The Role Of MiR-152and MiR-200S In IL-6-induced Hepatic Insulin Resistance

Part1miR-152and miR-200s contribute to IL-6-induced insulin resistance in mouse hepatocytesBackground&Aims:Insulin resistance, defined as a diminished ability of cells, such as adipocyte, skeletal muscle cells and hepatocytes, to respond to the action of insulin, plays a central role in the development of type2diabetes. MicroRNAs are small, noncoding20-to24-nt RNAs that negatively regulate their target gene expression at the posttranscriptional level by binding to specific, partially complementary regions in the3'-UTR of target mRNA. MicroRNAs express in all kinds of tissues. They can regulate the development and function of tissues. Recent studies have shown the involvement of microRNAs in the pathogenesis of type2diabetes and its complications. MicroRNAs also control insulin synthesis and secretion, insulin signal pathway and glucose and lipid metabolism. However, the role of microRNAs in the development of hepatic insulin resistance has not been reported. Previous study showed that miR-152and miR-200s are involved in the development of cancer. The role of miR-152and miR-200s in regulating insulin signal pathway has not been elucidated. In the present study, the IL-6-induced insulin resistance models in vivo and in vitro were established. The levels of miR-152and miR-200s were analyzed in order to determine the role of miR-152and miR-200s in IL-6-induced insulin resistance in hepatocytes. Our study aims to identify the mechanisms of miR-152and miR-200s in regulating PI3K/AKT signal pathway.Methods:1.12-week-old male db/db mice were used as insulin resistance mouse models. To profile the expression of miRNAs between two group mice, the miRNAs of liver samples from5db/db mice and5control mice were analyzed by the miRNA Array system. The results of miRNA Array were estimated by real-time PCR.2. NCTC1469cells and mouse primary hepatocytes were treated by10ng/ml IL-6for24h to induce insulin resistance. And the activation of PI3K/AKT signaling pathway and the levels of miR-152and miR-200s were analyzed. Implanting the IL-6osmotic pumps to the back subcuticle of mice was used to establish IL-6-induced insulin resistance models. And the activation of PI3K/AKT signal pathway and the levels of miR-152and miR-200s in the livers of mice were analyzed.3. miR-152and miR-200s mimics and inhibitors were transfected into NCTC1469cells. The activation of PI3K/AKT signal pathway and the level of hepatic glycogen were analyzed in order to determine the role of miR-152and miR-200s in the insulin signal pathway.4. The target genes of miR-200s were predicted by bio-informational software. The levels of target protein were analyzed by Western blot.5. To further assess the role of FOG2and PTEN in IL-6-induced hepatic insulin resistance, siRNAs targeting FOG2and PTEN mRNA were transfected into NCTC1469cells. The activation of PI3K/AKT signal pathway and the level of hepatic glycogen were analyzed.Results:1. The body weight and the levels of serum glucose and FFA were significantly increased in db/db mice. The concentration of IL-6in serum was also elevated in db/db mice. The activation of PI3K/AKT signal pathway and glycogen content were reduced in the livers of db/db mice. The results of miRNA array showed31increased miRNAs and88decreased miRNAs. The levels of miR-152and miR-200s were decreased in the livers of db/db mice.2. Because db/db mice are complex and accompanied by other factors such as elevated levels of serum FFA and glucose, it is difficult to determine the contribution of IL-6to down-regulation of miR-152and miR-200s. Therefore, we extended these observations from db/db mice to NCTC1469cells and mouse primary hepatocytes. The results showed that IL-6treatment decreased the level of miR-152and miR-200s, accompanied by inhibiting the activation of PI3K signal pathway and the level of glycogen. Moreover, down-regulation of miR-152and miR-200s by IL-6was assessed in the livers of C57BL/6J mice injected with IL-6. The activation of PI3K/AKT and the level of glycogen were decreased in the livers.3. There were several target sits for miR-200s in the3'-UTR of FOG2and PTEN. The results showed that miR-200s could regulate the protein levels of FOG2and PTEN.4. In order to determine the contribution of FOG2and PTEN to insulin signal