Hu Zhi-Xing, Geng Ju-Min, Liang Dao-Ming, Zhou Yi-Ping, Luo Min
Department of Pharmacology, Kunming Medical University, Kunming, China.
Sheng Li Xue Bao. 2009 Jun 25;61(3):247-54.
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 mumol/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 mumol/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 V-FITC/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.
肝细胞生长因子(HGF)预处理可保护多种细胞类型免受包括氧化应激在内的各种损伤所诱导的细胞凋亡。本研究旨在探讨HGF对培养的大鼠皮质神经元免受过氧化氢(H2O2)诱导的细胞凋亡的保护作用,进而探究HGF是否会影响细胞凋亡的线粒体途径。从Sprague-Dawley大鼠分离出原代大鼠皮质神经元,并在含有2%B27和Neurobasal-A的无血清培养基中培养。为模拟氧化应激损伤,将皮质神经元暴露于100μmol/L H2O2中4小时。为探究HGF对遭受H2O2损伤的神经元的影响,细胞分别用15、30、60 ng/mL HGF预处理24小时,然后暴露于100μmol/L H2O2中4小时。通过MTT比色法测定细胞活力,通过乳酸脱氢酶(LDH)泄漏率评估细胞损伤。通过Hoechst 33258染色和Annexin V-FITC/PI双标记流式细胞术检测凋亡细胞。通过比色法评估caspase-3活性。通过共聚焦激光扫描显微镜测定线粒体跨膜电位的变化。通过蛋白质印迹分析测量细胞色素C蛋白的表达。结果显示,H2O2处理显著降低细胞活力,增加LDH泄漏率和凋亡细胞百分比。不同浓度(15 - 60 ng/mL)的HGF预处理可显著提高神经元的细胞活力。与H2O2组(53.4%±7.4%)相比,用15、30和60 ng/mL HGF处理的神经元细胞活力分别显著提高至(69.3±6.4)%、(77.5±6.1)%和(82.9±9.3)%(P<0.05)。HGF预孵育也明显降低了受H2O2损伤的皮质神经元中的LDH泄漏率。Hoechst染色结果显示,HGF预处理可显著降低神经元的凋亡率。H2O2组的凋亡率为(62.8±7.1)%,而HGF组的凋亡率分别显著降低至(34.8±8.4)%、(23.5±3.2)%和(18.6±4.5)%(P<0.05)。来自caspase-3活性测定的数据表明,HGF预处理也可显著降低神经元的caspase-3活性。此外,在存在各种浓度HGF的情况下,H2O2损伤引起的神经元线粒体跨膜电位的降低可被逆转。而且,通过蛋白质印迹分析检测,HGF下调了神经元中细胞色素C蛋白的表达。这些结果表明,HGF对大鼠皮质神经元免受H2O2诱导的细胞凋亡具有保护作用,这可能与抑制线粒体凋亡途径和抑制caspase-3活性有关。