次氯酸钠消毒剂对人支气管上皮细胞的毒性效应

Toxic effects of sodium hypochlorite as disinfectants on human bronchial epithelial cells

[背景]在不同的环境空气中存在着多种微生物,一旦易感人群与环境中的致病微生物接触,很容易被感染。为避免致病菌的传播,采取消毒的方式将环境中的致病菌杀死以阻断致病菌与人类接触,是目前最简单有效的办法。次氯酸钠(NaClO)是目前应用最广泛的消毒剂,然而NaClO应用于环境空气消毒的安全性尚不明确。[目的]建立NaClO诱导支气管上皮细胞(BEAS-2B)损伤模型,探讨NaClO消毒剂对BEAS-2B细胞的毒性作用机制。[方法]将100 mmol·L^(-1)NaClO标准溶液用0.9%NaCl溶液梯度稀释至0、25、50、100、200、400μmol·L^(-1)处理细胞,分别在15、30 min后细胞计数试剂盒-8(CCK-8)法检测细胞活性,选择0、25、50μmol·L^(-1)NaClO处理的细胞在倒置显微镜下观察细胞形态,流式细胞术Annexin V FITC/PI双染法检测细胞凋亡情况,确定最终实验浓度。透射电镜观察线粒体等细胞器的形态,JC-1染色检测细胞线粒体膜电位,Fluo-4 AM荧光探针法检测细胞内Ca^(2+)浓度,2′,7′-二氯荧光素二乙酸酯(DCFH-DA)荧光探针检测细胞总活性氧(ROS),二氢乙锭(DHE)荧光探针检测细胞线粒体ROS,试剂盒检测脂质过氧化中间产物丙二醛(MDA)。[结果]与0μmol·L^(-1)NaClO处理组细胞相比,25μmol·L^(-1)NaClO处理30 min后细胞形态变化不大,50μmol·L^(-1)NaClO处理30 min后细胞活力下降至正常细胞活力的70%(P<0.01),细胞形态开始皱缩变圆,最终选择50μmol·L^(-1)NaClO处理30 min作为后期实验浓度。实验结果发现,与0μmol·L^(-1)NaClO处理组细胞相比,NaClO处理组凋亡细胞数量增加(P<0.05),线粒体膜电位降低(P<0.01),细胞内Ca^(2+)浓度升高(P<0.05),细胞ROS水平升高(P<0.05),线粒体ROS水平升高(P<0.01),MDA含量升高(P<0.01)。[结论]本研究成功建立了NaClO诱导BEAS-2B细胞损伤模型,并发现NaClO可通过诱导BEAS-2B细胞凋亡和氧化应激导致细胞损伤。根据结果推测可能的原因有2种,一是NaClO溶于水形成次氯酸(HClO),HClO具有氧化性,进入细胞后使细胞内ROS水平升高,导致细胞氧化应激。二是HClO进入细胞直接攻击线粒体膜,导致线粒体膜内外电位失衡,Ca^(2+)外流引起细胞凋亡。

[Background]There are a variety of microorganisms in ambient air,and susceptible people can be infected once contact with pathogenic microorganisms in the environment.In order to avoid the spread of pathogenic bacteria,disinfection is the simplest and most effective way of killing pathogenic bacteria in the environment to block the contact between pathogenic bacteria and humans.Sodium hypochlorite(NaClO)is the most widely used disinfectant,but its safety in ambient air disinfection is not clear yet.[Objective]To establish a model of bronchial epithelial cell(BEAS-2B)injury induced by NaClO,and to explore the mechanism of the toxic effect of NaClO disinfectants on BEAS-2B.[Methods]Cells were treated with concentration gradients of 0,25,50,100,200,and 400μmol·L^(−1)of the diluted NaClO(100 mmol·L^(−1))standard solution,respectively,and cell activity was measured by cell counting kit-8(CCK-8)assay after 15 and 30 min.Cells treated with 0,25,and 50μmol·L^(−1)NaClO were selected to observe the cell morphology under an inverted microscope,apoptosis was determined by flow cytometry Annexin V FITC/PI double staining to determine the final experimental concentration.The morphology of organelles such as mitochondria was observed under a transmission electron microscope.Mitochondrial membrane potential of the cells was detected by JC-1 staining.Intracellular Ca^(2+)concentration was measured with a Fluo-4 AM fluorescent probe.Total cellular reactive oxygen species(ROS)was detected with a 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe,cell mitochondrial ROS with a dihydroethidium(DHE)fluorescent probe,and lipid peroxidation intermediate malondialdehyde(MDA)with a commercial kit.[Results]Compared with 0μmol·L^(−1),NaClO treatment group,cell morphology did not change a lot after 25μmol·L^(−1)NaClO treatment for 30 min,and the cells began to wrinkle and become round after 30 min treatment with 50μmol·L^(−1)NaClO,showing about 70%of normal cell viability(P<0.01).So 30 min 50μmol·L^(−1)NaClO treatment was selected for the subsequent experiment.The experimental results found that compared with the 0μmol·L^(−1)NaClO treatment group,the number of apoptotic cells increased(P<0.05),the mitochondrial membrane potential decreased(P<0.01),the intracellular Ca^(2+)concentration increased(P<0.05),the cellular ROS level increased(P<0.05),the mitochondrial ROS level increased(P<0.01),and the MDA content increased(P<0.01)in the NaClO treatment group..[Conclusion]The study has successfully established a model of BEAS-2B injury induced by NaClO,and found that NaClO can lead to cell damage by inducing apoptosis and oxidative stress in BEAS-2B cells.According to the results,there are two possible reasons.First,NaClO solves in water to form hypochlorous acid(HClO)which is oxidative and increases the intracellular ROS level after entering cells,leading to cellular oxidative stress.Second,HClO enters cells to directly attack the mitochondrial membrane,resulting in the imbalance of potential inside and outside the mitochondrial membrane,and apoptosis caused by Ca^(2+)efflux.