昆布多糖对大鼠心肌缺血/再灌注模型葡萄糖调节蛋白-78及半胱氨酸天冬氨酸蛋白水解酶12蛋白表达含量的影响

Effect of laminarin on GRP78 and Caspase 12 expression in rat model of myocardial ischemia-reperfusion injury

摘要:

目的 研究昆布多糖(Lam)对大鼠心肌缺血/再灌注(MI/RI)模型中葡萄糖调节蛋白-78(GRP-78)及半胱氨酸天冬氨酸蛋白水解酶12(Caspase 12)蛋白表达含量的影响,探讨其对大鼠心肌是否有保护作用。方法 选取32只雄性SD大鼠,随机分为4组:假手术组(sham组)、MI/RI组、Lam低剂量组、Lam高剂量组。采用左冠状动脉结扎法制作大鼠MI/RI模型。酶联免疫吸附试验(ELISA)测定肌酸激酶同工酶MB(CK-MB),比色法测定髓过氧化物酶(MPO)的表达量。应用蛋白质免疫印迹(Western blotting)检测各组中大鼠心肌GRP-78、Caspase 12、B淋巴细胞瘤-2蛋白(Bcl-2)蛋白的表达情况。光镜下观察心肌组织的病理形态学改变。伊文思蓝(Evans)、氯化三苯基四氮唑红(TTC)双染色法测定心肌梗死面积。采用SPSS 21.0软件进行统计分析。结果 与MI/RI组相比,低、高剂量组的CK-MB[(93.74±5.37)和(72.71±5.63)和(59.79±9.67)U/L]、MPO[(72.66±3.40)和(58.92±4.88)和(46.06±5.74)U/L]、GRP-78[(1.13±0.02)和(0.81±0.01)和(0.66±0.01)]、Caspase 12[(1.45±0.10)和(0.82±0.06)和(0.62±0.02)]表达显著减少(P<0.01),Bcl-2[(1.01±0.04)和(1.19±1.10)和(1.41±0.02)]表达显著升高(P<0.01)。与MI/RI组相比,Lam各处理组的心肌组织损伤有不同程度的减轻。与MI/RI组比较,Lam各处理组心肌梗死面积显著减少[(55.36±2.47)%和(46.63±2.10)%和(38.40±2.07)%,P<0.05]。结论 Lam能减轻大鼠MI/RI,对MI/RI的心肌有保护作用。其机制可能是Lam抑制GRP-78的过度表达,减轻了由内质网应激介导细胞凋亡途径的激活,下调Caspase 12凋亡通路,增强Bcl-2的表达,从而抑制心肌细胞凋亡。

Abstract:

Objective To investigate the effect of laminarin (Lam) on the protein expression of glucose regulated protein-78 (GRP-78) and cysteine-dependent aspartate-specific protrases 12 (Caspase12) in rat model of myocardial ischemia-reperfusion injury (MI/RI) and whether it has protective effect on rat myocardium. Methods Thirty-two male SD rats were randomly divided into 4 groups:sham operation group, MI/RI group, low- and high-dose Lam treatment groups. The rat MI/RI model was established by left coronary artery ligation. The serum content of creatine kinase isoenzyme MB (CK-MB) was measured by enzyme-linked immunosorbent assay (ELISA), the content of myeloperoxidase (MPO) in the myocardial tissue was measured by colorimetric assay, and the protein expression levels of GRP78, Caspase 12, B-cell lymphoma-2 (Bcl-2) in myocardium was detected by Western blotting. The pathomorphological changes of myocardial tissue were observed by light microscopy. Myocardial infarction area was measured by Evans Blue and 2,3,5-triphenyltetrazolium chloride (TTC) double staining. SPSS statistics 21.0 was used to perform the statistical analysis. Results The low- and high-dose Lam treatment groups had significantly decreased serum level of CK-MB [(72.71±5.63) vs (59.79±9.67) vs (93.74±5.37)U/L], myocardial content of MPO [(58.92±4.88) vs(46.06±5.74) vs(72.66±3.40)U/L] and protein levels of GRP-78 [(0.81±0.01) vs (0.66±0.01) vs (1.13±0.02)] and Caspase 12 [(0.82±0.06) vs (0.62±0.02) vs (1.45±0.10)], but obviously enhanced expression of Bcl-2 [(1.19±1.10) vs (1.41±0.02) vs (1.01±0.04)] when compared with the MI/RI group (all P<0.01). The myocardial tissue damage in the Lam treatment groups was alleviated to different extents than that in the MI/RI group. And the area of myocardial infarction was remarkably reduced in the low-and high-dose Lam treatment groups than the MI/RI group [(55.36±2.47)% vs (46.63±2.10)% vs (38.40±2.07)%, P<0.05]. Conclusion Lam can alleviate MI/RI and exerts protective effect on the injury in rats. The mechanism may be that Lam can inhibit the overexpression of GRP-78, reduce the activation of apoptosis pathway mediated by endoplasmic reticulum stress, down-regulate the apoptosis pathway of Caspase 12 and enhance the expression of Bcl-2, and thus inhibit the apoptosis of cardiomyocytes.