Jiang Xue, Guo Cai-xia, Zeng Xiang-jun, Li Hui-hua, Chen Bu-xing, Du Feng-he
Department of Cardiology, Beijing Tian Tan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing, 100050, China.
Apoptosis. 2015 Aug;20(8):1033-47. doi: 10.1007/s10495-015-1130-4.
sRAGE can protect cardiomyocytes from apoptosis induced by ischemia/reperfusion (I/R). However, the signaling mechanisms in cardioprotection by sRAGE are currently unknown. We investigated the cardioprotective effect and potential molecular mechanisms of sRAGE inhibition on apoptosis in the mouse myocardial I/R as an in vivo model and neonatal rat cardiomyocyte subjected to ischemic buffer as an in vitro model. Cardiac function and myocardial infarct size following by I/R were evaluated with echocardiography and Evans blue/2,3,5-triphenyltetrazolium chloride. Apoptosis was detected by TUNEL staining and caspase-3 activity. Expression of the apoptosis-related proteins p53, Bax, Bcl-2, JAK2/p-JAK2, STAT3/p-STAT3, AKT/p-AKT, ERK/p-ERK, STAT5A/p-STAT5A and STAT6/p-STAT6 were detected by western blot analysis in the presence and absence of the JAK2 inhibitor AG 490. sRAGE (100 µg/day) improved the heart function in mice with I/R: the left ventricular ejection fraction and fractional shortening were increased by 42 and 57%, respectively; the infarct size was decreased by 52%, the TUNEL-positive myocytes by 66%, and activity of caspase-3 by 24%, the protein expression of p53 and ratio of Bax to Bcl-2 by 29 and 88%, respectively; protein expression of the p-JAK2, p-STAT3 and p-AKT were increased by 92, 280 and 31%, respectively. sRAGE have no effect on protein expression of p-ERK1/2, p-STAT5A and p-STAT6 following by I/R. sRAGE (900 nmol/L) exhibited anti-apoptotic effects in cardiomyocytes by decreasing TUNEL-positive myocytes by 67% and caspase-3 activity by 20%, p53 protein level and the Bax/Bcl-2 ratio by 58 and 86%, respectively; increasing protein expression of the p-JAK2 and p-STAT3 by 26 and 156%, respectively, p-AKT protein level by 33%. The anti-apoptotic effects of sRAGE following I/R were blocked by JAK2 inhibitor AG 490. The effect of sRAGE reduction on TUNEL-positive myocytes and caspase-3 activity were abolished by PI3K inhibitor LY294002, but not ERK 1/2 inhibitor PD98059. These results suggest that sRAGE protects cardiomyocytes from apoptosis induced by I/R in vitro and in vivo by activating the JAK2/STAT3 signaling pathway.
可溶性晚期糖基化终末产物受体（sRAGE）可保护心肌细胞免受缺血/再灌注（I/R）诱导的凋亡。然而，sRAGE发挥心脏保护作用的信号机制目前尚不清楚。我们以小鼠心肌I/R作为体内模型，以缺血缓冲处理的新生大鼠心肌细胞作为体外模型，研究了sRAGE抑制对凋亡的心脏保护作用及其潜在分子机制。通过超声心动图和伊文思蓝/2,3,5-三苯基氯化四氮唑评估I/R后的心脏功能和心肌梗死面积。通过TUNEL染色和半胱天冬酶-3活性检测凋亡情况。在存在和不存在JAK2抑制剂AG 490的情况下，通过蛋白质印迹分析检测凋亡相关蛋白p53、Bax、Bcl-2、JAK2/p-JAK2、STAT3/p-STAT3、AKT/p-AKT、ERK/p-ERK、STAT5A/p-STAT5A和STAT6/p-STAT6的表达。sRAGE（100μg/天）改善了I/R小鼠的心脏功能：左心室射血分数和缩短分数分别增加了42%和57%；梗死面积减少了52%，TUNEL阳性心肌细胞减少了66%，半胱天冬酶-3活性降低了24%，p53蛋白表达和Bax与Bcl-2的比值分别降低了29%和88%；p-JAK2、p-STAT3和p-AKT的蛋白表达分别增加了92%、280%和31%。I/R后，sRAGE对p-ERK1/2、p-STAT5A和p-STAT6的蛋白表达无影响。sRAGE（900nmol/L）在心肌细胞中表现出抗凋亡作用，使TUNEL阳性心肌细胞减少67%，半胱天冬酶-3活性降低20%，p53蛋白水平和Bax/Bcl-2比值分别降低58%和86%；p-JAK2和p-STAT3的蛋白表达分别增加26%和156%，p-AKT蛋白水平增加33%。I/R后sRAGE的抗凋亡作用被JAK2抑制剂AG 490阻断。PI3K抑制剂LY294002消除了sRAGE减少对TUNEL阳性心肌细胞和半胱天冬酶-3活性的影响，但ERK 1/2抑制剂PD98059没有。这些结果表明，sRAGE通过激活JAK2/STAT3信号通路在体外和体内保护心肌细胞免受I/R诱导的凋亡。
