二甲双胍降低2型糖尿病大鼠主动脉磷酸化丝裂原活化蛋白激酶的蛋白表达

摘要

Objective: To explore the relationship between p38 MAPK protein expression and macro vascular lesions in type 2 diabetes mellitus (DM) rats with the effect of metformin intervention. Methods: A total of 25 healthy male Wistar rats were randomly divided into 2 groups: Control group, the rats had normal diet for 4 weeks and then received citrate buffer solution for modeling control, n =6. Experimental group, the rats receivedhigh fat diet for 4 weeks and then received streptozotocin injection for DM modeling, n =19; there were 15 rats successfullydeveloped DM and randomly divided into 2 subgroups: DM control (DM-C) group, n =7 and DM + metformin group, n =8,in which DM rats received metformin 200 mg/kg·d for 8 weeks. At the end of intervention, intraperitoneal injections ofglucose tolerance test ( IPGTT ) was performed, laboratory biochemical indexes and fasting insulin level were detected,protein expressions of nueclear factor (NF-κB), monocyte chemo-attractant protein-1(MCP-1) in the thoracic aorta wereexamined by immunohistochemistry.Results: ① HE staining showed that DM-C group had increased tunica intimae thickness, endothelia cell swollen, mediasmooth muscle disorder and collagen fiber hyperplasia. ② Compared with Control group, DM-C group had increased levelsof fasting insulin (20.00 ± 5.91) mIU/L vs (11.34 ± 3.88) mIU/L, NF-κB (170.22 ± 21.53) μmol/L vs (78.68 ± 12.15) μmol/L,ICAM-1 (130.59 ± 16.00) pg/ml vs (75.63 ± 19.52) pg/ml, VCAM-1 (990.19 ± 119.18) ng/ml vs (616.54 ±54.51) ng/ml, andincreased TC (4.15 ± 0.41) mmol/L vs (2.18 ± 0.39) mmol/L, TG (1.83 ± 0.40) mmol/L vs (0.81 ± 0.27) mmol/L, LDL(2.53 ± 0.44) mmol/L vs (1.24 ± 0.47) mmol/L, P <0.05; while decreased AUCi (30.78 ± 11.25) mIU·L-1·h vs (47.55 ± 5.23)mIU·L-1·h, P <0.05; increased protein expressions of p38 MAPK, NF-κB and MCP-1, P <0.05. ③ Compared with DM-Cgroup, DM + metformin group presented decreased levels of fasting insulin, NF-κB, ICAM-1, VCAM-1, TC, TG, P <0.05;increased aortic protein expressions of p38 MAPK, NF-κB and MCP-1; while AUCi was similar between 2 groups, P>0.05.Conclusion: The activation of p38 MAPK pathway plays an important role in DM macro angiopathy, metformin protectsblood vessel via decreasing p38 MAPK level, inhibiting inflammatory reaction and regulating lipid metabolism in DM rats.%目的:探讨p38丝裂原活化蛋白激酶( MAPK)与2型糖尿病大鼠主动脉病变的关系及二甲双胍的干预作用. 方法:25只雄性Wistar大鼠随机分为对照组和实验组,实验组采用高脂高糖饲料喂养联合链脲佐菌素注射建立2型糖尿病大鼠模型.将成模糖尿病大鼠随机分为2组:2型糖尿病空白对照组(2型糖尿病组,n=7)、2型糖尿病+二甲双胍干预组(二甲双胍组,n=8).二甲双胍组给予二甲双胍200 mg/kg灌胃8周,于干预末行腹腔葡萄糖耐量试验,次晨心尖取血并分离血清,全自动生化分析仪测定血糖、血脂等生化指标;放免法测定胰岛素水平;采用酶联免疫吸附方法测定血清细胞间黏附分子-1( ICAM-1), 血管细胞黏附分子-1(VCAM-1)和核因子-κB水平;取胸主动脉进行苏木素伊红(HE)染色,光镜下观察血管病理变化;免疫组化法检测大鼠胸主动脉磷酸化p38 MAPK、核因子-κB、单核细胞趋化蛋白-1蛋白的表达.结果(:1)HE染色可见2型糖尿病组大鼠主动脉管壁结构层次不清,内膜增厚,内皮细胞肿大变性,中膜平滑肌细胞排列紊乱,胶原纤维增生.(2)与对照组相比,2型糖尿病组大鼠空腹胰岛素[(20.00 ± 5.91) mIU/L vs(11.34 ± 3.88)mIU/L]、核因子-κB [(170.22±21.53)μmol/L vs(78.68±12.15)μmol/L]、ICAM-1[(130.59±16.00) pg/ml vs(75.63±19.52)pg/ml]、VCAM-1 [(990.19±119.18) ng/ml vs(616.54±54.51) ng/ml]、总胆固醇 [(4.15±0.41) mmol/L vs(2.18±0. 93 ) mmol/L]、甘油三酯 [(1.83±0.40) mmol/L vs(0.81±0.27)mmol/L]、低密度脂蛋白胆固醇[(2.53±0.44) mmol/L vs (1.24±0.47)mmol/L]水平明显升高(P<0.05),胰岛素曲线下面积(30.78 ± 11.25)mIU·L-1·h vs(47.55 ± 5.23)mIU·L-1·h明显降低(P<0.05),主动脉磷酸化p38 MAPK、核因子-κB、MCP-1蛋白表达水平升高(P<0.05),差异均有统计学意义.(3)实验结束时二甲双胍组大鼠空腹血糖、空腹胰岛素、核因子-κB、单核细胞趋化蛋白-1、VCAM-1、甘油三酯、总胆固醇水平较2型糖尿病组明显降低(P< 0.05);二甲双胍组和2型糖尿病组间胰岛素曲线下面积无明显差异(P> 0.05).二甲双胍组大鼠主动脉磷酸化p38 MAPK、核因子-κB、单核细胞趋化蛋白-1蛋白表达水平也较二甲双胍组明显降低(P<0.05),差异有统计学意义.结论:p38 MAPK信号通路的激活在糖尿病大血管病变发生发展中起重要作用,二甲双胍通过降低p38 MAPK磷酸化水平、抑制炎症反应、调节脂代谢而起到血管保护作用.