| Background and purpose:Preliminary studies in this laboratory have proved that Glycyrrhiza Uralensis’ miRNA 156 affected the composition of intestinal microbiota and the expression of immune-related genes.At the same time,Glycyrrhiza Uralensis’ miRNA 156 in the co-housing group showed the same change trend in the intestine of mice in the gavage group.However,we don’t know the way in which Glycyrrhiza Uralensis’ miRNA 156 works and the reason for the changes in the intestines of mice in the co-housing group.Therefore,this study mainly explores the ways in which miRNA 156 exerts biological effects and the reasons for the changes in microbes and intestinal immune genes in the co-housing.And on this basis,to explore whether there are differences in the expression of immune related genes in the intestinal tract of mice after intragastric administration of Glycyrrhiza Uralensis’ miRNA156 at intervals of 1.5 h,3 h,6 h and 24 h.Methods:1.Synthesized miRNA 156 according to the results of highthroughput sequencing in the early stage,and dissected the mice by gavage according to the experimental requirements,collected the serum of each group of mice,extracted the total RNA in the serum,and then performed high-throughput sequencing to explore whether there was miRNA 156 in mouse serum after gavage miRNA 156 at different time intervals,and then determined the way of miRNA 156 drug effect.2.Took the fecal of mice in the miRNA 156 gavage group and the control group,extracted the miRNA in the feces and performed highthroughput sequencing to detect whether the miRNA 156 sequence was contained in the feces of the mice in the miRNA 156 gavage group,and explore a series of reasons for changes in the intestines of mice in cohousing group.3.Took the ileum tissue of each group of mice,a part of it was quickfrozen in liquid nitrogen,and Trizol was added to grind to extract the tissue RNA to verify the differential gene,and the other part was placed in a fixative containing 4% paraformaldehyde,and then embedded in paraffin.Do HE staining to observe the pathological conditions of the intestines in each group.4.Collected the ileum of each group of mice,after partial extraction of tissue protein,the expression of IgA protein in the tissue was detected by Western blot,and tissue sections were taken at the same time,the expression of IgA protein in the ileum tissue was qualitatively determined by immunohistochemistry.Results:1.The serum RNA was extracted from the serum of mice in the 3 h,6h group,the 6 h group and control group after gavage of miRNA 156 with Glycyrrhiza Uralensis’ extract and then subjected to high-throughput sequencing.The results showed that miRNA 156 was not detected in the serum samples.This indicated that the way that miRNA 156 works may not be the blood-entry pathway.2.Took the miRNA 156 gavage group and the control group to extract miRNA from the feces of mice for high-throughput sequencing.The results showed that after gavage the mice with miRNA 156,there was no miRNA156,which proved that the changes in the intestinal flora and intestinal immune status in the intestines of co-housing mice in the previous experiment might not directly caused by the unabsorbed miRNA 156 in the intestines of mice in the gavage group.It was related to the altered intestinal flora in mice.3.The ileum tissues of the four time points of miRNA 156,the Glycyrrhiza Uralensis’ extract,and other groups of mice were verified by real-time quantitative PCR for differential genes.The results showed that after gavage the mice with the extract and miRNA,from 1.5 h to 24 h,the expression of multiple genes showed an upward trend,among them,the expression level of each gene at 24 h was consistent with the transcriptome sequencing results.At the same time,it could be seen by comparing the HE staining results of the ileal tissue at three time points after gavage and 24 h after gavage.After gavage of the extract,there were capillary congestion in the mucosal bottom of the mouse ileum,and local edema appears.As the extract entered the body for a longer time,edema gradually became serious;and after gavage of miRNA 156,red blood cells were seen in the capillary lumen of the tissue at each time point,but there was no edema.4.The protein level of IgA in the ileum tissue at different time points was verified.Western blot results showed that compared with the control group,the expression level of the extract after intragastric administration had little difference,but gradually the expression level showed an upward trend.With the extension of time after the mice were gavage with miRNA156,the expression of IgA in the ileum tissue of the mice in the miRNA group gradually increased and reached a maximum at 24 h.This result was consistent with the immunohistochemical results.Conclusions:The presence of miRNA 156 was not detected in the serum of mice in the 3 h and 6 h groups of Glycyrrhiza Uralensis’ and miRNA 156 after gavage.This proved that the way of miRNA 156 works was might not through absorption into the blood,but presumably after gavage,it passed through the stomach and reached the intestine to be absorbed,and then it played a role.At the same time,we found that there was no miRNA 156 in the feces of the miRNA 156 gavage group,which might indicate the changes in the intestinal microbiota and the expression of immune-related genes in the gut of mice in the miRNA 156 co-housing group were not directly caused by the unabsorbed miRNA 156.It was speculated that the changed gut flora of mice in the gavage group was eaten by the co-housing mice and caused related changes.And through the verification of the molecular level and protein level,we found each mouse gavage miRNA156 and the extract at different time intervals have different degrees of influence on the expression of immune genes and IgA protein in the intestine. |
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