MRL-45696减轻2型糖尿病大鼠心肌缺血再灌注后的DNA损伤

Poly(ADP-ribose) polymerases-1 inhibitor MRL-45696 alleviates DNA damage after myocardial ischemia-reperfusion in diabetic rats

目的研究摄食二磷酸腺苷核糖聚合酶抑制剂MRL-45696是否减轻2型糖尿病大鼠心肌缺血/再灌注后DNA的损伤。方法大鼠高糖高脂饲料喂食8周后腹腔注射链脲佐菌素(STZ)(30 mg/kg),构建2型糖尿病大鼠模型,选取2型糖尿病大鼠40只,随机分为糖尿病组(DM)、假手术组(S)、MRL-45696治疗+假手术组(NO)、缺血再灌注损伤模型组(MI/R)、MRL-45696治疗+缺血再灌注损伤组(MRL),每组各8只。灌胃给予2型糖尿病大鼠MRL-45696(50 mg/kg·d),1周后以大鼠冠状动脉左前降支结扎30 min再灌注120 min的方法制作心肌缺血再灌注损伤模型。检测大鼠血浆心肌肌钙蛋白Ⅰ(cTnI)、血清肌酸激酶(CK)、血清乳酸脱氢酶(LDH)活性和心肌梗死范围、细胞凋亡比例、丙二醛(MDA)、超氧化物歧化酶活性(SOD)。Western blot检测γ-H2AX、cleaved caspase-3、PARP-1、PAR;比色法检测大鼠心肌组织中烟酰胺腺嘌呤二核苷酸(NAD)水平(以NAD+/NADH比例代表)。结果 MRL组心肌梗死面积显著小于MI/R组(P<0.05),MI/R组cTnI、CK、LDH、MDA、γ-H2AX、cleaved caspase-3表达水平及细胞凋亡较其它组显著升高(P<0.05),而SOD水平明显降低(P<0.05);与MI/R组相比,MRL组大鼠cTnI、CK、LDH、MDA、γ-H2AX、cleaved caspase-3、PARP-1、PAR表达水平及细胞凋亡明显降低,SOD及NAD表达水平明显升高,差异有统计学意义(P<0.05)。结论糖尿病加重大鼠心肌细胞缺血再灌注后DNA损伤,MRL-45696可能通过抑制糖尿病大鼠PARP-1的过度激活,减轻心肌细胞缺血再灌注损伤,减少心肌梗死面积。

Objective To study the protective effect of MRL-45696, an inhibitor of poly（ADP-ribose） polymerase-1（PARP-1）,against DNA damage after myocardial ischemia/reperfusion（I/R） in diabetic rats. Methods Rat models of type 2 diabetes mellitus were established by high-fat feeding and a single peritoneal dose of streptozotocin. Forty diabetic rats were randomized equally into diabetic group, sham-operated group, sham-operated group with MRL-45696 treatment, I/R injury model group and I/R injury group with MRL-45696 treatment. The rats in MRL-45696-treated groups were subjected to daily intragastric administration of MRL-45696（50 mg/kg） for 7 consecutive days, after which sham operation was performed or myocardial I/R injury was induced by ligation of the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. The range of myocardial infarction, plasma cardiac troponin I（cTnI）, serum creatine kinase（CK）,lactate dehydrogenase（LDH） activity, malondialdehyde（MDA）, superoxide dismutase（SOD） activity, and cardiac myocyte apoptosis were detected. The levels of γ-H2 AX, cleaved caspase-3, PARP-1, and PAR were detected with Western blotting, and the level of NAD was detected using colorimetry. Results The infarct size was significantly smaller in MRL-45696 treatment group than in I/R injury group（P〈0.05）. In I/R model group, the levels of cTnI, CK, and LDH in the plasma or serum and MDA, γ-H2 AX, cleaved caspase-3 and apoptotic rate in the cardiac myocytes were significantly higher than those in the other groups（P〈0.05）, and SOD activity was significantly decreased（P〈0.05）. Compared with I/R model group, the rats with MRL-45696 treatment showed significantly decreased levels of cTnI, CK, LDH, MDA, γ-H2 AX, cleaved caspase-3, PARP-1, PAR expression and cell apoptosis with significantly increased levels of SOD and NAD（P〈0.05）. Conclusion MRL-45696 can inhibit excessive activation of PARP-1, increase intracellular level of NAD and inhibit cardiac myocyte apoptosis to alleviate myocardial I/R-induced DNA damage and reduce myocardial infarct size in diabetic rats.