| Objective: Type2diabetes mellitus (T2DM) is a polygenic inheritancemetabolic diseases, about80%of T2DM patients died of macrovascularcomplications, such as myocardial infarction and stroke, which is the leadingcause of morbidly and mortality in patients with DM. The remarkablepathological change of diabetic macroangiopathy is atherosclerosis, DM canlead to premature and accelerate atherosclerosis. Several mechanisms havebeen shown to be involved in the development of Diabetic macroangiopathy,however, the exact mechanism is not yet fully understood. Recent insightssupport that the activation of p38mitogen activated protein kinases (p38MAPK) pathway may constitute a common downstream mechanism fordiabetic macroangiopathy. p38MAPK pathway belong to the MAPK family, astress activated serine/threonine protein kinase, which is the downstreamtarget of proinflammatory cytokines and oxidative stress, closely associatedwith endothelial cell injury. Metformin can effectively reduce blood glucoseand plasma insulin level, increase insulin sensitivity, anti-inflammatory,antioxidant stress, and improve endothelial function. In the present study, wedeveloped a rat model of type2diabetes to explore the relationship betweenphosphorylated p38MAPK(p-p38MAPK) and diabetic macroangiopathyand the intervention effect of metformin.Methods: A total of25healthy male Wistar rats (4weeks old) were housedunder specific pathogen-free conditions at the Animal Facility of the3rdhospital of Hebei medical University. They were maintained on freelyprovided standard rodent chow and tap water, and housed in a temperature(22±2℃) and humidity controlled (55±10%) room under a12-hourdark/light cycle. After one week of adaptive feed, All rats were randomlydivided into normal control group (NC, n=6) and experiment group (EX, n=19), they were fed with standard rodent chow diet or high-fat, high-sugardiet, which composed of (by mg)20%sugar,10%lard,2.5%cholesterol,1%cholic and66.5%standard chow diet, respectively. Each rat remained on theassigned diet throughout the whole experiment. Four weeks later, the EXgroup was intraperitoneally injected with STZ (30mg/kg), the NC group wassimultaneously injected with sodium citrate buffer. Two weeks later, tail veinblood glucose was measured, and those with blood glucose≥7.8mmol/Lwere considered DM rats. DM rats were randomly divided into two subgroups:diabetic control group (DM-C, n=7) and metformin-treated diabetic group(DM-T, n=8), DM-T group was treated with metformin (200mg·kg-1·d-1)through intragastric administration for8weeks. Body weight was measuredweekly.At the end of14week, intraperitoneal injections of glucose tolerance test(IPGTT) was performed to calculate the area under the curve of glucose(AUCg) and insulin (AUCi), HOMA-IR[HOMA-IR=FPG (mmol/L)×FINS (mIU/L)/22.5]，and HOMA-β[HOMA-β=20×FINS/(FPG–3.5)].Then the rats were executed to collect blood and thoracic aorta, totalcholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol (HDL), the level ofintercellular adhesion molecule (ICAM), vascular cell adhesion molecule(VCAM) and nuclear transcription factor (NF)-kappa B were detected. Thethoracic aorta was observed by HE staining. The protein expression ofphosphorylated p38MAPK, NF-κB, and MCP-1in the thoracic aorta wasmeasure by immunohistochemistry. The experimental data was presented asx±s, all statistical analyses were conducted with the use of SPSS software,version16.0, P <0.05was considered statistically significant.All the animal studies were conducted under a protocol approved by theInstitutional Research Animal Care Committee, and the Institute ReviewBoard of Hebei medical university granted ethical permission to this study.Results:1Experimental data at the end of6week: The fasting blood glucose (FBG) in experiment group was significantly higher than control group(16.56±5.20mmol/L V.S.6.50±0.52mmol/L，P=0.003)(Table1, Fig.1).2Biochemical indexes at the end of14-week:2.1The level of FBG in DM-C and DM-T group was significantly higherthan that of NC group (12.99±2.07mmol/L,9.03±1.49mmol/L V.S.6.33±0.69mmol/L, P <0.05). Compared with DM-C group, the level of FBGwas significantly decreased in DM-T group (P <0.05). The AUCg in DM-Cand DM-T group was higher than that of NC group（34.34±7.63mmol.L-1.h,32.38±6.65mmol.L-1.h V.S.18.72±3.27mmol.L-1.h, P=0.001）, however,there was no statistically difference between DM-C and DM-T group in AUGg(P>0.05)(Table2, Fig.2,3).2.2The fasting insulin (FINS) in DM-C group was higher than that ofNC and DM-T group （20.00±5.91mIU/L V.S.11.34±3.88mIU/L,13.61±7.60mIU/L, P=0.003）, whereas there was no statistically differencebetween NC and DM-T group in FINS (P>0.05). The AUCi in NC group washigher than that of DM-C and DM-T group（47.55±5.23mIU.L-1.h V.S.30.78±11.25mIU.L-1.h,32.78±11.64mIU.L-1.h, P=0.006）, whereas therewas no statistically difference between DM-C and DM-T group in AUCi (P>0.05). HOMA-IR was significantly higher in DM-C group than that of NC andDM-T group (11.20±2.22V.S.3.13±0.88,5.67±3.73, P <0.05), whereas therewas no statistically difference between NC and DM-T group inHOMA-IR(P>0.05). There was no statistically difference in HOMA-βamong3groups (P>0.05).(Table2, Fig.4,5,6).2.3The levels of serum TG, TC, ICAM-1, VCAM-1and NF-κB weresignificantly higher in DM-C group than those of DM-T and NC group (P <0.05), which were significant higher in DM-T group than those of NC group.Compared with NC group, the level of serum HDL-C was decreased in DM-Cand DM-T group（P <0.05）, but the level of serum HDL-C between DM-Cand DM-T group had no statistically difference(P>0.05). Compared withNC group, the level of serum LDL-C was increased in DM-C and DM-Tgroup（P <0.05）, whereas there was no statistically difference between DM-C and DM-T group in LDL-C (P>0.05)(Table2-3, Fig.7-10).3Pathological changes of thoracic aortic: the vascular tissue section afterHE staining showed there was complete visible vascular endothelial cell, neatrows, intimal smooth and neat membrane in smooth muscle cells in NC group;there was aortic intimal thickening, swollen endothelial cell degeneration,nucleus shrivel, intimal thickening, elastic plate fracture, disorderedarrangement of membrane in the smooth muscle cells and collagen fiberhyperplasia in DM-C group. After8weeks of metformin intervention, theaortic pathological changes were ameliorated compared with DM-C group(Fig.11-13).4Immunohistochemical results: Compared with NC group, theexpression of phosphorylation of p38MAPK, NF-κB and MCP-1wassignificantly increased in DM-C and DM-T group (P <0.05). After8weeksof metformin treatment, the expression of phosphorylation of p38MAPK,NF-κB and MCP-1was significantly decreased than that of DM-C group (P <0.05)(Table4, Fig.14-16).Conclusions:1Phosphorylated p38MAPK pathway may involved in the developmentof diabetic macrovascular complication. The progression of diabeticmacrovascular complication may be ameliorated by intervening p38MAPKsignal transduction pathway.2Metformin may have protective effect on diabetic macrovascularcomplication, which partly by reducing the expression of p-p38MAPK. |
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