The Research Of Mechanism About MiR 17 Improves Insulin Sensitivity In Heat Pathogenesis Diabetes

Objectives:Symptom-complex of excessive eating in traditional Chinese medicine mainly refers to modern diabetes.Classical Chinese medicine theory believes that the athogenesis is yin deficiency and heat.However,in today’s actual situation,typical Yin deficiency patients are rare,so the internal heat-induced disease-elimination machine is benefited.Pay attention to^[1].Recent research on traditional Chinese medicine theory pointed out that the entire process of the type 2 diabetes mellitus caused by the fibrosis is the root cause of internal heat syndromes^[2].MicroRNAs play a role in type 2 diabetes and many other pathological processes.One of the triggers of type 2 diabetes and insulin resistance is chronic inflammation of adipose tissue.MiR-17 plays an anti-inflammatory role in many biochemical reactions.We hypothesized that miR-17 inhibits the biological function of inflammatory macrophages and has an ameliorating effect on insulin resistance in type 2 diabetes,which is confirmed by this experiment.In addition,software predicts that ASK1 is the anti-inflammatory target of miR-17.This study further clarified this viewpoint and observed that ASK1 and miR-17 are involved in macrophage migration,inflammatory cytokine secretion,and insulin-stimulated glucose uptake.Methods:Part Ⅰ MiR-17 Regulates Macrophage Inflammation and Insulin Resistance1.The WT and ob/ob mice were divided into two groups of 6 mice.The macrophages in the peritoneal tissues of the mice were isolated and cultured.The qPCR method was used to detect the miR-17 gene expression in macrophages in both groups.2.Macrophages from mouse WT and standard RAW 264 macrophages were used as study subjects.Two macrophage samples were treated with LPS and HG respectively and then grouped into NG,HG,control,and LPS（n=6）.QPCR technique was used to detect the expression of miR-17 in macrophages under different intervention conditions.3.Macrophages from WT and RAW 264 macrophages were used as study subjects.Two macrophages were transfected with pre-miRNAs of miR-17（miR-17）and NC-pre-miRNAs（miR-NC）,respectively.Hours（n=6）.QPCR then detected the amount of miR-17 expression in the four samples.4.The sample from the previous step was treated with LPS,followed by transwell cell migration assay and ELISA assay to evaluate macrophage migration and inflammatory cytokines under different miR-17 expression levels.5.The 3T3-L1 adipocytes were divided into CM,CM-LPS,CM-LPS+miR-NC and CM-LPS+miR-17.The medium was switched to KRBH buffer containing insulin,tritiated glucose was added and incubated,and the radioactivity of tritiated glucose was detected.To evaluate the insulin-stimulated glucose uptake of adipocytes under different miR-17 overexpression conditions.Part Ⅱ The Regulation of ASK1 Expression by MiR-17 and Its Effects on Inflammation and Glucose Metabolism1.Use the TargetScan Database to predict that the 3′UTR of ASK1 is the target of miR-17.This gene fragment is GCACUUUA,and a mutant gene fragment corresponding to its complementary sequence is created.The 3′UTR fragment containing wild-type or mutant ASK1,miR-17 or its associated miR-NC and Renilla luciferase reporter vector were co-transfected into RAW264.7 macrophages and the luciferase activity was measured.2.Detect the gene expression level of ASK1 in peritoneal macrophages of diabetic mice and non-diabetic mice.The expression level of ASK1 gene and protein in macrophages treated with LPS or HG was detected by qPCR and western blot.The effect of miR-17 overexpression and low expression on ASK1 was observed.Transfection of pre-miRNAs of miR-17（miR-17）,NC-pre-miRNAs（miR-NC）,miR-17 inhibitor（anti-miR-17）,and miR-17 inhibitor NC（anti-NC）To RAW264 macrophages,qPCR was used to detect miR-17 expression levels,Western blot was used to detect ASK1 protein expression,andβ-actin was used as an internal reference.3.The expression of ASK1 was knocked down by infection of lentivirus containing shRNA.The gene fragment or control RNA was transfected into RAW264macrophages for 48 hours and became ASK1 sh1,ASK1 sh2,and scramble groups,respectively.The protein expression level of phagocytic ASK1 was detected by using cell migration assay and macrophages secreted inflammatory cytokines by ELISA.The 3T3-L1 adipocytes were then treated with a variety of conditioned mediums using different types of culture media（CM-LPS-scramble,CM-LPS-sh1,and CM-LPS-sh2）in the previous step to detect insulin stimulation.Glucose intake.4.ASK1 and/or miR-17 were co-transfected into Raw264.7 macrophages,which were pre-miRNAs of miR-17（miR-17）and NC-pre-miRNAs（miR-NC）;pre-MiRNAs of miR-17+ASK1（miR-17+ASK1）and pre-miRNAs of miR-17+vector control（miR-17+vector）were detected after macrophage migration and secretion of inflammatory cytokines.The corresponding four conditioned media were prepared to treat 3T3-L1 adipocytes and then incubated in KRBH buffer containing insulin,after which insulin-stimulated glucose uptake was measured.Results:Part Ⅰ MiR-17 Regulates Macrophage Inflammation and Insulin Resistance1.The expression of miR-17 in macrophages of diabetic mice was significantly lower than that of non-diabetic macrophages.The expression level of miR-17 is significantly down-regulated after macrophage cells are incubated with lipopolysaccharide or high glucose for 24 hours.MiR-17 plays an anti-inflammatory role in the inflammatory response induced by diabetic macrophages.2.miR-17 inhibits macrophage migration.We observed that the addition of LPS promoted the migration of macrophages and that miR-17 overexpressed the complete termination of migratory movement.The results indicate that miR-17 can inhibit LPS-mediated macrophage migration.3.miR-17 inhibits the secretion of inflammatory factors by macrophages.MiR-17 can inhibit the secretion of IL-6,IL-1βand TNF-αby LPS-mediated macrophages.Part Ⅱ The Regulation of ASK1 Expression by MiR-17 and Its Effect on Inflammation and Glucose Metabolism1.ASK1 is a biological target of miR-17.MiR-17 significantly reduced the luciferase activity associated with the 3′UTR of wild-type ASK1,but had no significant effect on the mutant luciferase activity.miR-17 directly restricts the activity of the ASK1 3′UTR mutation site in this experiment.2.The mRNA levels of ASK1 in macrophages of diabetic mice were significantly upregulated compared with non-diabetic mice.Compared with the control group,LPS or HG treatment increased the ASK1 protein level,which was contrary to the expression of miR-17 and ASK1.3.Up-regulation of miR-17 decreased ASK1 protein levels;on the contrary,down-regulation of miR-17 increased ASK1 expression.MiR-17 inhibits its expression by targeting the 3’UTR of ASK1.4.Knockdown of ASK1 significantly inhibited the migration of macrophages,reduced the secretion of IL-6,IL-1β,and TNF-αfrom macrophages and increased insulin-stimulated glucose uptake by adipocytes.The role of miR-17 and ASK1 in diabetic inflammation and insulin resistance is the opposite.5.Overexpression of ASK1 does not affect the expression level of miR-17.Overexpression of ASK1 can reverse the anti-inflammatory and anti-insulin effects of miR-17.The inhibitory effect of miR-17 on macrophage migration and secretion of inflammatory cytokines is antagonized by ASK1 overexpression.Conclusion:MiR-17 can improve inflammation and its associated insulin resistance by inhibiting ASK1 expression in macrophages.However,when ASK1 is overexpressed,the anti-inflammatory and anti-insulin resistance of miR-17 will be inhibited.MiR-17can improve type 2 diabetes by regulating heat pathogenesis indicators.