The Role And Mechanism Of Exosome MiR-146a-5p In The Development Of NASH

Research objectives:1.The GEO database was used to analyze the expression level of miR-146a-5p in serum exosomes in the non-alcoholic steatohepatitis(NASH).2.Construct a NASH mouse model by feeding a Methionine-and choline deficient(MCD)diet and explore the expression level of miR-146a-5p in plasma exosomes of NASH mice.3.The exosomes with high expression of miR-146a-5p were prepared by ultracentrifugation,and their effects on M1 polarization of macrophages were investigated at the cellular level,which provided a theoretical basis for further in vivo experimental treatment of NASH.Methods:1.Retrieve data sets related to the project through GEO database;use "Analyze with GEO2R" to analyze the series data online;after obtaining the specific values of all samples of the gene,analyze the data through "Graph Pad prism 9".2.The 7-week-old mice were randomly divided into 2 groups(6 mice/group),which were fed normal diet and MCD diet for 5 weeks respectively,and their body weight was recorded weekly.After the experimental period,the content of triglycerides(TG)total cholesterol(TC)and the expression of CD80 in liver tissues of mice in both groups were detected;the content of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)and IL-6 in fasting plasma were detected.The mice were dissected,the livers were taken out,the liver morphology of the two groups of mice was observed by taking pictures and weighed,and a little liver tissue was taken for pathological sections.After mouse serum exosomes were extracted,the expression levels of miR-146a-5p in serum exosomes of the two groups were detected.Finally,primary cell extraction was performed to further clarify the expression of miR-146a-5p in liver cells and liver macrophages of NASH mice.3.50 nmol/L miR-146a-5p mimic or negative control was transfected into induced differentiation macrophages,and cells were collected 48 h after transfection.Western blot was used to detect the expression of M1 macrophage marker protein CD86 in macrophages.Total RNA was isolated from macrophages and the m RNA was reversely transcribed into c DNA.The expression of CD86 was quantified using GAPDH as internal reference.Through immunofluorescence labeling of CD86,the role of miR-146a-5p in the polarization of M1 was further clarified.4.Hep G2 cells were transfected with 50 nmol/L Mir-146A-5p mimic or negative control,respectively.After transfection 48 h,cell culture medium supernatant was collected,and exosomes with high expression of miR-146a-5p and control exosomes were prepared by supercentrifugation method The morphology of exosomes was observed by transmission electron microscope.Western blot was used to detect the expression of exosome labeled protein CD63.Total RNA was isolated from exosomes and miRNA was reverse transcribed into c DNA.The expression of miR-146a-5p was quantified using U6 as an internal reference.5.Exosomes with high expression of miR-146a-5p or control group were added into macrophages for incubation for 48 hours;Using U6 as internal reference,the expression of miR-146a-5p in macrophages was quantified.Using 18 S as internal reference,the expression of MCP-1 TNF-α IL-6 in macrophages was quantified.The expression of CD86 in macrophages was detected by Western blot.Target genes were predicted by bioinformatics and related mechanisms were studiedResults:1.The expression of miR-146a-5p expression was down-regulated in serum exosomes of NASH patients and mice.2.The expression of miR-146a-5p in serum exosomes was negatively correlated with serum IL-6 content;the expression of miR-146a-5p in primary liver cells and liver macrophages of mice in NASH group was significantly decreased.3.Overexpression of miR-146a-5p in macrophages reduced the expression of M1-macrophage marker protein CD86 at both protein and m RNA levels;In addition,LPS-induced release of M1-macrophage proinflammatory cytokines TNF-α IL-1β and IL-6 was reversed to a certain extent4.After 48 h co-incubation of exosomes with high expression of miR-146a-5p with macrophages,the expression of miR-146a-5p in macrophages was significantly increased;Exosomes with high expression of miR-146a-5p can reduce the expression of CD86 and the release of MCP-1,TNF-α,IL-6 of M1 macrophage pro-inflammatory factor.Exosomes with high expression of miR-146a-5p can inhibit M1 polarization of macrophages by targeting CD80Conclusions:The expression of miR-146a-5p in plasma exosomes of NASH patients and mice was significantly down-regulated;the expression of miR-146a-5p in primary hepatocytes and hepatic macrophages of mice in NASH group was significantly decreased.Cellular experimental studies have shown that overexpression of miR-146a-5p in macrophages can inhibit M1 polarization;preparation of exosomes with high expression of miR-146a-5p and co-incubation of macrophages in vitro can target CD80 to inhibit M1 polarization of macrophages and reduce the release of pro-inflammatory cytokines MCP-1,TNF-α,and IL-6.