The Role Of Ndrg2 And IL-6 In Pathogenesis Of Diabetes Related Microvascular Disease

Objective:This study aimed to explore the association and related mechanisms between Ndrg2,IL-6 and diabetic microvascular complications,and to further clarify the function of Ndrg2 in type 2 diabetic mellitus,which can provide evidence for the prevention and control of diabetic microvascular complications.Methods:(1)8 ~ 10 weeks old male specific pathogen-free(SPF)C57BL/6 mice were used in our study.After 7 days of adaptive feeding,these mice were randomly divided into normal diet group and high-fat diet group according to the method of random number table.After12 weeks of high-fat diet feeding,the blood of the mice in high-fat diet group was taken from the tail and blood glucose was measured.The diabetic mice were successfully induced when the blood glucose met the following standards: fasting glucose level was higher than 7mmol/L and 2-hour postprandial glucose was higher than 11.1mmol/L.After that,the weight,blood glucose,blood lipid and HOMA-IR were detected or calculated for in both groups.After euthanasia,Pancreas tissues of mice were collected and fixed in 4%paraformaldehyde(pH 7.4).Paraffin embedded tissue was sectioned into 5-?m thick slices.The expression of Ndrg2 in the pancreas was then dectected using quantitative PCR technique and immunofluorescence method.(2)Clinical research: 58 patients free of any diabetic microvascular complications(NDR group),49 cases of non-proliferative diabetic retinopathy(NPDR group),42 patients of non-proliferative diabetic retinopathy(PDR group),and 40 cases of normal control were recruited in our study.Their levels of IL-6,TNF-? were measured by ELISA method using the blood samples obtained from the subjects.The correlation between these inflammatory cytokines and HOMA-IR,blood lipid series and duration of the disease was analyzed.Animal studies: Diabetic mice model was constructed.The serum IL-6 levels in diabetic mice and control mice were detected by ELISA method.The ultrastructuralchanges of the heart,kidney,and retina,brain tissue of both groups were observed by transmission electron microscopy.Immunohistochemical method was used to detect the expression of Ndrg2 in mouse islet.The role of IL-6 in diabetic microvascular complications and its correlation with Ndrg2 expression in islet were then investigated.(3)The expression of Ndrg2,IL-6,STAT3,p-STAT3 proteins of pancreatic tissues were detected using Western Blot method in both groups and the expression of STAT3 and p-STAT3 in mouse islet were measured using immunohistochemical method.The roles of Ndrg2/IL-6/STAT3 signaling passway in the pathogenesis and development of diabetes were also explored.Results:(1)Significant differences of weight,blood glucose,blood lipid,HOMA-IR were found between the high-fat mice and normal mice.The expression of Ndrg2 was significantly increased in mice islets,and mostly expressed in pancreatic ? cells.(2)Clinical results demonstrated that serum IL-6,TNF-? level were significantly increased in patients with diabetic retinopathy,and it was related with the severity of microvascular complications.Serum IL-6,TNF-? level were positively correlated with HOMA-IR,triglyceride and low-density lipoprotein cholesterol.Serum IL-6 level was significantly higher in diabetic mice compared to normal-diet group,and it was also positively correlated with HOMA-IR and triglyceride.The brain tissue,kidney,retina and myocardium exhibited obvious microstructure change in diabetic mice compared with control.The level of Ndrg2 protein,which mainly located in the islet of diabetic mice,was significantly higher than control mice,and it was positively correlated with IL-6.(3)Protein expression of Ndrg2,IL-6,STAT3,p-STAT3 was significantly higher in diabetic mouse islets than normal-diet group.Immunohistochemical test showed a significant increase of p-STAT3 expression in diabetic mice when compared with normal-diet group,however no difference of STAT3 expression was found in both groups.Ndrg2 exprssion was positively correlated with p-STAT3 expression in diabetic mouse islets,and serum IL-6 of diabetic mice was positively correlated with p-STAT3 expression in islets.Conclusions:(1)Diabetic mice showed significant insulin resistance.Ndrg2 expression was significantly increased in diabetic mouse islets,and mostly expressed in ? cells.(2)The level of inflammatory factors can be one of the test factors for diabetic retinopathy.The inflammatory level is correlated with insulin resistance level,which may lead to the occurrence and development of diabetic microvascular disease.Ndrg2 is also associated with IL-6 and may be involved in the pathogenesis of diabetes and its further complications.(3)Ndrg2 may aggravate the occurrence and development of diabetes through activating IL-6/STAT3 signaling pathway.