pathway and glycogenesis, siRNAs targeting to FOG2and PTEN were transfected into NCTC1469cells. The results showed that inhibition of FOG2and PTEN could reverse the effects of IL-6on PI3K/AKT signal pathway and glycogenesis.5. In order to determine the role of miR-152in regulating miR-200s, the miR-152inhibitor was transfected into NCTC1469cells. The results showed that inhibition of miR-152could decrease the level of miR-200s.Conclusions:1. The levels of miR-152and miR-200s, the activation of PI3K/AKT signal pathway and the level of glycogen were decreased by IL-6in mouse hepatocytes.2. Overexpression of miR-152and miR-200s could reverse the effects of IL-6on PI3K/AKT signal pathway and glycogenesis in hepatocytes.3. miR-200s could regulate the protein level of FOG2and PTEN.4. Inhibition of miR-152decreased the level of miR-200s. Part2Panax notoginseng saponins suppress development of atherosclerotic plaques in apolipoprotein-E-deficient miceBackground&Aims:Atherosclerosis is a chronic inflammatory disease. It has been suggested that endothelial dysfunction and inflammation is an early marker of atherosclerosis. Panax notogingseng saponins (PNS) are effective free radical-scavenger, which have an antioxidant property and are able to suppress adhesion molecules and chemokines. Receptor for advanced glycation end products (RAGE) is involved in the development of early atherosclerosis plaques. RAGE contributes to chronic inflammation and endothelial dysfunction. Recent studies showed that PNS can inhibit the progress of atherosclerosis. However, the precise mechanisms of by which PNS reduce expression of adhesion molecules in atherosclerotic plaques have not been elucidated. In present study, we examined the role of PNS in the sizes and contents of atherosclerosis plaques. To explore the effects of PNS on the progression of atherosclerosis, the activation of NF-kB, the expression of RAGE and the activation of JNK, ERK and p38MAPK were analyzed in atherosclerotic plaques of apoE-deficient mice administered with PNS.Methods:1.10-week-old apoE-/-mice were fed with atherogenic diet for8weeks to establish atherosclerosis models. PNS were dissolved in distilled water and administered daily for4weeks. Blood samples were collected from the mice for measurement of glucose, lipids. In order to determine the antioxidant property of PNS, the levels of malonidialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) in serum and ROS product in plaques were analyzed.2. The sizes of branchiocephalic arteries (BCA) and aortic root were analyzed by Movat staining. The sections from the BCA were used for immunohistochemistry staining to identify the macrophages, collagen content and smooth muscle cells (SMC).3. The expression of CD68, Galectin-3, RAGE, p38MAPK, JNK, ERK1/2, NF-kB, MCP-1, ICAM-1and VCAM-1in atherosclerotic plaques were analyzed by Western blot. Results:1. PNS did not affect the levels of serum glucose and serum lipid in apoE-/mice. The apoE-/-mice administered with PNS for4weeks displayed increased activity of serum SOD and GSH, and decreased MDA levels, accompanied by decreased ROS product in plaques of BCA. Our results suggested that PNS suppressed oxidative stress under hyperlipidemic conditions.2. The results of Movat staining showed that PNS significantly decreased the lesion area of BCA in apoE-/-mice, but not the aortic root. The results of immunohistochemistry staining indicated a decreased of macrophage amounts in atherosclerosis lesions, but not the content of collagen and the amount of SMC. It was suggested that PNS inhibited the progression of atherosclerosis.3. The descending arteries were dissected and used to analyze the protein levels by Western blot. The results showed that PNS inhibited the activation of NF-kB, JNK, ERK1/2and p38MAPK and RAGE expression as well as down-regulated expression of VCAM-1, ICAM-1and MCP-1. Conclusions:1. PNS significantly decrease the levels of serum MDA and increased the activity of serum SOD and GSH, which supposed that PNS have an antioxidant property.2. PNS inhibited the activation of NF-kB.3. PNS down-regulated the expression of RAGE.4. PNS suppressed the expression of RAGE, MAPK signal pathways and NF-kB activation, leading to reduced expression of pro-inflammatory factors including VCAM-1, ICAM-1and MCP-1in the lesions of apoE-/-mice.