【摘要】 目的 探讨克洛索蛋白对自然衰老大鼠心肌缺血再灌注损伤（MIRI）的影响及其作用机制。方法 将27只SD雄性大鼠在SPF环境中饲养18个月构建自然衰老大鼠模型，采用随机数字表法将其分为假手术组、造模组及克洛索干预组，每组9只。克洛索干预组按0.01 mg/kg剂量腹腔注射克洛索蛋白（2 d/次），假手术组与造模组注射等量生理盐水，连续干预14 d。克洛索干预组与造模组通过结扎左冠状动脉前降支30 min，再灌注2 h构建MIRI模型。采用TTC染色测定心肌梗死面积；HE与Masson染色观察心肌组织形态与胶原沉积情况；酶联免疫吸附试验法检测血清肌酸激酶MB型同工酶（CK-MB）、乳酸脱氢酶（LDH）活性及心肌组织超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平；TUNEL染色检测心肌细胞凋亡率；RT-qPCR和Western blot法检测心肌组织PI3K、Akt、Bax、Bcl-2、Caspase-3 mRNA和蛋白表达。结果 与假手术组比较，造模组心肌梗死面积增加，血清CK-MB、LDH活性升高（P<0.05）；与造模组比较，克洛索干预组心肌梗死面积，血清CK-MB、LDH活性降低（P<0.05）。与假手术组比较，造模组心肌组织MDA含量升高、SOD活性降低（P<0.05）；与造模组比较，克洛索干预组心肌组织MDA含量降低、SOD活性升高（P<0.05）。与假手术组比较，造模组心肌病理损伤评分、心肌胶原沉积评分升高（P<0.05）；与造模组比较，克洛索干预组心肌病理损伤评分、心肌胶原沉积评分降低（P<0.05）。与假手术组比较，造模组心肌细胞凋亡率升高（P<0.05）；与造模组比较，克洛索干预组心肌细胞凋亡率降低（P<0.05）。与假手术组比较，造模组心肌组织Bax mRNA表达升高，Bcl-2 mRNA表达降低（P<0.05）；与造模组比较，克洛索干预组心肌组织Bax mRNA表达降低；Bcl-2 mRNA表达升高（P<0.05）。与假手术组比较，造模组心肌组织Bax蛋白表达升高；p-PI3K、Akt、p-Akt、Bcl-2蛋白表达降低（P<0.05）。与造模组比较，克洛索干预组心肌组织PI3K、p-PI3K、Akt、p-Akt、Bcl-2蛋白表达升高；Bax、cleaved-Caspase-3蛋白表达降低（P<0.05）。结论 克洛索对自然衰老大鼠MIRI具有保护作用，其机制可能与激活PI3K/Akt通路，进而抑制氧化应激与心肌细胞凋亡有关，但与该通路的直接性需进一步验证。更多还原

【Abstract】 Objective To investigate the effect and mechanism of Klotho protein on myocardial ischemia/reperfusion injury(MIRI) in naturally aging rats. Methods A total of 27 male SD rats were raised in an SPF environment for 18 months to establish a naturally aging rat model. The model rats were divided into sham-operated group, model group, and Klotho intervention group, with nine rats in each groups. The Klotho intervention group was intraperitoneally injected with Klotho protein at a dose of 0.01 mg/kg(2 days each time); while the sham-operated group and the model group received an equal volume of normal saline via the same route and schedule, for 14 consecutive days. MIRI was induced in the Klotho intervention group and model group by ligating the left anterior descending coronary artery for 30 min, followed by 2 hours of reperfusion. Myocardial infarct size was measured by TTC staining; myocardial tissues morphology and collagen deposition were observed by HE and Masson staining; the activities of serum creatine kinase MB isoenzyme(CK-MB) and lactate dehydrogenase(LDH), as well as the activities of superoxide dismutase(SOD) and the content of malondialdehyde(MDA) in myocardial tissue were detected by enzyme-linked immunosorbent assay; the apoptosis rate of myocardial cell was detected by TUNEL staining; and the mRNA and protein expressions of PI3K, Akt, Bax, Bcl-2, and Caspase-3 in myocardial tissues were detected by RT-qPCR and Western blot. Results Compared with the sham-operated group, the Myocardial infarct size and the activities of serum CK-MB and LDH in the model group increased(P<0.05); compared with the model group, the myocardial infarct size and the activities of serum CK-MB and LDH in the Klotho intervention group decreased(P<0.05). Compared with the sham-operated group, the MDA content in myocardial tissue of the model group increased and the SOD activity decreased(P<0.05); compared with the model group, the MDA content in the myocardial tissue in the Klotho intervention group decreased and the SOD activity increased(P<0.05). Compared with the sham-operated group, the myocardial pathological injury score and myocardial collagen deposition score in the model group increased(P<0.05); compared with the model group, the myocardial pathological injury score and myocardial collagen deposition score in the Klotho intervention group were decreased(P<0.05). Compared with the sham-operated group, the apoptosis rate of myocardial cells in the model group increased(P<0.05); compared with the model group, the apoptosis rate of myocardial cells in the Klotho intervention group decreased(P<0.05). Compared with the sham-operated group, the expression of Bax mRNA in myocardial tissues of the model group increased, and the expression of Bcl-2 mRNA decreased(P<0.05); compared with the model group, the expression of Bax mRNA in myocardial tissues of the Klotho intervention group decreased; the expression of Bcl-2 mRNA increased(P<0.05). Compared with the sham-operated group, the expression of Bax protein in myocardial tissues of the model group increased; while the expressions of p-PI3K, Akt, p-Akt, and Bcl-2 protein decreased(P<0.05); compared with the model group, the expressions of PI3K, p-PI3K, Akt, p-Akt, and Bcl-2 protein in myocardial tissues of the Klotho intervention group increased; while the expressions of Bax and cleaved-Caspase-3 protein decreased(P<0.05). Conclusion Klotho has a protective effect on MIRI in naturally aging rats. The mechanism may be related to the activation of the PI3K/Akt pathway, which in turn inhibits oxidative stress and cardiomyocyte apoptosis. However, the directness to this pathway needs further verification.更多还原