| Background and purpose:Previous studies have shown that miRNA156 was abundant in Glycyrrhiza uralensis decoction,and the effects of Glycyrrhiza uralensis' s miRNA on human immune cells were investigated by cell morphology,cell proliferation and immune-related gene expression.In this study,the effects of Glycyrrhiza uralensis Fisch and its miRNA on mice were studied from the observation of intestinal flora,immunogene expression of lymphocyte,transcription sequencing of intestinal tissue and intestinal tissue's morphology,which laid a foundation for the development of new research direction of traditional Chinese medicine.Methods:1.We prepared Glycyrrhiza uralensis decoction and honeysuckle decoction as control group.According to the early high-throughput sequencing results of miRNA156,we designed and synthesized the corresponding miRNA156 mimic and its reverse transcription and detection primers,gavaged different doses of the decoction and miRNA156 mimic respectively and injected miRNA156 mimics on tail vein.Blood was taken from eyes and centrifuged to obtain serum and detected the miRNA in serum by qRT-PCR.2.genomic DNA was extracted from fresh feces of mice,and 16 s rDNA V4V5 hypervariable region sequencing was carried out to analyze the differences of intestinal flora in each group.The relationship between each group and the role of miRNA156 in Glycyrrhiza uralensis were determined by ? diversity and ? diversity analysis.3.The ileum tissue of mice were collected,part of which was used for RNA extracting by liquid nitrogen grinding for RNA-seq and bioinformatic analysis,the other part was fixed by 4 % paraformaldehyde solution,dehydrated,paraffin embedded,tabletted(4?m),HE stained,optical microscope observed and photographed.4.the spleen tissues of mice were collected,and the lymphocytes were obtained by filter screen grinding and erythrocyte-breaking treatment.The total RNA was extracted,the different expressions of transcription factors,cytokines and genes detected in the sequence analysis of the transcriptome were detected by qRT-PCR.Results:1.Blood,feces,spleen and ileum of mice were obtained by animal experiments.The miRNA in serum was detected by qRT-PCR,and the result showed that miRNA156 was not in serum.2.16 s rDNA V4V5 hypervariable region sequencing results showed that the intestinal microbes in mice were mainly composed of Firmicutes,Bacteroidetes and Proteobacteria.Compared with the blank group,the abundance of Bacteroidetes and Proteobacteria decreased with the increase of the concentration of Glycyrrhiza uralensis decoction,while the abundance of Firmicutes increased significantly.Glycyrrhiza uralensis' s miRNA has little effect on the Firmicutes,but reduces the Bacteroidetes and increases the Proteobacteria,and the change of some bacteria that have been shown to be associated with inflammation or nutrient absorption.3.In the sequence analysis of the transcriptome,the different expression of genes of the ileum related to immune function were found by the combination of GO function enrichment and KEGG pathway analysis.By observing the histopathological sections of the ileum after HE staining,it was found that the whole villus structure was maintained in the low dose group,the miRNA tail vein injection group and the blank group,while the high dose group and so on caused inflammation.4.By detecting the expression of cytokines,transcription factors and genes detected in the sequence analysis of the transcriptome in mouse spleen lymphocytes,it was found that the expression of mBAX,mIL12p35,mNF?B1,mSTAT1,mTGF-?,mIFN-?,mP53,mCd72,mCd79 b,mCr2,mSell were significantly different.Conclusion:Combined with previous experimental results,Glycyrrhiza uralensis' s miRNA not only has a significant immunomodulatory effect on immune cells,but also has a significant regulatory effect on intestinal flora and intestinal tissue immune system,which has far-reaching significance in the research field of the pharmacodynamic mechanism of Glycyrrhiza uralensis,and brings Glycyrrhiza uralensis new research ideas for the further research of clinical application,mechanism and new drug research and development. |
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