饱和氢气生理盐水通过调控B细胞淋巴瘤/白血病-2/B细胞淋巴瘤/白血病-2相关X蛋白/半胱氨酰天冬氨酸特异性蛋白酶-3通路减轻幼鼠高氧环境所致的肺损伤

Hydrogen-saturated saline alleviates lung injury induced by hyperoxia in young rats by regulating the B cell lymphoma/leukemia-2/B cell lymphoma/leukemia-2 associated X protein/cysteinyl aspartate-specific protease-3 pathway

目的探讨饱和氢气生理盐水对幼鼠高氧所致肺损伤的影响及其分子机制。方法幼年、雄性SD大鼠63只,2周龄,体重30~50 g,将其随机数字表法分为3组:高氧组(HO组,n=21),连续7 d吸入高浓度氧气并腹腔注射生理盐水0.5 ml;高氧并饱和氢气生理盐水干预组(HO+HRS组,n=21),连续7 d吸入高浓度氧气并腹腔注射饱和氢气生理盐水0.5 ml;空白对照组(C组,n=21),连续7 d吸入空气并腹腔注射生理盐水0.5 ml。7 d观察结束后处死幼鼠,收集眼球血和肺组织,检测幼鼠血浆中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的浓度,测定幼鼠肺脏组织湿/干重(W/D)比值、丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性、B细胞淋巴瘤/白血病-2相关X蛋白(bax)和B细胞淋巴瘤/白血病-2(bcl-2)蛋白的相对表达量以及半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3蛋白的表达,原位缺口末端标记法(TUNEL)观察肺脏组织细胞凋亡,计算凋亡指数(AI)。各指标组间比采用单因素方差分析。结果HO、HO+HRS组W/D比值(5.68±0.18、5.43±0.21)、血浆TNF-α[(801±183)、(611±173)pg/ml]、血浆IL-6[(519±101)、(399±95)pg/ml]、肺组织MDA含量[(8.11±2.35)、(7.01±1.56)nmol/mg蛋白]、bax蛋白的相对表达量(0.59±0.08、0.39±0.04)、Caspase-3蛋白的免疫组织化学评分[(6.01±1.01)、(3.55±1.43)分]、AI[(55.6±4.7)%、(32.5±5.4)%]高于C组,差异有统计学意义(F=7.658、9.894、10.236、5.673、120.121、5.034、85.310,P<0.05);HO组、HO+HRS组肺组织SOD含量[(9.96±2.36)、(11.21±2.26)U/mg蛋白]、bcl-2蛋白的相对表达量(0.16±0.34、0.27±0.09)、bcl-2/bax值(0.27±0.06、0.72±0.05)低于C组,差异有统计学意义(F=7.088、150.901、72.522,P<0.05);HO+HRS组W/D比值、血浆TNF-α、血浆IL-6、肺组织MDA含量、bax蛋白的相对表达量、Caspase-3蛋白的免疫组织化学评分、AI低于HO组,差异有统计学意义(F=7.658、9.894、10.236、5.673、120.121、5.034、85.310,P<0.05),肺组织SOD含量、bcl-2蛋白的相对表达量、bcl-2/bax值高于HO组,差异有统计学意义(F=7.088、150.901、72.522,P<0.05)。结论幼鼠在高氧环境下导致肺损伤,饱和氢气生理盐水通过调控bcl-2/bax/Caspase-3通路降低幼鼠高氧肺损伤。

Objective To investigate the effects of hydrogen-rich saline(HRS)on lung injury induced by hyperxia in young rats and its related molecular mechanism.Methods Totally,63 clean young male SD rats,aged 2 weeks and with body weight of 30-50 g,were randomly divided into three groups according to random number table method:hyperoxic group(HO group,n=21),inhaled high concentration of oxygen and intraperitoneally injected 0.5 ml of normal saline for consecutive 7 days;hyperoxic and hydrogen-rich saline intervention group(HO+HRS group,n=21),inhaled high concentration of oxygen and intraperitoneally injected 0.5 ml saturated hydrogen saline for consecutive 7 days;the blank control group(sham group,n=21),given air inhalation and intraperitoneal injection of 0.5 ml normal saline for consecutive 7 days.At the end of the 7-day observation,the rats were sacrificed,the eyeball blood and lung tissue were collected,and the concentrations of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in plasma of the young rats were determined.Wet/dry ratio(W/D ratio),concentration of malondialdehyde(MDA),activity of superoxide dismutase(SOD),relative expression levels of B cell lymphoma/leukemia-2 associated X protein(bax)and B cell lymphoma/leukemia-2(bcl-2)and expression levels of cysteinyl aspartate-specific protease(Caspase)-3 in lung tissues of young rats were measured.The apoptosis of lung tissue cells was observed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)method,and the apoptosis index(AI)was calculated.One-way analysis of variance(ANOVA)was used for inter-group ratio of each indicator.Results In HO group and HO+HRS group,W/D(5.68±0.18,5.43±0.21),plasma TNF-α[(801±183),(611±173)pg/ml],plasma IL-6[(519±101),(399±95)pg/ml],lung tissue MDA content[(8.11±2.35),(7.01±1.56)nmol/mg protein],relative expression level of bax protein(0.59±0.08,0.39±0.04),immunohistochemical score of Caspase-3 protein[(6.01±1.01),(3.55±1.43)points],AI[(55.6±4.7)%,(32.5±5.4)%]were significantly higher than those in the sham operation group(F=7.658,9.894,10.236,5.673,120.121,5.034,85.310,P<0.05).In the HO group and HO+HRS group,SOD content in lung tissue[(9.96±2.36),(11.21±2.26)U/mg protein],the relative expression of bcl-2 protein(0.16±0.34,0.27±0.09)and the bcl-2/bax value(0.27±0.06,0.72±0.05)were significantly lower than those in the sham operation group(F=7.088,150.901,72.522,P<0.05).As compared with the HO group,the W/D value,plasma TNF-α,plasma IL-6,the content of MDA in lung tissue,the relative expression level of bax protein,the immunohistochemical score of Caspase-3 protein and the AI in HO+HRS group were significantly decreased(F=7.658,9.894,10.236,5.673,120.121,5.034,85.310,P<0.05),the SOD content,the relative expression level of bcl-2 protein and the bcl-2/bax value in lung tissue were significantly increased(F=7.088,150.901,72.522,P<0.05).Conclusion Hyperoxic environment can cause lung injury in young rats,and hydrogen-saturated saline can reduce the lung injury in young rats by regulating the bcl-2/bax/Caspase-3 pathway.