Yan Ling, Zhu Tie-Bing, Wang Lian-Sheng, Tao Zheng-Xian, Pan Shi-Yang, Yang Zhi-Jian, Cao Ke-Jiang
Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2010 Nov;38(11):1019-24.
To examine whether the anti-apoptotic effect of hepatocyte growth factor (HGF) in cardiomyocytes underwent ischemia/reperfusion (I/R) is associated with downregulation of calcium sensing receptor (CaSR) mRNA expression.
Neonatal rat cardiomyocytes were isolated and randomly divided into 7 groups: control, I/R, GdCl(3), GdCl(3) + NiCl(2) + CdCl(2), GdCl(3) + LY294002, GdCl(3) + HGF, GdCl(3) + HGF + LY294002.I/R was established by incubating primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2 h, then reincubated in normal culture medium for 24 h. Cardiomyocyte apoptosis was detected by TUNEL. The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of Caspase-3, Bcl-2 and Phosphoinositide-3 Kinase (PI3K) was analyzed by Western blot.
I/R enhanced the expression of CaSR mRNA (I/R: 2.62 ± 0.41, control: 1.00 ± 0.31, P < 0.01) and cardiomyocyte apoptosis [I/R: (15.32 ± 2.54)%, control: (2.90 ± 1.45)%, P < 0.01]. GdCl(3) further increased the expression of CaSR mRNA (GdCl(3): 4.46 ± 0.62, I/R: 2.62 ± 0.41, P < 0.01) and cardiomyocyte apoptosis [GdCl(3): (25.36 ± 2.60)%, I/R: (15.32 ± 2.54)%, P < 0.01], along with upregulation of Caspase-3 (GdCl(3): 1.93 ± 0.28, I/R: 1.50 ± 0.21, P < 0.01), downregulation of Bcl-2 (GdCl(3): 0.82 ± 0.18, I/R: 1.71 ± 0.30, P < 0.01) and PI3K phosphorylation inhibition (I/R: 0.87 ± 0.08, GdCl(3): 0.61 ± 0.07, P < 0.01). Combination of GdCl(3) with LY294002 further enhanced cardiomyocytes apoptosis [GdCl(3) + LY294002: (32.6 ± 3.42)%, GdCl(3): (25.36 ± 2.60)%, P < 0.01] but did not affect CaSR mRNA expression (GdCl(3) + LY294002: 4.27 ± 0.56, GdCl(3): 4.46 ± 0.62, P > 0.05). HGF decreased I/R- and GdCl(3)-induced apoptosis [GdCl(3) + HGF: (11.8 ± 1.89)%, GdCl(3): (25.36 ± 2.60)%, P < 0.05] by suppressing Caspase-3 (GdCl(3) + HGF: 1.12 ± 0.23, (GdCl(3): 1.93 ± 0.28, P < 0.05; GdCl(3) + HGF + LY294002: 1.87 ± 0.31, GdCl(3) + LY294002: 3.86 ± 0.47, P < 0.05) and promoting Bcl-2 (GdCl(3) + HGF: 2.56 ± 0.54, GdCl(3): 0.82 ± 0.18, P < 0.05; GdCl(3) + HGF + LY294002: 1.68 ± 0.28, GdCl(3) + LY294002: 0.68 ± 0.13, P < 0.05) and PI3K phosphorylation expression (GdCl(3) + HGF: 2.87 ± 0.21, GdCl(3): 0.61 ± 0.07, P < 0.05; GdCl(3) + HGF + LY294002: 2.01 ± 0.14, GdCl(3) + LY294002: 0.44 ± 0.10, P < 0.05) in accordance with downregulation of CaSR mRNA expression (GdCl(3) + HGF: 1.46 ± 0.37, GdCl(3): 4.46 ± 0.62, P < 0.01).
HGF exerts protective role in I/R-induced apoptosis at least in part by inhibiting CaSR mRNA expression along with promoting Bcl-2, suppressing Caspase-3 expression and stimulating PI3K phosphorylation signaling pathway.
研究肝细胞生长因子（HGF）对缺血/再灌注（I/R）损伤心肌细胞的抗凋亡作用是否与钙敏感受体（CaSR）mRNA表达下调有关。
分离新生大鼠心肌细胞，随机分为7组：对照组、I/R组、GdCl₃组、GdCl₃+NiCl₂+CdCl₂组、GdCl₃+LY294002组、GdCl₃+HGF组、GdCl₃+HGF+LY294002组。通过将原代新生大鼠心室肌细胞置于模拟缺血溶液中孵育2小时，然后再置于正常培养基中孵育24小时建立I/R模型。采用TUNEL法检测心肌细胞凋亡。通过逆转录聚合酶链反应（RT-PCR）检测CaSR mRNA的表达。采用蛋白质免疫印迹法分析Caspase-3、Bcl-2和磷酸肌醇-3激酶（PI3K）的表达。
I/R增强了CaSR mRNA的表达（I/R组：2.62±0.41，对照组：1.00±0.31，P<0.01）和心肌细胞凋亡[I/R组：（15.32±2.54）%，对照组：（2.90±1.45）%，P<0.01]。GdCl₃进一步增加了CaSR mRNA的表达（GdCl₃组：4.46±0.62，I/R组：2.62±0.41，P<0.01）和心肌细胞凋亡[GdCl₃组：（25.36±2.60）%，I/R组：（15.32±2.54）%，P<0.01]，同时上调了Caspase-3（GdCl₃组：1.93±0.28，I/R组：1.50±0.21，P<0.01），下调了Bcl-2（GdCl₃组：0.82±0.18，I/R组：1.71±0.30，P<0.01）并抑制了PI3K磷酸化（I/R组：0.87±0.08，GdCl₃组：0.61±0.07，P<0.01）。GdCl₃与LY294002联合使用进一步增强了心肌细胞凋亡[GdCl₃+LY294002组：（32.6±3.42）%，GdCl₃组：（25.36±2.60）%，P<0.01]，但不影响CaSR mRNA的表达（GdCl₃+LY294002组：4.27±0.56，GdCl₃组：4.46±0.62，P>0.05）。HGF通过抑制Caspase-3（GdCl₃+HGF组：1.12±0.23，GdCl₃组：1.93±0.28，P<0.05；GdCl₃+HGF+LY294002组：1.87±0.31，GdCl₃+LY294002组：3.86±0.47，P<0.05）、促进Bcl-2（GdCl₃+HGF组：2.56±0.54，GdCl₃组：0.82±0.18，P<0.05；GdCl₃+HGF+LY294002组：1.68±0.28，GdCl₃+LY294002组：0.68±0.13，P<0.05）和PI3K磷酸化表达（GdCl₃+HGF组：2.87±0.21，GdCl₃组：0.61±0.07，P<0.05；GdCl₃+HGF+LY294002组：2.01±0.14，GdCl₃+LY294002组：0.44±0.10，P<0.05），降低了I/R和GdCl₃诱导的凋亡[GdCl₃+HGF组：（11.8±1.89）%，GdCl₃组：（25.36±2.60）%，P<0.05]，这与CaSR mRNA表达下调一致（GdCl₃+HGF组：1.46±0.37，GdCl₃组：4.46±0.62，P<0.01）。
HGF至少部分通过抑制CaSR mRNA表达、促进Bcl-2、抑制Caspase-3表达和刺激PI3K磷酸化信号通路，对I/R诱导的凋亡发挥保护作用。
