摘要目的:研究氢气对肺腺癌A549细胞凋亡的影响及作用机制。方法:本研究为实验研究。常规培养A549细胞，当细胞数量达到实验需要时，采用随机数字表法将细胞分为对照组、20%氢气组、40%氢气组、60%氢气组。对各氢气组细胞分别使用20%、40%、60%氢气干预，干预结束后收集各组细胞使用流式细胞仪检测细胞凋亡情况。使用蛋白质印迹法检测各组A549细胞中X连锁凋亡抑制因子(XLP2)、胱天蛋白酶3(Caspase3)、胱天蛋白酶7(Caspase7)的表达。9只5~6周雄性裸鼠皮下成瘤造模，采用随机数字表法将荷瘤鼠分为空白对照组、氢气组(给予60%浓度氢气吸入，1次/d，4 h/次)、顺铂组(4 mg/kg腹腔注射顺铂，1次/周)，每组3只。免疫组织化学比较3组肿瘤组织中XLP2、Caspase3、Caspase7的表达。结果:对照组、20%氢气组、40%氢气组、60%氢气组细胞凋亡率分别为(1.270±0.096)%、(2.250±0.076)%、(3.080±0.040)%、(7.760±0.050)%，差异有统计学意义( F＝5 186.17， P＝0.001)；与对照组比较，其余3组的细胞凋亡率均升高(均 P<0.001)。对照组、20%氢气组、40%氢气组、60%氢气组中XLP2的相对表达量分别为(0.355±0.012)、(0.300±0.016)、(0.303±0.019)、(0.231±0.022)，差异有统计学意义( F＝7.33， P＝0.011)；与对照组比较，其余3组XLP2的相对表达量均降低(均 P<0.05)。对照组、20%氢气组、40%氢气组、60%氢气组中Caspase7的相对表达量分别为(0.123±0.008)、(0.225±0.009)、(0.335±0.002)、(0.449±0.007)，差异有统计学意义( F＝1 181.95， P<0.001)；与对照组比较，其余3组XLP2的相对表达量均增高(均 P<0.05)。对照组、20%氢气组、40%氢气组、60%氢气组中Caspase3的相对表达量分别为(1.174±0.130)、(1.136±0.167)、(1.194±0.111)、(1.169±0.179)，差异无统计学意义( F＝0.05， P＝0.984)。XLP2主要在细胞质中表达，氢气组、顺铂组、空白对照组中XLP2的表达量分别为(0.041±0.012)、(0.024±0.001)、(0.090±0.033)，差异有统计学意义( F＝8.44， P＝0.018)；氢气组、顺铂组XLP2的表达量均较空白对照组降低(均 P<0.05)，氢气组与顺铂组之间XLP2的表达量比较差异无统计学意义( P>0.05)。Caspase7主要在细胞质中表达，在氢气组、顺铂组、空白对照组中Caspase7的表达量分别为(0.204±0.004)、(0.234±0.007)、(0.122±0.003)，差异有统计学意义( F＝372.42， P<0.001)；氢气组、顺铂组Caspase7的表达量均较空白对照组升高(均 P<0.05)，顺铂组Caspase7的表达量较氢气组升高( P<0.05)。Caspase3主要在胞浆中表达，空白对照组、氢气组、顺铂组中Caspase3的表达量分别为(0.255±0.024)、(0.273±0.109)、(0.287±0.046)，差异无统计学意义( F＝0.16， P＝0.853)。 结论:氢气可促进肺腺癌A549细胞凋亡，降低XLP2在肺腺癌A549细胞和瘤组织中的表达，提高Caspase7在肺腺癌A549细胞和瘤组织中表达。氢气可能通过影响XLP2和Caspase7蛋白表达量，发挥其促进肺癌A549细胞凋亡的作用。

abstractsObjective:To investigate the effect of hydrogen on regulating the apoptosis of the lung adenocarcinoma cell line A549 and the underlying mechanism.Methods:It was an experimental study.A549 cells were routinely cultured to required cell number, and then they were treated with blank control, 20% hydrogen, 40% hydrogen and 60% hydrogen.Cell apoptosis was determined by flow cytometry.Protein expressions of X-linked inhibitor of apoptosis (XLP2), Caspase-3 and Caspase-7 in A549 cells were detected by Western blot.A total of 9 male nude mice with 5-6 weeks old were subjected to subcutaneous xenograft modeling.They were randomly divided into blank control group, hydrogen group (4-h inhalation of 60% hydrogen daily) and cisplatin group (intraperitoneal injection of 4 mg/kg cisplatin once a week), with 3 mice per group.After sacrifice, positive expressions of XLP2, Caspase-3 and Caspase-7 in tumor xenografts were detected by immunohistochemical staining.Results:A significant difference was detected in the apoptosis rate of A549 cells treated with blank control, 20% hydrogen, 40% hydrogen and 60% hydrogen ( F=5 186.17, P=0.001). The