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目的:观察苓桂术甘汤含药血清对转化生长因子-β1(Transforming growth factor,TGF-β1)诱导的大鼠心肌细胞H9c2凋亡的影响,探讨苓桂术甘汤抗心肌细胞凋亡的机制。方法 :分别用苓桂术甘汤(2.1、4.2、8.4 g/kg)大鼠含药血清(10%)与大鼠心肌细胞H9c2共培养12 h后,加入TGF-β1(20 ng/ml)继续培养12 h。用MTT法检测心肌细胞H9c2的存活率、Hoechst 33258荧光染色法观察细胞形态学变化、流式细胞术(Annexin V/PI双染色法)检测细胞凋亡率、ELISA法检测H9c2细胞半胱氨酸蛋白酶-3和半胱氨酸蛋白酶-8的表达量。结果:与正常对照组比较,TGF-β1模型组H9c2细胞存活率明显降低,细胞染色质边聚,细胞核致密浓染且固缩并呈现蓝白色,细胞凋亡率明显升高,半胱氨酸蛋白酶-3、半胱氨酸蛋白酶-8表达量明显升高;与TGF-β1模型组比较,苓桂术甘汤(4.2、8.4 g/kg)含药血清组细胞存活率均明显升高,苓桂术甘汤(2.1、4.2、8.4 g/kg)含药血清组凋亡率由(25.7±1.1)%分别下降至(22.2±1.0)%、(14.7±0.9)%和(10.1±1.3)%;苓桂术甘汤(2.1、4.2、8.4 g/kg)含药血清组半胱氨酸蛋白酶-3的表达量由(33.1±1.0)pmol/l分别明显下降至28.9±2.0、27.5±2.8和(24.2±2.3)pmol/L,苓桂术甘汤(4.2、8.4 g/kg)含药血清组半胱氨酸蛋白酶-8的表达量由(20.8±2.4)pmol/L分别明显下降至(16.4±1.3)、(13.7±0.7)pmol/L。结论:苓桂术甘汤含药血清能够明显抑制TGF-β1诱导的大鼠心肌细胞H9c2的凋亡,该作用与其抑制半胱氨酸蛋白酶-3和半胱氨酸蛋白酶-8表达有关。
AIM: To observe the effect of Lingguizhugan decoction on the apoptosis of H9c2 induced by transforming growth factor-β1 (TGF-β1) in rat cardiomyocytes, and to explore the mechanism of Lingguizhugan decoction on cardiomyocyte apoptosis. Methods: Co-cultured rat myocardial cells H9c2 with the serum containing 10% (2.1,4.2,8.4 g / kg) of Lingguizhugan Decoction for 12 h, and then treated with TGF-β1 (20 ng / ml) Cultivate for 12 h. The survival rate of H9c2 cells was detected by MTT assay. The morphological changes of H9c2 cells were observed by Hoechst 33258 fluorescence staining. The apoptosis rate was detected by flow cytometry (Annexin V / PI double staining), and the cysteine Protease-3 and Caspase-8 expression levels. Results: Compared with the normal control group, the survival rate of H9c2 cells in TGF-β1 model group was significantly lower than that in normal control group. The chromatin of the cells aggregated, the nucleus was densely stained and blue-white, the cell apoptosis rate was significantly increased, and the cysteine Compared with TGF-β1 model group, the survival rate of cells in serum of Lingguizhugan Decoction (4.2, 8.4 g / kg) were significantly increased, and the expression of protease-3 and caspase- The apoptotic rates of Shugan Decoction (2.1,4.2,8.4 g / kg) decreased from (25.7 ± 1.1)% to (22.2 ± 1.0)%, (14.7 ± 0.9)% and (10.1 ± 1.3)%, ; The expression of caspase-3 in the serum containing Lingzhi Shugan Decoction (2.1,4.2,8.4 g / kg) decreased significantly from (33.1 ± 1.0) pmol / l to 28.9 ± 2.0,27.5 ± 2.8 and (24.2 ± 2.3) pmol / L, and the expression of caspase-8 in the serum of Lingguizhugan Decoction (4.2,8.4 g / kg) was significantly decreased from (20.8 ± 2.4) pmol / L to ± 1.3), (13.7 ± 0.7) pmol / L. Conclusion: The serum of Lingguizhugan Decoction can significantly inhibit the apoptosis of H9c2 induced by TGF-β1 in rat cardiomyocytes, which is related to the inhibition of the expression of caspase-3 and caspase-8.