硫化氢对尼古丁诱导的人支气管上皮细胞内质网应激及细胞凋亡的影响

Hydrogen sulfide attenuates bronchial epithelial cell apoptosis by inhibiting endoplasmic reticulum stress

目的 探讨硫化氢对尼古丁诱导的人支气管上皮细胞内质网应激及细胞凋亡的影响.方法 应用尼古丁诱导人支气管上皮细胞16HBE细胞来模拟香烟烟雾引起的细胞凋亡模型.浓度梯度分组为对照组与5、10、20、40、80 μmol/L尼古丁组,均处理72 h;时间梯度分组为对照组与尼古丁24、48、72 h组,除对照组以外尼古丁浓度均为40 μmol/L.应用Western印迹法检测尼古丁不同浓度和不同作用时间对16HBE细胞凋亡指标CCAAT/增强子结合蛋白同源蛋白（CHOP）蛋白表达的影响.为观察硫化氢对尼古丁诱导的内质网应激相关凋亡的作用,16HBE细胞分为对照组、40 μmol/L尼古丁组、100 μmol/L硫氢化钠＋40 μmol/L尼古丁组、200 μmol/L硫氢化钠＋40 μmol/L尼古丁组、400 μmol/L硫氢化钠＋40 μmol/L尼古丁组、10 mmol/L牛磺酸＋40 μmol/L尼古丁组,硫氢化钠或牛磺酸均需预孵育30 min后加入尼古丁处理72 h.应用Western印迹法检测16HBE细胞中内质网应激指标GRP78以及内质网应激相关凋亡指标剪切型兔抗半胱氨酸天冬氨酸蛋白酶（caspase）-12和CHOP蛋白表达情况,应用Hoechst 33258染色观察和分析16HBE细胞核凋亡形态学改变,计算细胞的凋亡指数.结果 尼古丁可浓度依赖性和时间依赖性地上调16HBE细胞CHOP的表达,40 μmol/L尼古丁组的CHOP与GAPDH的平均吸光度值比值显著高于对照组（1.04±0.32比0.30±0.17,P＜0.05）.200 μmol/L硫氢化钠＋40μmol/L尼古丁组的GRP78与β-肌动蛋白平均吸光度值比值（0.59 ±0.19比1.00±0.08）、剪切型caspase-12与caspase-12前体平均吸光度比值[0.06（0.01,6.06）比20.30（12.79,23.78）]、CHOP与β-肌动蛋白平均吸光度比值（0.18±0.10比0.53±0.09）均显著低于40 μmol/L尼古丁组（均P＜0.05）.200 μmol/L硫氢化钠＋40 μmol/L尼古丁组的凋亡指数（3.04±1.83比16.60 ±3.32）与10 mmol/L牛磺酸＋40 μmol/L尼古丁组的凋亡指数（4.08±2.04比16.60 ±3.32）均显著低于40 μmol/L尼古丁组（均P＜0.01）.结论 硫化氢可显著抑制尼古丁引起的人支气管上皮细胞凋亡,其机制与硫化氢缓解内质网应激,尤其是下调支气管上皮细胞中内质网应激相关凋亡指标CHOP、剪切型caspase-12有关.

Objective To explore the effects of hydrogen sulfide on nicotine-induced bronchial epithelial cell endoplasmic reticulum （ER） stress and apoptosis.Methods Nicotine was used to establish the apoptotic model in human bronchial epithelial cell line （16HBE） for mimicing the effect of cigarette smoke on apoptosis.The 16HBE cells were grouped by the concentration gradients of 0 （control）,5,10,20,40,80 μmol/L nicotine dosing.All groups were treated for 72 h.And 16HBE cells were grouped by the time gradients of 0 （control）,24,48,72 h of nicotine dosing.For control group,the nicotine concentration was 40 μmol/L.Then the protein expression level of CCAAT/enhancer binding protein homologous protein （CHOP） was measured by Western blot to define the effect of various concentrations of nicotine and different dosing periods of nicotine on the protein expression level of CHOP.For observing the role of hydrogen sulfide in ER stress-mediated apoptosis,16HBE cells were divided into 6 groups of control,40 μmol/L nicotine,100 μmol/L sodium hydrosulfide （NaHS） ＋ 40 μmol/L nicotine,200 μmol/L NaHS ＋ 40 μmol/L nicotine,400 μmol/L NaHS ＋ 40 μ mol/L nicotine and 10 mmol/L taurine ＋ 40 μmol/L nicotine.NaHS or taurine was pretreated for 30 min and then nicotine dosed for 72 h.The protein expression levels of GRP78 and ER stress-mediated apoptosis markers,such as cleaved caspase-12 and CHOP,were measured by Western blot.And chromatin dye Hoechst 33258 was used to detect the morphological changes of apoptotic 16HBE cells and apoptotic index calculated.Results Nicotine could concentration and time-dependently improve the expression of CHOP in 16HBE cells.The ratio of CHOP to average absorbance of glyceraldehyde phosphate dehydrogenase （GAPDH） was significantly higher in 40 μ mol/L nicotine group than that in control group （1.04 ± 0.32 vs 0.30 ± 0.17,P ＜ 0.05）.The ratio of GRP78 to average absorbance of β-actin （0.59 ± 0.19 vs 1.00 ±0.08）,cleaved caspase-12 to average absorbance of procaspase-12 （0.06（0.01,6.06） vs 20.30（12.79,23.78）） and CHOP to average absorbance of β-actin （0.18 ±0.10 vs 0.53 ±0.09） in 200 μmol/L NaHS ＋ 40 μmol/L nicotine group were all significantly lower than those in 40 μ mol/L nicotine group （all P ＜ 0.05）.The apoptotic index in 200 μmol/L NaHS ＋ 40 μmol/L nicotine group （3.04 ± 1.83 vs 16.60 ±3.32） and apoptotic index in 10 mmol/L taurine ＋40 μmol/L nicotine group （4.08 ±2.04 vs 16.60 ± 3.32） were significantly lower than those in 40 μ mol/L nicotine group （all P ＜ 0.01）.Conclusions NaHS exerts its protection against nicotine-induced bronchial epithelial cell apoptosis through suppressing ER stress.And the underlying mechanism may be through a down-regulation of ER stressmediated apoptosis markers of cleaved caspase-12 and CHOP.