Intervention Of Ginseng Decoction And Its MicroRNAs On Qi-Deficient Fatigue Mice And Study On Cross-Kingdom Regulation Of Ginseng MicroRNAs

Objective:A qi-deficiency fatigue mouse model was constructed to explore the effects of ginseng decoction and its microRNA on qi-deficiency fatigue mice,to explore the possibility of ginseng miRNA as a medicinal substance,and to explore possible mechanism of cross-regulation regulation of miRNAs in anti-fatigue effect of ginseng.Methods:1.The verification of some ginseng miRNAs in ginseng decoction:According to the sequence of miR159,mi R166,miR5072,miR5658 and miR8175 in the previous high-throughput sequencing results,corresponding reverse transcription and amplification primers were designed and synthesized,and these miRNAs were confirmed in ginseng decoction by RT-qPCR,which were identified in the ginseng decoction by high-throughput sequencing technology before.2.Negative gravity exhausted swimming experiment of qi deficiency fatigue mouse model:120 Kunming mice were divided into 10groups?blank control group-BC,model group-MD,ginseng decoction group-GD,ginseng decoction with RNase group-NC,ginseng total RNA group-RNA,miR159 group-159,miR166 group-166,mi R5072 group-5072,miR5658 group-5658,miR8175 group-8175?,12 mice in each group.Qi deficiency fatigue mouse model was built a by limiting daily food intake+negative gravity exhaustion swimming for 21 consecutive days.After preparing ginseng decoction,ginseng decoction with RNase,ginseng total RNA,designing and synthesizing mimic of mi R159,miR166,mi R5072,miR5658 and miR8175,these drugs were administered to corresponding group mice once a day for 21 consecutive days.On day 22,each material was collected and biochemical indicators such as blood urea nitrogen?BUN?,lactate dehydrogenase?LDH?,liver glycogen and muscle glycogen,liver superoxide dismutase?SOD?and malondialdehyde?MDA?,muscle ATPase activity were detected.Enzyme linked immunosorbent assay?ELISA?was used to detect the levels of serum inflammatory factors interleukin-4?IL-4?and tumor necrosisfactor-??TNF-??in mice.3.Identification of ginseng miRNAs in mouse blood and prediction of target genes:After extracting RNA from the blood of mice administered with ginseng decoction,the purity and integrity of total RNA were detected by agarose gel electrophoresis,Nano Drop and Agient2100 Bioanalyzer.Illumina HiSeq^TM 2500 sequencer was used for high-throughput small RNA sequencing to identify the ginseng mi RNAs.Target gene prediction was performed on the identified ginseng mi RNAs,and GO and KEGG enrichment analysis were performed.4.Validation of ginseng miRNAs and expression of target genes in some tissues of mice:The blood,spleen and thymus of model group?MD?,ginseng decoction group?GD?,ginseng decoction with RNase group?NC?and ginseng total RNA group?RNA?were were selected for RT-qPCR verification of mi R159,miR166 and miR8175.At the same time,RT-q PCR was used to detect the changes of immune gene expression in blood,spleen and thymus of qi-deficiency fatigue mice.Results:1.RNA was extracted from the prepared ginseng decoction,and the target miRNA was detected by RT-qPCR.It was found that miR159,miR166,mi R5072,miR5658 and mi R8175 were indeed existed in the total RNA of the ginseng decoction,which validated the previous high-throughput sequencing results,and it will lay a solid foundation for subsequent research.2.Compared with the mice in the blank control group,the mice in the model group had reduced activity and showed fatigue characteristics.Body weight,exhausted swimming time,liver glycogen,muscle glycogen content,SOD activity were significantly reduced,BUN level and MDA level were significantly increased,indicating that the mouse model of qi deficiency fatigue was successfully established in the experiment.The mice in the ginseng decoction group,ginseng total RNA group,miR159group,miR166 group,miR5072 group and miR5658 group had longer exhausted swimming time,higher liver glycogen and muscle glycogen level,lower BUN levels and higher LDH activity when compared with the model group,showing certain anti-fatigue activity of ginseng and its miRNAs.The ginseng decoction group,ginseng total RNA group,miR5072 group,miR5658 group and miR8175 group significantly increased SOD activity,reduced MDA content,increased gastrocnemius Na⁺K⁺-ATPase activity,significantly increased serum IL-4 levels,and decreased serum TNF-?levels,indicating that ginseng decoction and its miRNA may relieve fatigue by improving energy metabolism,reducing oxidative stress and regulating body immunity.3.High-throughput sequencing was performed on the blood of mice fed with ginseng decoction,and 30 ginseng miRNAs from 23 families were identified by comparing the ginseng reference genome.The target genes of ginseng miRNAs were predicted,and 2955 target genes which related to immunity were predicted.The target genes were analyzed by GO and KEGG enrichment.It was found that the target genes were enriched in aspects of cellular process,biological regulation,and immune system process.These results indicate to some extent that ginseng miRNA may have an effect on the immune function of mice.This makes it possible in theory that miRNA may regulate target genes cross-dingdom.4.Mi R159,mi R166 and miR8175 were detected in mice blood,spleen and thymus by RT-q PCR,indicating that ginseng mi RNA can be taken orally into mouse tissues and organs.At the same time,it was verified by RT-qPCR that the expression of immune genes in the mice in the RNA group significantly changed,and the expression of IL4R in the spleen decreased significantly,suggesting that ginseng mi RNA had a certain effect on the immune genes in mice.Conclusion:Ginseng decoction and its miRNAs can relieve fatigue by improving energy metabolism,anti-oxidative stress,and regulating body immunity,indicating that ginseng miRNA may participate in the qi-enhancing anti-fatigue effect of ginseng as a medicinal substance and may play a role in cross-kingdom regulation.The existence of ginseng miRNA in the ginseng decoction,blood,spleen,and thymus of mice administered with ginseng decoction was detected by high-throughput sequencing and RT-qPCR,indicating that ginseng miRNA can enter the mice through the gastrointestinal tract.The anti-fatigue effect of ginseng miRNA has a material basis for cross-kingdom regulation,which is conducive to further research in the future to reveal the mechanism of cross-kingdom regulation.