PI3K/Akt信号通路在氢气抑制大鼠心肌缺血再灌注时细胞凋亡中的作用

Role of PI3K/Akt signaling pathway in hydrogen-induced inhibition of cell apoptosis during myocardial ischemia/reperfusion in rats

目的 评价磷脂酰肌醇3-激酶/蛋白激酶B（PI3 K/Akt）信号通路在氢气抑制大鼠心肌缺血再灌注时细胞凋亡中的作用.方法 健康雄性SD大鼠40只,体重300 ～ 350 g,采用随机数字表法分为4组（n=10）：假手术组（S组）、心肌缺血再灌注组（I/R组）、氢气组（H组）和氢气＋PI3K特异性阻断剂LY294002组（HL组）.采用结扎左冠状动脉前降支30 min,再灌注120 min的方法制备心肌缺血再灌注损伤模型.H组和HL组分别于再灌注即刻腹腔注射浓度为99.6％的氢气5 ml/kg,HL组于氢气注射前鼠尾静脉注射LY294002 0.3 mg/kg.于再灌注120 min时取右颈总动脉血样2 ml,测定血清肌酸激酶同工酶（CK-MB）和乳酸脱氢酶（LDH）活性;采用TUNEL染色法检测心肌细胞凋亡情况,并计算凋亡指数（AI）,免疫组化法检测Bcl-2、Bax及caspase-3的表达.计算Bcl-2/Bax比值.结果 与S组比较,I/R组血清CK-MB、LDH活性及AI均升高,心肌Bcl-2、Bax、caspase-3表达上调,Bcl-2/Bax比值降低（P＜ 0.01）;与I/R组比较,H组血清CK-MB、LDH活性及AI均降低,心肌Bcl-2表达上调,Bax和caspase-3表达下调,Bcl-2/Bax比值升高（P＜0.01）,HL组上述指标差异无统计学意义（P＞0.05）;与H组比较,HL组血清CK-MB、LDH活性及AI升高,心肌Bcl-2表达下调,Bax和caspase-3表达上调,Bcl-2/Bax比值降低（P＜0.01）.结论 氢气可通过激活PI3K/Akt信号通路,进而上调抗凋亡蛋白Bcl-2表达及下调促凋亡蛋白Bax和caspase-3的表达,抑制大鼠心肌缺血再灌注时细胞凋亡.

Objective To evaluate the role of phosphoinositide 3 kinase/protein kinase B （PI3K/Akt） signaling pathway in hydrogen-induced inhibition of cell apoptosis during myocardial ischemia/reperfusion （I/R） in rats.Methods Forty healthy male Sprague-Dawley rats,weighing 300-350 g,were randomly allocated into 4 groups （n =10 each） using a random number table：sham operation group （S group）,I/R group,hydrogen group （group H）,and hydrogen ＋ LY294002 group （group HL）.Myocardial I/R was induced by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion.In H and HL groups,99.6 ％ hydrogen 5 ml/kg was injected intraperitoneally immediately after beginning of reperfusion,and in addition LY294002 （0.3 mg/kg） was injected through the caudal vein before hydrogen injection in group HL.Arterial blood samples were collected at the end of 120 min reperfusion for determination of serum creatine kinase isoenzyme-MB （CK-MB） and lactate dehydrogenase （LDH） activities.The rats were then sacrificed.Myocardial apoptosis was detected by TUNEL and apoptosis index （AI） was calculated.The expression of Bcl-2,Bax and caspase-3 was detected by immuno-histochemistry.Bcl-2/Bax ratio was calculated.Results The serum CK-MB and LDH activities and AI were significantly increased,the expression of myocardial Bcl-2,Bax and caspase-3 was upregulated,and the ratio of Bcl-2/Bax was decreased in group I/R as compared with group S.Compared with group I/R,the serum CK-MB and LDH activities and AI were significantly decreased,the expression of myocardial Bcl2 was up-regulated,while the expression of myocardial Bax and caspase-3 was down-regulated,and the ratio of Bcl-2/Bax was increased in group H,and no significant changes were found in the parameters mentioned above in group HL.The serum CK-MB and LDH activities and AI were significantly increased,the expression of myocardial Bcl-2 was down-regulated,while the expression of myocardial Bax and caspase-3 was up-regulated,and Bcl-2/Bax ratio was decreased in group HL as compared with group H.Conclusion Hydrogen can activate the PI3K/Akt signaling pathway,and further up-regulates Bcl-2 expression and down-regulates Bax and Caspase-3 expression,thus inhibiting cell apoptosis during myocardial I/R in rats.