Role Of Advanced Glycation End Products In Smooth Muscle Myopathy In Diabetic Colonic Motility Dysfunction

Background and aimsGastrointestinal motility dysfunction is common among diabetic patients, and the incidence is about 30-50%. Smooth muscle dysfunction could impair the gastrointestinal motility. Advanced glycation end products (AGEs) participate diabetic complications, and AGEs exist in diabetic rat colonic tissue. Nε-carboxymethyllysine (CML) is used as AGEs marker to evaluate AGEs levels in vivo. So far, no studies have reported AGEs is involved in diabetic colonic smooth muscle lesions. Smooth muscle cells (SMC) have specific proteins, such as myosin heavy chain, smooth muscle alpha-actin and caldesmon. These proteins are equipments and regulation factors of smooth muscle cell contraction. First, the aim of the present study was to provide a detailed description of the ultrastructural abnormalities in colonic smooth muscle of patients with diabetes, determine the AGEs levels in these patients’colon and the expression levels of SMC specific proteins, and if there are correlation between AGEs levels and SMC landmark proteins. Then, we aim to determine whether these changes of SMC occur as a result of AGEs and AGEs has a relationship with diabetic colon dysmotility.SMC have two phenotype, contractile phenotype and synthesis phenotype, and SMC could transform from one to another. The SMC in gastrointestinal tract are contractile phenotype. AGEs involve in oxidative stress and activation of JNK or p38 MAPK during diabetic complication development. Myocardin is a key regulator of smooth muscle specific gene expression. Finally, we will establish a culture system to maintain SMC in contractile phenotype in vitro and demonstrate the mechanisms of AGEs-induced smooth muscle specific genes expression in contractile phenotype SMC.Methods1. Clinical research of correlation between diabetic colonic smooth muscle pathology advanced glycation end products(1) Colonic muscle tissues were collected from patients with colon surgical in the First Affiliated Hospital of Nanjing Medical University from August 2012 to January 2010. Samples were resected 5cm away from the edge of the colon lesions.(2) CML is an AGEs marker. The levels of CML in colonic muscle tissues samples were tested by western blot.(3) Transmission electron microscopy was used to determine ultrastructural abnormalities in colonic smooth muscle of patients with diabetes.(4) SMC specific proteins in diabetic colon were measured by western blot and real-time PCR.(5) The correlations between the CML levels and mRNA expression of SMC specific proteins were analyzed by person analysis.2. Role of advanced glycation end products in intestinal smooth muscle lesions of diabetic rat with colonic motility dysfunction(1) Diabetic rats were induced by STZ injection, and AG was administrated as an AGEs inhibitor. There were four groups in this study:(1) Control group; (2) Control+AG group; (3) DM group; (4) DM+AG group.(2) AGEs (CML) in serum and colonic muscle of rats were tested by ELISA and western blot respectively.(3) Colonic transit time and contractility of colonic muscle strips were measured.(4) General situation of colon and the pathology of smooth muscle layer in colon were determined. (5) Smooth muscle specific genes expression in colon was tested by western blot and real-time PCR.3. Mechanism of Advanced glycation end products promote smooth muscle specific gene expression(1) Established improved culture system to maintain SMC in contractile phenotype. Then confirmed this system was effective.(2) The effect of AGEs on cell cycle in contractile phenotype SMC was tested by flow cytometry.(3) The dose-dependent effect and time-dependent effect of AGEs on smooth muscle specific genes expression in contractile phenotype SMC were measured by western blot and real-time PCR.(4) Effect of AGEs on ROS formation in contractile phenotype SMC was tested by flow cytometry.(5) Role of INK and p38 MAPK in effect of AGEs on smooth muscle specific genes expression in contractile phenotype SMC were measured by western blot.