apoptosis rate of A549 cells induced with 20%, 40% and 60% hydrogen was significantly higher than that of blank control ([2.250±0.076]%, [3.080±0.040]% and [7.760±0.050]% vs [1.270±0.096]%; all P<0.001). A significant difference was detected in the protein expression of XLP2 in A549 cells treated with blank control, 20% hydrogen, 40% hydrogen and 60% hydrogen ( F=7.33, P=0.011). The protein expression of XLP2 in A549 cells induced with 20%, 40% and 60% hydrogen was significantly lower than that of blank control ([0.300±0.016], [0.303±0.019] and [0.231±0.022] vs [0.355±0.012]; all P<0.05). A significant difference was detected in the protein expression of Caspase-7 in A549 cells treated with blank control, 20% hydrogen, 40% hydrogen and 60% hydrogen ( F=1 181.95, P<0.001). The protein expression of Caspase-7 in A549 cells induced with 20%, 40% and 60% hydrogen was significantly higher than that of blank control ([0.225±0.009], [0.335±0.002] and [0.449±0.007] vs [0.123±0.008]; all P<0.05). There was no significant difference in the expression of Caspase-3 in A549 cells induced with blank control, and 20%, 40% and 60% hydrogen ([1.174±0.130], [1.136±0.167], [1.194±0.111] and [1.169±0.179], respectively; F=0.05, P=0.984). XLP2 was mainly expressed in the cytoplasm.There was a significant difference in the cytoplasmic expression of XLP2 among rats in hydrogen group, cisplatin group and blank control group ( F=8.44, P=0.018). Its cytoplasmic expression in hydrogen group and cisplatin group was significantly lower than that in blank control group ([0.041±0.012], [0.024±0.001] vs [0.090±0.033]; both P<0.05), which was similar between the former two groups ( P>0.05). Caspase-7 was mainly expressed in the cytoplasm.There was a significant difference in the cytoplasmic expression of Caspase-7 among rats in hydrogen group, cisplatin group and blank control group ( F=372.42, P<0.001). Cisplatin group was significantly higher than that in hydrogen group ([0.234±0.007] vs [0.204 ±0.004]; P<0.05), which was significantly higher in hydrogen group than in blank control group ([0.204±0.004] vs [0.122±0.003]; P<0.05). Caspase-3 was mainly expressed in the cytoplasm.There was no significant difference in the expression of Caspase-3 among rats in hydrogen group and cisplatin group ([0.255±0.024], [0.273±0.109] and [0.287±0.046], respectively; F=0.16, P=0.853). Conclusions:Hydrogen can promote the apoptosis of the lung adenocarcinoma cell line A549, reduce the expression of XLP2 in lung adenocarcinoma cell line A549 and tumor tissues, and increase the expression of Caspase7.Hydrogen induces the apoptosis of the lung adenocarcinoma cell line A549 by downregulating XLP2 and upregulating Caspase-7.