胡智兴, 耿菊敏, 梁道明, 周轶平, 罗敏
昆明医学院药理教研室，昆明 650031；云南省疾病预防控制中心毒理室，昆明 650022；昆明医学院第二附属医院， 昆明 650101；昆明医学院云南省天然药物药理重点实验室，昆明 650031
肝细胞生长因子(hepatocyte growth factor, HGF)能对抗多种因素引起的细胞凋亡，从而起到保护细胞的作用。为 了研究HGF 对过氧化氢(H2O2)诱导的大鼠皮质神经元凋亡的对抗作用及其对凋亡的线粒体途径的影响，我们分离培养原代 大鼠皮质神经元，HGF (15~60 ng/mL)预处理24 h 后，用100 μmol/L H2O2 诱导神经元凋亡。采用MTT 比色法检测细胞存 活率；应用Hoechst 33258 染色和流式细胞仪检测神经元的凋亡；比色法检测细胞乳酸脱氢酶(lactate dehydrogenase, LDH) 漏出率和caspase-3 活性；激光共聚焦检测线粒体跨膜电位变化，Western blot 检测神经元细胞色素C 蛋白表达的变化。结 果显示，100 μmol/L H2O2 处理4 h 的大鼠神经元的细胞存活率下降为正常对照组的(53.4±7.4)%，LDH 漏出率上升为 (37.8±5.3)%，细胞凋亡率为(62.8±7.1)%，细胞caspase-3 活性显著升高(P<0.05)。与H2O2 处理组相比，用15~60 ng/mL HGF 预处理24 h 的大鼠神经元的细胞存活率升高，细胞凋亡率、LDH 漏出率以及caspase-3 活性均显著降低(P<0.05)；而 且HGF 预处理可逆转H2O2 诱导的神经元线粒体跨膜电位降低，使神经元的细胞色素C 蛋白表达减少。上述结果提示，HGF 具有对抗H2O2 诱导大鼠皮质神经元凋亡的作用，其作用机制可能与HGF抑制凋亡的线粒体途径，降低神经元caspase-3 活性 有关。
[Protection of hepatocyte growth factor against hydrogen peroxide-induced mitochondria-mediated apoptosis in rat cortical neurons.] [Ariticle in Chinese]
HU Zhi-Xing, GENG Ju-Min, LIANG Dao-Ming, ZHOU Yi-Ping, LUO Min
Department of Pharmacology, Kunming Medical University, Kunming 650031, China； Laboratory of Toxicology, Yunnan Center for Disease Control and Prevention, Kunming 650022, China； The Second Affiliated Hospital, Kunming Medical University, Kunming 650101, China； Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming 650031, China
Hepatocyte growth factor (HGF) pretreatment could protect multiple cell types from apoptosis induced by various damages including oxidative stress. The present study was designed to investigate the protective effect of HGF on rat cortical neurons against apoptosis induced by hydrogen peroxide (H2O2) in culture, and then to explore whether HGF could influence the mitochondrial pathway of apoptosis. Primary rat cortical neurons were isolated from Sprague-Dawley rats and cultured in serum free medium containing 2% B27 and Neurobasal-A. To mimic the oxidative stress damage, cortical neurons were exposed to 100 μmol/L H2O2 for 4 h. To explore the effects of HGF on the neurons subjected to H2O2 injury, cells were pretreated with HGF 15, 30, 60 ng/mL for 24 h, respectively, and then exposed to 100 μmol/L H2O2 for 4 h. The cell viability was measured by MTT colorimetric assay and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. Apoptotic cells were detected by Hoechst 33258 staining and Annexin VFITC/ PI double labeled flow cytometry. The caspase-3 activity was assessed by colorimetry. The alteration of transmembrane potential of mitochondria was determined by confocal laser scanning microscopy. The expression of cytochrome C protein was measured by Western blot analysis. The results showed that H2O2 treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apoptotic cells. Pretreatment of HGF at different concentrations (15-60 ng/mL) could remarkably increase the cell viability of neurons. Compared with that of H2O2 group (53.4%±7.4%), the cell viabilities of neurons treated with 15, 30, and 60 ng/mL HGF significantly increased to (69.3±6.4)%, (77.5±6.1)% and (82.9±9.3)% (P<0.05), respectively. HGF preincubation also evidently decreased the LDH leakage rate in cortical neurons damaged by H2O2. The results of Hoechst staining revealed that HGF pretreatment could significantly reduce the apoptotic rate of neurons. The apoptotic rate of H2O2 group was (62.8±7.1)%, while that of HGF groups decreased significantly to (34.8±8.4)%, (23.5±3.2)% and (18.6±4.5)% (P<0.05), respectively. The data from caspase-3 activity assay indicated that HGF preconditioning could also remarkably decrease the caspase-3 activity of neurons. In addition, in the presence of various concentrations of HGF, the decrease of transmembrane potential of mitochondria in neurons caused by H2O2 injury could be reversed. Moreover, as detected by Western blot analysis, HGF downregulated the expression of cytochrome C protein in neurons. These results suggest that HGF has a protective effect on rat cortical neurons against apoptosis induced by H2O2, which might be related to the inhibition of the mitochondrial apoptotic pathway and the suppression of the caspase-3 activity.
通讯作者：胡智兴 E-mail: email@example.com
胡智兴, 耿菊敏, 梁道明, 周轶平, 罗敏. 肝细胞生长因子对抗过氧化氢诱导的大鼠皮质神经元线粒体途径凋亡[J]. 生理学报 2009; 61 (3): 247-254.
HU Zhi-Xing, GENG Ju-Min, LIANG Dao-Ming, ZHOU Yi-Ping, LUO Min. [Protection of hepatocyte growth factor against hydrogen peroxide-induced mitochondria-mediated apoptosis in rat cortical neurons.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (3): 247-254 (in Chinese with English abstract).