Studies On Immunity And Metabolism Of Panax Ginseng MiRNAs In Vitro And In Vivo And Functional Analysis Of Differential MiRNAs

Objective:In this study,ginseng miRNA was applied to qi-deficiency and fatigue mice,macrophages(RAW264.7)and liver cancer(HEPG2)cells,and then to study the immune and energy metabolism regulation of ginseng miRNA,in order to further explore ginseng miRNA as a medicinal substance possibilities to offer new perspectives.Methods:1.Establishment of the Qi-deficiency and fatigue mice model: Kunming(KM)mice were divided into blank group(BC),model group(MD),ginseng water decoction group(GD),ginseng water decoction plus enzyme group(NC),ginseng total RNA group(RNA),miR156 group(156),miR159 group(159),miR166 group(166),10 mice in each group.Methods of Food intake restriction and forced swimming were used for modeling fatigue and Qi-deficiency on mice.Mice in each group were intragastric and swimming once a day for 21 consecutive days.blood samples was collected from the orbit,Serum lactate(LA),glucose,creatine kinase(CK),blood urea nitrogen(BUN),lactic dehydrogenase(LDH),malondialdehyde(MDA),uperoxide dismutase(SOD),liver glycogen(LG),muscle glycogen(MG),IL-4,MMP9,PEPCK,FBPase levels were determined.liver and gastrocnemius muscle were taken to measure the levels of liver and muscle glycogen.2.The serum and liver of mouse model group,ginseng decoction group and ginseng total RNA group were sequenced.The differential miRNAs in model group,ginseng decoction group and ginseng total RNA group were screened.Target genes of the selected differential miRNAs were predicted,and GO and KEGG enrichment analysis was performed.3.CCK8 method was used to detect the effects of miR156,miR159 and miR166 simulants on RAW264.7 and HEPG2 cell proliferation.The proliferation rate and neutral red growth rate of RAW264.7 cells,the contents of NO and TNF-α and the mobility of HEPG2 cells were determined by miRNA mimics.Results:1.Compared with model group,the endurance swimming times of mice in ginseng decoction group,ginseng total RNA group,ginseng RNA removal group,miR156 group,miR159 group and miR166 group was prolonged.It reduced the metabolites urea nitrogen and lactic acid,and reduced the extravasation of lactate dehydrogenase and creatine kinase.Except for 159 grpup,liver glycogen content was increased in other administration groups,and muscle glycogen content was increased in ginseng decoction group,miR156 group,miR159 group and miR166 group.SOD activity in ginseng total RNA group and miR159 group significantly increased,reduced MDA content.The levels of MMP9 in the ginseng decoction group,the ginseng total RNA group,the ginseng RNA-removed group,and the miR156 group decreased,suggesting that the ginseng decoction and some miRNAs may improve energy metabolism,reduce the accumulation of metabolites in the body,and regulate the body’s immunity to relieve fatigue effect.2.High-throughput sequencing was performed on the tissues of ginseng decoction and ginseng total RNA of mice.The differential mirnas were identified by comparison of ginseng reference genome.In serum and liver,compared with the model group,the ginseng decoction group had 5 and 13 up-regulated miRNAs,and 2and 17 down-regulated miRNAs,respectively.Compared with the model group,the ginseng total RNA group had 8 and 17 up-regulated miRNAs,and 5 and 14 downregulated miRNAs,respectively.we analyzed the molecular functions of differentially expressed m RNAs using GO and KEGG databases,Most genes are related to "cellular process","metabolic process","biological regulation" and "immune system process".In the "cellular components" section,most genes are related to "cell part" and "organelle part",and a few genes are related to "cell junction" and "extracellular matrix" related.In the "cellular components" section,most genes are related to "cell part" and "organelle part",and a few genes are related to "cell junction" and "extracellular matrix")related.In the "Molecular Function" section,most genes are related to "binding" and "catalytic activity".In the "Molecular Function" section,most genes are related to "binding" and "catalytic activity".KEGG pathway enrichment analysis were performed to the target gene annotation of ginseng miRNA is mainly enriched in MAPK signaling pathway,Wnt signaling pathway,FOXO signaling pathway,AMPK signaling pathway,c AMP signaling pathway,etc.These results indicate that ginseng miRNA may have a certain regulatory effect on energy metabolism and immune function in mice with Qi deficiency and fatigue.3.In vitro studies showed that miRNA mimics could significantly promote the proliferation of RAW264.7 cells and inhibit the proliferation and migration of HEPG2 cells.The content of TNF-α in supernatant of RAW264.7 cells was increased,but miRNA mimics had NO significant difference in promoting NO release from macrophages,suggesting that miRNA mimics may have a poor effect on activating the signal transduction pathway of macrophages or releasing NO,which needs further study to confirm.These results suggest that miRNA mimics may have potential immunomodulatory effects.Conclusion:1.In vivo studies showed that ginseng decoction and some of its miRNA could relieve fatigue by improving energy metabolism and regulating immunity in mice,suggesting that ginseng miRNA may be involved in the qi replenifying and antifatigue effect of ginseng as a pharmacodynamic substance,and ginseng miRNA may play a cross-border regulatory role.Through high-throughput sequencing of serum and liver,it was found that panax ginseng miRNA could be absorbed into mice through gastrointestinal tract through ingestion,but the enrichment degree of miRNA in different tissues was different.2.In vitro results showed that miRNA mimics could regulate cell viability,phagocytosis,NO production,and TNF-α secretion in RAW264.7 macrophages,and at the same time,inhibited HEPG2 cell proliferation and migration.Therefore,it is speculated that ginseng decoction and its miRNA may be one of the effective material bases for ginseng to regulate energy metabolism and improve immune function.In addition,the regulatory role of plant miRNAs in human health and disease remains to be further explored.Although we have evidence that ginseng decoction and its miRNAs can improve exercise performance,studies on human miRNAs are currently lacking.Therefore,these results should be validated in human studies for their bioavailability.