(1.哈尔滨医科大学附属第一医院 心内科,黑龙江省哈尔滨市 150001;2.哈尔滨医科大学附属第一医院 甲状腺外科,黑龙江省哈尔滨市 150001)
1.Department of Cardiology,Harbin, Heilongjiang 150001, China ;2.Department of Thyroid Surgery, the First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China)
目的]探究组织因子途径抑制物(TFPI)对大鼠心肌缺血再灌注(I/R)及心肌细胞缺氧复氧(H/R)损伤的影响,并从心肌细胞凋亡的变化探索其机制。 [方法]在体内实验中,通过SD大鼠心脏原位结扎法可逆阻断前降支建立大鼠心肌I/R模型。将大鼠随机分为对照组、I/R组和I/R+rTFPI(重组TFPI)组,再灌注后3天采用HE染色观察大鼠心肌组织形态学变化,TTC染色法评估心肌梗死区范围,扫描透射电镜观察心肌超微结构损伤情况,Western blot法检测各组大鼠心肌组织中Bcl-2、Bax和cleaved Caspase-3蛋白的表达。在体外实验中,采用胰酶消化法及差速贴壁法培养SD乳鼠原代心肌细胞,用MIC101系统模拟心肌细胞I/R损伤,缺氧2 h、复氧12 h后建立体外心肌细胞H/R模型。将心肌细胞分为对照组、H/R组和H/R+rTFPI(10 μg/L)组,用CCK-8法检测心肌细胞活力,TUNEL法检测心肌细胞凋亡率,Western blot法检测心肌细胞中Bax、Bcl-2及cleaved Caspase-3蛋白的表达水平。 [结果]体内实验中,成功建立大鼠在体心肌I/R模型。HE染色结果显示I/R组较对照组心肌细胞坏死程度加重,I/R+rTFPI组较I/R组心肌细胞坏死程度减低；TTC染色示I/R+rTFPI组较I/R组心肌梗死范围减少了39.76%(P＜0.05)；扫描透射电镜观察显示I/R组凋亡及损伤程度较对照组加重,I/R+rTFPI组凋亡及损伤较I/R组减轻；Western blot结果示,再灌注3天后I/R组心肌组织Bcl-2的表达较对照组降低了53.43%(P＜0.05),Bax和cleaved Caspase-3的表达较对照组分别增加了29.05%和73.25%(P＜0.05),而I/R+rTFPI组Bcl-2的表达水平较I/R组升高了55.01%(P＜0.05),Bax和cleaved Caspase-3的表达水平较I/R组分别降低了13.77%和24.25%(P＜0.05)。在体外实验中,CCK-8检测结果显示H/R组细胞活力较对照组下降了29.70%(P＜0.05),H/R+rTFPI组细胞活力较H/R组升高了19.77%(P＜0.05)。TUNEL结果显示H/R组较对照组凋亡率增加了56.76%,H/R+rTFPI组细胞凋亡率较H/R组降低了24.55%(P＜0.05)。Western blot结果示,H/R组细胞Bcl-2表达较对照组降低了46.92%,Bax表达较对照组增加了41.90%(P＜0.05),cleaved Caspase-3表达较对照组升高了2.68倍(P＜0.05)；H/R+rTFPI组Bcl-2表达较H/R组增加了28.24%(P＜0.05),Bax及cleaved Caspase-3表达较H/R组分别降低了26.34%和57.60%(P＜0.05)。 [结论]TFPI可显著拮抗心肌I/R和心肌细胞H/R损伤,此效应与其抑制心肌细胞凋亡有关。
Aim To investigate the effects of tissue factor pathway inhibitor (TFPI) on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) injury. Methods I/R model was established by ligating the anterior descending coronary artery in vivo. Rats were randomly divided into three groups:the control, I/R, and I/R+rTFPI groups. HE staining was used to evaluate the morphological changes of rats myocardial tissue after reperfusion for 3 days. TTC staining was used to detect the myocardial infarct size. Ultrastructural damage in cardiomyocytes was measured by transmission electron microscopy. The expression levels of Bcl-2, Bax and cleaved Caspase-3 in rats myocardial tissue were detected by Western blot analysis. Cardiomyocytes of Sprague Dawley (SD) rats were cultured by enzyme digestion and differential adherent method in vitro experiments. Cardiomyocytes I/R injury in vitro model was established by 2 h of hypoxia and 12 h of reoxygenation using MIC101 system. Cardiomyocytes were divided into control group, H/R group, and H/R+rTFPI group (10 μg/L). Cell viability was detected by CCK-8 assay. Detection of cardiomyocytes apoptosis was performed by TUNEL technique. Western blot analysis was used to detect the expression of Bax, Bcl-2 and cleaved Caspase-3. Results A myocardial I/R model was successfully established in vivo. HE staining showed myocardiumin exhibited a higher degree of necrosis than that in control group, which was milder in I/R+rTFPI group than that in I/R group (P＜0.05). TTC staining showed that, compared with I/R group, myocardial infarct size reduced 39.76% in I/R+rTFPI group (P＜0.05). The data of transmission electron microscopy showed that the degree of apoptosis and injury was severer in I/R group than that in control group, whereas it was milder in I/R+rTFPI group than that in I/R group. Western blot detection results showed that compared with control group, the expression of Bcl-2 decreased 53.43% in I/R group(P＜0.05), but the expression of Bax and cleaved Caspase-3 increased 29.05% and 73.25% respectively (P＜0.05). After adding rTFPI, the expression of Bax and cleaved Caspase-3 in myocardial tissue decreased 13.77% and 24.25% respectively compared with I/R group(P＜0.05), whereas the expression of Bcl-2 increased 55.01% compared with I/R group (P＜0.05). In in vitro experiment, the CCK-8 results showed the cell viability of H/R group decreased 29.70% compared with control group(P＜0.05), but it increased 19.77% after adding rTFPI compared with H/R group(P＜0.05). The TUNEL detection showed that the apoptotic rate of H/R group increased 56.76% compared with control group, whereas the apoptotic rate of H/R+rTFPI group decreased 24.55% compared with H/R group(P＜0.05). Western blot results showed that compared with control group, the expression of Bcl-2 in cardiomyocytes decreased 46.92% in H/R group (P＜0.05), but the expression of Bax increased 41.90%(P＜0.05) and the expression of cleaved Caspase-3 increased 2.68 fold (P＜0.05). After adding rTFPI, the expressions of Bax and cleaved Caspase-3 in the cardiomyocytes decreased 26.34% and 57.60% respectively when compared with H/R group(P＜0.05), whereas the expression of Bcl-2 increased 28.24% compared with H/R group (P＜0.05). Conclusion TFPI served a protective role against I/R and H/R-induced cardiomyocyte injury by inhibiting apoptosis.