:Firstly,this study is to verify the existence of microRNAs in Changbai mountain ginseng decoction.Secondly,to verify the miRNA expression changes of ginseng in blank group?normal rat?and treatment group?rats that i.g ginseng decoction?.Thirdly,to verify the effects of miRNA on its target gene by establishing the model of Qi deficiency fatigue type in rats.Method:1.The general plant miRNA extraction kit was used to extract the miRNA from ginseng water decoction.The quality,purity and integrity of RNA were tested by agarose gel and Agilent2100 Bioanalyzer.The Illumina HiSeq^TM 2500 sequencer was used to sequence and analyze the ginseng decoction and to predict whether there were known miRNAs and new miRNAs.RT-qPCR was used to verify known miRNAs.2.Six rats were divided into 2 groups?the blank group and the treatment group?,3 in each group.Except the normal group,ginseng water decoction was orally administered to the rats in the treatment group twice a day,and the volume of gastric perfusion was 1ml per 100g a day.On the 31st day,no ginseng water decoction was administered,blood was collected from each group,and blood RNA was extracted by using the HiPure Liquid RNA/miRNA Kit.By comparing ginseng miRNA contained in rat blood verified by RT-qPCR and miRNAs in the decoction of the ginseng obtained by sequencing with bioinformatics methods,it was subjected to filter and analyze target gene.3.60 rats were divided into 6 groups?ginseng decoction group-R,de-ginseng miRNA group-Q,ginseng miRNA group-M,ginsenoside group-Z,PBS group-P,and blank group-K?,10 in each group.The Qi-deficiency fatigue rat model was made by using dietary distress and exhausted swimming method for 20 consecutive days.On the 21st day,the rats were administered but not swimming.Various materials from rats were taken,and BUN,LDH in serum and the content of liver glycogen were measured.The effect of each group of drugs on the rats was observed.RT-qPCR was used to verify whether the ginseng miRNAs affected the expression of immune genes in rats with Qi deficiency fatigue.The extracted ginseng decoction miRNA was used to act on rats with Qi-deficiency fatigue,and the expression of immune genes of ginseng miRNA in rats with Qi-deficiency fatigue was verified by RT-qPCR.Results:1.It was proved that miRNA can still be effectively extracted from ginseng water decoction which were tested by agarose gel and Agilent2100 Bioanalyzer.71 miRNA families were finally obtained in the ginseng decoction by sequencing.The presence and expression of miR159,miR167,miR396,miR166,miR168,miR156,miR165,miR162and miR394 in decoction of ginseng were verified by RT-qPCR,and these miRNAs had decreased expression when compared with the one extracted from fresh ginseng.2.RT-qPCR were used to further verify the presence of ginseng miRNAs in rat blood,such as miR159,miR167,miR393,miR156,miR8175,and miR5072.And through the analysis of relevant software for the prediction of target genes,it was found that some miRNAs in ginseng can achieve high degree of match for some genes in rats,such as miR159 and Mmp9,miR167 and Bcl2l1,miR8175 and Il4r,miR5072 and Bcl2,miR158 and Mapk14 and so on.3.With verification of various indicators,we found that this experiment succeeded in establishing a rat model of Qi-deficiency syndrome.After drugs were administered to each group,in the ginseng miRNA group,the expression level of immune genes had significant changes was vertified by RT-qPCR.Ginseng miRNA could affect the mRNA expression of INF-8,IL10,MMP9,IL4R,MARK14,TNF-?and BCL2L1,which may affect multiple immune response signal transduction pathways,and then regulate the expression of a variety of cytokines to prevent Qi deficiency.Conclusion:It was the first time to vertify the presence of miRNAs in ginseng decoction and the regulative effects of ginseng miRNAs on immune genes in rats with qi deficiency fatigue which provided us with a new way to recognize and study ginseng.On the other hand,it also reveals the mechanism of the curative effect of traditional Chinese medicine from the gene level.On the other hand,it also affects the research and development of ginseng new medicines,health care products and related products.