| Objective: The prevalence of diabetes which is characterized by increased plasma glucose continues to rise worldwide, causing serious health problems and imposing a substantial economic burden on societies. Type 2 diabetes mellitus(T2DM) is the most common type of diabetes and accounts for more than 90% of all diabetes. Diabetic macrovascular complication is the leading cause of mortality in diabetic patients, about 80% of patients with T2 DM died of macrovascular disease, such as storke, myocardial infarction, etc. The remarkable pathological change of diabetic macroangiopathy is atherosclerosis(AS), and T2 DM can accelerate AS. Unfortunately, the exact mechanism is not yet fully understood. Recent insights support that AS is related to the excessive activation of mitogen-activated protein kinases(MAPK). p38 MAPK pathway belongs to the MAPK family, a stress activated serine/threonine protein kinase, which is the downstream target of proinflammatory cytokines and oxidative stress, closely associated with endothelial cell injury. Presumably, phosphorylated p38 MAPK maybe involved in the pathogenesis of T2 DM macroangiopathy. Liraglutide is a glucagon-like peptide-1(GLP-1) analog used for the treatment of Type 2 diabetes. Similar to the actions of endogenous GLP-1, liraglutide potentiates the post-prandial release of insulin, inhibits glucagon release and increases satiety. Recently, it was reported that treatment with GLP-1 analog may also decrease the risk of cardiovascular disease in diabetic patients. The mechanism responsible for this effect has to be determined. In the present study, we developed a rat model of type 2 diabetes to explore the relationship between phosphorylated p38 MAPK(p-p38 MAPK) and diabetic macrovascular disease and the intervention effect of liraglutide.Methods: 24 healthy male Wistar rats(4 weeks old) were randomly divided into normal control group(NC, n=6) and experiment group(EXP, n=18). NC group fed with standard rodent chow diet, while the EXP group fed with high fat and high sugar diet, which composed of(by mg) 20% sugar, 10% lard, 2.5% cholesterol, 1% cholic acid and 66.5% standard chow diet. 4 weeks later, the EXP group was intraperitoneally injected with 1% STZ(30 mg/kg), NC group was simultaneously injected with sodium citrate buffer. 2 weeks later, blood glucose was measured, and those with blood glucose≥7.8 mmol/L were considered DM rats(n=14). DM rats were randomly divided into two subgroups: diabetic control group(DM, n=7) and liraglutide-treated diabetic group(LIR, n=7), LIR group was treated with liraglutide(100 μg·kg-1, ih, twice daily) for 8 weeks. Body weight was measured weekly.At the end of the experiment, the rats were executed to measure body weight, perform intraperitoneal glucose tolerance test(IPGTT), then euthanized and collected blood and thoracic aorta, the levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein-cholesterol(LDL-C), high-density lipoprotein-cholesterol(HDL-c), intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1) and nuclear factor(NF)-kappa B were detected. HE staining of thoracic aorta, the pathological changes of thoracic aorta were observed under optical microscope. Immunohistochemical method was performed to measure the expression of the phosphorylated p38 MAPK, NF-κB, and monocyte chemoattractant protein-1(MCP-1) in the thoracic aorta. All statistical analyses were conducted with the use of SPSS software, version 13.0, P<0.05 was considered statistically significant.All the animal studies were conducted under a protocol approved by the Institutional Research Animal Care Committee, and the Institute Review Board of Hebei medical university granted ethical permission to this study. Result: 1 Biochemical characteristic of rats 1.1 Indexes at the end of 6 weekThe level of fasting plasma glucose(FBG) was significantly higher in EXP group than NC group(15.90±4.85 mmol/L V.S. 6.50±0.52 mmol/L,P <0.05)(Table1, Fig.1). 1.2 Indexes at the end of 14 weekThe level of FBG was significantly higher in DM and LIR group than NC group(12.99±2.07 mmol/L, 8.87±1.82 mmol/L V.S. 6.33±0.69 mmol/L, P < 0.05). Compared with DM group, the level of FBG was significantly decreased in LIR group(P < 0.05). AUCg was significantly higher in DM and LIR group than that of NC group [(34.34±7.63) mmol·L-1·h,(27.67±4.93) mmol·L-1·h V.S.(18.72±3.27) mmol·L-1·h, P < 0.05], and which was lower in LIR group than DM group(P < 0.05)(Table2, Fig.2,3). 1.3 The level of fasting insulin(FINS) was significantly lower in DM and LIR group than NC group [(6.49±1.99) m IU/L,(11.13 ± 1.92)m IU/L V.S.(18.02±2.52) m IU/L, P < 0.05], and which was higher in LIR group than DM group(P < 0.05). AUCINS was significantly decreased in DM group than that of NC and LIR group [(27.40±10.32)m IU·L-1·h V.S.(49.22±4.44) m IU·L-1·h,(40.42 ± 6.71) m IU·L-1·h, P<0.05], whereas there was no statistically difference between NC and LIR group in AUCINS(P > 0.05). There was no statistically difference among 3 groups in the HOMA-IR(P > 0.05). HOMA-β was significantly lower in DM and LIR group than NC group(14.04±3.87, 45.95 ± 20.65 V.S. 132.44±30.30, P < 0.05), and which was higher in LIR group than DM group(P < 0.05)(Table2, Fig.4-Fig.6). 1.4 The levels of serum TG, TC, LDL-C, ICAM-1, VCAM-1 and NF-κB were significantly higher in DM group than those of LIR and NC group(P < 0.05), and which was significantly higher in LIR group than NC group(P < 0.05). Compared with NC group, the level of serum HDL-C was decreased in DM and LIR group(P < 0.05), whereas there was no statistically difference between DM and LIR group(Table2, Fig.7-Fig.10).2 Pathological changes of thoracic aorta:NC group: Under microscopy, HE staining showed there were complete visible vascular endothelial cells, neat rows and intima smooth. The structure of inner membrane, medical membrane, outer membrane and smooth muscle cells was clear and intact.DM group:Structural destructions were observed in rat aorta tissue by HE staining, which characterized by endothelia cells swollen and degeneration and intima thickness. Smooth muscle disorders and collagen fiber hyperplasia in tunica media were also found.LIR group: All those pathological alterations in rat aorta tissue were ameliorated compared with DM group(Fig.11). 3 Immunohistochemical analysis:The expression of phosphorylation of p38 MAPK, NF-κB and MCP-1 was significantly increased in DM and LIR group than NC group(P < 0.05). The expression of phosphorylation of p38 MAPK, NF-κB and MCP-1 were significantly decreased compared with DM group(P < 0.05). There was a positive correlation between the expression of phosphorylation of p38 MAPK and NF-κB, MCP-1(r=0.458, 0.318, P<0.05)(Table4, Fig.12-14).Conclusions:1 Phosphorylated p38 MAPK pathway may involved in the development of diabetic macrovascular complication in the experiment of rats.2 Liraglutide may ameliorate diabetic macrovascular disease through decreasing p38 MAPK pathway, and inhibiting vascular inflammatory response. |
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