缺血预处理通过下调MMPs表达减轻2型糖尿病大鼠心肌缺血再灌注损伤

Protective effects of ischemic preconditioning on diabetic rats'myocardium ischemia reperfusion injury

目的探讨缺血预处理对2型糖尿病大鼠心肌缺血再灌注损伤的保护作用及机制。方法雄性SD大鼠42只随机分为六组:假手术组(CC组)、缺血再灌注组(CI组)、缺血预处理组(CIPC组)、糖尿病假手术组(DC组)、糖尿病缺血再灌注组(DI组)、糖尿病缺血预处理组(DIPC组)。高脂高糖饮食复合腹腔注射链脲佐菌素的方法制备2型糖尿病模型;结扎左冠状动脉前降支30 min再灌注2 h制备心肌缺血再灌注损伤模型;预处理组于缺血前30 min,反复三次缺血5 min,再灌注5 min操作后,再行缺血再灌注处理。光镜下观察心肌细胞形态学改变,酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)法检测血清肿瘤坏死因子(tumor necrosis factor,TNF)-α、白细胞介素(interleukin,IL)-6水平,Western blot检测心肌组织基质金属蛋白酶(matrix metallo proteinase,MMP)-2、MMP-9表达水平。结果与DC组相比,再灌注后DI组血清TNF-α、IL-6水平升高[(213.81±8.73)比(114.66±7.07),(175.25±7.72)比(89.77±6.42),t值分别为23.25、17.78,P值均<0.05],与CC组相比,再灌注后CI组血清TNF-α、IL-6水平升高[(153.59±6.68)比(84.56±6.54);(158.64±6.18)比(75.15±4.08),t值分别为19.54、29.83,P值均<0.05]。与DC组相比,再灌注后DI组心肌MMP-2、MMP-9表达升高[(1.50±0.07)比(0.28±0.05);(1.85±0.14)比(0.33±0.08),t值分别为36.46、27.68,P值均<0.05],与CC组相比,再灌注后CI组心肌MMP-2、MMP-9表达升高[(1.44±0.08)比(0.14±0.05);(1.45±0.08)比(0.19±0.09),t值分别为37.52、24.94,P值均<0.05]。与DI组相比,再灌注后DIPC组血清TNF-α、IL-6水平降低[(140.14±7.24)比(213.81±8.73);(129.60±7.48)比(175.25±7.72),t值分别为17.19、6.81,P值均<0.05];与CI组相比,再灌注后CIPC组血清TNF-α、IL-6水平降低[(112.36±5.36)比(153.59±6.68);(92.64±6.58)比(158.64±6.18),t值分别为12.74、19.34,P值均<0.05]。与DI组相比,再灌注后DIPC组心肌MMP-2、MMP-9表达降低[(0.94±0.06)比(1.50±0.07);(0.97±0.08)比(1.85±0.14),t值分别为8.89、10.52,P值均<0.05],与CI组相比,再灌注后CIPC组心肌MMP-2、MMP-9表达降低[(1.06±0.08)比(1.44±0.08);(0.99±0.08)比(1.45±0.08),t值分别为16.07、14.44,P值均<0.05]。与非糖尿病组大鼠相比,糖尿病组大鼠在基础水平的TNF-α、IL-6、MMP-2和MMP-9差异均无统计学意义(P>0.05)。结论缺血预处理减轻正常大鼠和糖尿病大鼠心肌缺血再灌注损伤,其机制与抑制MMPs表达及减轻炎症反应有关。

Objective To explore whether ischemia preconditioning has a protective effect on myocardial ischemia-reperfusion injury in type 2 diabetic rats.Methods A total of 42 male SD rats were randomly divided into six groups,sham group(CC group),ischemia reperfusion group(CI group),ischemic preconditioning group(CIPC group),diabetic sham group(DC group),diabetic ischemia reperfusion group(DI group)and diabetic ischemic preconditioning group(DIPC group).The model of type 2 diabetes mellitus was established by high fat and high sugar diet combined with intraperitoneal injection of streptozotocin.The myocardial ischemia-reperfusion injury model was prepared by ligation of the anterior descending branch of the left coronary artery for 30 min and reperfusion for 2 h.In ischemic preconditioning group,30 min before ischemia was repeated for 5 min ischemia and 5 min reperfusion for three cycles,and then ischemia-reperfusion was performed again.The morphological changes of myocardial cells were observed under light microscope,the levels of tumor necrosis factor(TNF)-αand interleukin(IL)-6 in serum were detected by enzyme linked immunosorbent assay(ELISA),and the expression levels of MMP-2 and MMP-9 in myocardial tissue were detected by Western blot.Results After reperfusion,the level of serum TNF-αIL-6 in DI group was increased compared with DC group[(213.81±8.73)vs(114.66±7.07);(175.25±7.72)vs(89.77±6.42),t values were 23.25,17.78,both P values<0.05].Serum TNF-αIL-6 level was increased in CI group after reperfusion compared with CC group[(153.59±6.68)vs(84.56±6.54);(158.64±6.18)vs(75.15±4.08),t values were 19.54,29.83,both P values<0.05].Compared with DC group,the expression of MMP-2 mMP-9 was increased in DI group after reperfusion[(1.50±0.07)vs(0.28±0.05);(1.85±0.14)vs(0.33±0.08),t values were 36.46,27.68,both P values<0.05].Compared with CC group,myocardial matrix metallo proteinase(MMP)-2 and MMP-9 expression increased in CI group after reperfusion[(1.44±0.08)vs(0.14±0.05);(1.45±0.08)vs(0.19±0.09),t values were 37.52,24.94,both P values<0.05].After reperfusion,serum TNF-αIL-6 levels were decreased in the DIPC group compared with the DI group[(140.14±7.24)vs(213.81±8.73);(129.60±7.48)vs(175.25±7.72),t values were17.19,6.81,both P values<0.05].Compared with CI group,serum TNF-αIL-6 level was decreased in CIPC group after reperfusion[(112.36±5.36)vs(153.59±6.68);(92.64±6.58)vs(158.64±6.18),t values were 12.74,19.34,both P values<0.05].Compared with DI group,myocardial MMP-2 and MMP-9 expression in DIPC group was decreased after reperfusion[(0.94±0.06)vs(1.50±0.07);(0.97±0.08)vs(1.85±0.14),t values were 8.89,10.52,both P values<0.05].Compared with CI group,myocardial MMP-2 and MMP-9 expression in CIPC group was decreased after reperfusion[(1.06±0.08)vs(1.44±0.08);(0.99±0.08)vs(1.45±0.08),t values were 16.07,14.44,both P values<0.05].Compared with the non-diabetic group,there was no significant difference in TNF-α,IL-6,MMP-2 and MMP-9 of the diabetc group(P>0.05).ConclusionIs chemic preconditioning alleviates myocardial ischemia-reperfusion injury in normal and diabetic rats by inhibiting MMPs expression and reducing inflammatory response.