Glycyrrhiza Uralensis's MiRNA156 Influence Intestinal Immunity Via Gut Microbiota

Background and purpose:Previous studies found that Glycyrrhiza uralensis Fisch contain abundant microRNAs.High throughput sequencing showed that microRNA156 ranked first in the richness of Glycyrrhiza uralensis Fisch.Moreover,experiments in vitro showed that Glycyrrhiza uralensis microRNAs could regulate the gene expression of human immune cells to a certain extent.experiments in vivo showed that Glycyrrhiza uralensis microRNAs also had certain regulatory effects on intestinal bacteria and intestinal immune system.The purpose of this study was to investigate how Glycyrrhiza uralensis microRNA156 regulates the immune system of mice,and what role intestinal bacteria play in it.Methods:1.In vitro experiments: NCBI was used to detect microRNA156 mimic sequence target bacteria of licorice,and co-cultured with microRNA156 mimic,microRNA156 mutant and microRNA156 inhibitor respectively.The growth curve was observed by detecting 0D600.2.In vivo experiment: Mice were randomly divided into 6 groups,which were divided into gavage groups and cage groups.Six groups were fed with Glycyrrhiza uralensis decoction(1.5g/kg)and glycyrrhizic acid(0.375g/kg),microRNA156 mimic,microRNA156 mutant and microRNA156 inhibitor respectively.The other six groups were fed in the same cage with the six groups,and feces were collected 7 days later for 16 S V3-V4 region sequencing to analyze the changes of intestinal microorganisms in each group.3.Some of the ileum and colon tissues of mice were dehydrated with liquid nitrogen.RNA was extracted by Trizol grinding and used for transcriptome sequencing.Differential genes were analyzed.Some of the ileum and colon were fixed with 4% polyformaldehyde,then paraffin embedded,HE staining,and microscopic photographs were taken to analyze the differences between the groups.4.After extracting RNA from mouse spleen,RT-QPCR was used to confirmation the expression of differentially expressed genes in spleen.Results:1.The growth curve of Acinetobacter baumannii and Escherichia coli under free growth conditions was determined.The growth curve of Acinetobacter baumannii and Escherichia coli co-cultured with microRNA156 mimic,microRNA156 mutant and microRNA156 inhibitor to plateau stage was determined.From the growth curves,we can see that microRNA156 mimic can regulate the growth of Acinetobacter baumannii,but there is no difference in co-culture of Escherichia coli.2.The results of 16 S stool sequencing showed that the intestinal flora of the Glycyrrhiza uralensis decoction group,glycyrrhizic acid group and microRNA156 micmic group were similar to that of the same cage group.The content of lactic acid bacteria increased significantly in the microRNA156 mimic group and the same cage group,while the content of lactic acid bacteria decreased significantly in the microRNA156 mutant,microRNA156 inhibitor group and the same cage group.3.The results of ileal transcriptome sequencing showed that the immune-related signaling pathways were enriched in the Glycyrrhiza uralensis decoction group,the glycyrrhizic acid group and the microRNA156 mimic group,but there was no significant difference in the immune-related signaling pathways between the microRNA156 mutant group and the microRNA156 inhibitor group.The results of HE staining of ileum and colon showed that the intestinal structure of the microRNA 156 micmic group was more complete,and the Glycyrrhiza uralensis decoction group had no significant difference in immune-related signaling pathways.The Glycyrrhiza uralensis decoction group and glycyrrhizic acid group had local slight edema,while the microRNA156 mutant group and the microRNA156 inhibitor group had small amount of intestinal gland necrosis and local edema.The pathological condition of the cage cage group and the gavage group tended to be consistent.The intestinal state of the microRNA156 mimic gavage group and cage group showed a good state.The gastrointestinal status of the Glycyrrhiza uralensis decoction gavage group and microRNA156 mimic gavage group tended to be similar,while the difference between the Glycyrrhiza uralensis decoction gavage cage group and cage group was obvious.The gastrointestinal status of the microRNA156 mimic gavage group and cage group also tended to be the consistent.It indicated that the microRNA156 of Glycyrrhiza uralensis was an effective component of the Glycyrrhiza uralensis decoction,and microRNA156 could affect the intestinal immunity of mice via intestinal bacteria.4.The results of RT-QPCR showed that intragastric administration of Glycyrrhiza uralensis microRNA156 not only affected intestinal immunity of mice,but also probably affected splenic immune system,indicating that the effect of intragastric administration of Glycyrrhiza uralensis microRNA156 on immune system was systemic,not limited.Conclusion:The results of 16 S sequencing in feces showed that intestinal bacteria in the gavage group and the cage group tended to be identical under the co-culture conditions and the number of beneficial intestinal bacteria increased significantly in the microRNA156 mimic group.The results of intestinal transcription and intestinal pathological section showed that the effective components in the Glycyrrhiza uralensis decoction were not only glycyrrhizic acid,but also microRNA156,and Glycyrrhiza uralensis microRNA156 could improve the intestinal immunity of mice by regulating intestinal bacteria,even probably affect the expression level of immune genes in spleen of mice.