(6) siRNA interference and luciferase test were used to observe the role of myocardin in AGEs-induced smooth muscle specific genes expression in contractile phenotype SMC.Results1. Clinical research of correlation between diabetic colonic smooth muscle pathology advanced glycation end products(1) Fifteen cases were included in control and diabetic group respectively. There were no significant differences in average age and gender composition between diabetic group and control group. The HbAlc level was higher in diabetic group.(2) CML (AGEs marker) levels increased in colon muscle layer of diabetic patients.(3) Ultrastructural abnormalities in colonic smooth muscle of patients with diabetes are:swollen mitochondrial, increased dense band and dense body, increased caeolae in cell membrane and broken gap junction. The distance between SMC did not widen, and there were no redundant collagen fibers in intercellular space.(4) Expression levels of SMC specific proteins (SM MHC and SM alpha-actin) were increased in diabetic colon tissues.(5) There were positive correlation between CML levels and SMC specific genes mRNA expression levels.2. Role of advanced glycation end products in intestinal smooth muscle lesions of diabetic rat with colonic motility dysfunction(1) Diabetes rat model were successfully established, and AG did not affect blood glucose level.(2) AGEs (CML) level in serum and colonic tissue of diabetic rats was increased significantly, but there was no difference between AG+DM and control group.(3) Diabetic rats showed a prolonged colonic transit time and weak contractility of smooth muscle strips. But inhibition of AGEs prevented diabetic rats’colon motility function. There was no difference in colonic transit time and contractility of smooth muscle between AG+DM and control group.(4) Pathologies of diabetic rats were longer colon length, thickened muscle layer and larger smooth muscle cells. AGEs inhibitor (AG) reversed these changes of diabetic colon.(5) Smooth muscle marker genes expression were increased in diabetic rats’colon, but these genes expression did not increased in AG administrated diabetic rats. There was no difference in smooth muscle marker genes expression level between control group and control+AG group.3. Mechanism of Advanced glycation end products promote smooth muscle specific gene expression(1) The cultural system with laminin coated dish, DMEM supplied with low concentration fetal bovine serum and insulin could maintain SMC in contractile phenotype for 3 days at least.(2) AGEs had no effect on cell cycle in contractile phenotype SMC.(3) AGEs induced smooth muscle specific gene expression as a dose-dependent and time-dependent manner.(4) AGEs did not promote ROS formation in contractile phenotype SMC(5) AGEs activated phosphorylation of JNK and p38 MAPK, but only p38 MAPK inhibitor (SB239063) could block the effect of AGEs in SMC, suggested that AGEs promote smooth muscle specific genes expression through p38 MAPK, but not JNK.(6) ① AGEs induced increased expression of myocardin in contractile phenotype SMC. Myocardin siRNA could inhibit the effect of AGEs-induced smooth muscle specific genes expression, which suggested that myocardin participate in this effect.② p38 MAPK inhibitor could block two effects:AGEs-induced myocardin expression in SMC; AGEs-induced myocardin promoter activity. Therefore, AGEs-induced myocaredin expression through p38 MAPK signal.Conclusions1. Ultrastructural changes existed in colonic SMC of diabetic patients, and these changes could be the basis of diabetic colonic motility disorders. There was an increased AGEs levels and expression of smooth muscle cell specific protein in diabetic patients’ colonic muscle tissue. There was a positive correlation between AGEs and Expression of smooth muscle cell specific protein, which suggested that AGEs may involve in diabetic colonic smooth muscle lesions.2. Diabetic rat showed a declined colonic transit function and muscle contractility, increased size of smooth muscle cells and expression of smooth muscle marker genes. AGEs were an upstream factor of diabetic colonic motility dysfunction and impaired muscle contractility and increased expression of smooth muscle marker genes in diabetic colon.3. AGEs did not promote contractile phenotype SMC proliferation, and AGEs did not induce ROS formation either. AGEs promote smooth muscle specific genes expression in contractile phenotype SMC. The mechanism of this effect was p38 MAPK induced increased promoter activity and expression of myocardin gene, which in turn up-regulated smooth muscle specific genes expression.