N-乙酰半胱氨酸在百草枯诱导A549细胞转分化过程中的保护作用  

N-Acetyl-cysteine protects A549 cells in paraquat-induced epithelial-mesenchymal transition

作　　者：宋扬 孙昊 余国琴 张晓敏 吴雨璇 王军[3] 张劲松 Song Yang;Sun Hao;Yu Guoqin;Zhang Xiaomin;Wu Yuxuan;Wang Jun;Zhang Jinsong(Department of Emergency Medicine,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China;Department of Neurology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China;Key Lab of Modern Toxicology (NJMU),Ministry of Education,Department of Toxicology,School of Public Health,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学第一附属医院急诊医学科,210029 [2]南京医科大学第一附属医院神经内科,210029 [3]南京医科大学公共卫生学院卫生毒理系,现代毒理学教育部重点实验室,211166

出　　处：《中华急诊医学杂志》2019年第6期712-716,共5页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金(81671899,81772057);江苏省自然科学基金(BK20171500);江苏省医学创新团队(CXTDA2017007).

摘　　要：目的探讨N-乙酰半胱氨酸(N-Acetyl-cysteine, NAC)在百草枯(paraquat, PQ)诱导人非小细胞肺癌细胞(A549细胞)发生上皮间充质细胞转分化(epithelial-mesenchymal transition,EMT)过程中的保护作用以及机制。方法利用A549细胞暴露于不同浓度的PQ溶液,构建拟肺泡上皮细胞EMT模型。分别于第0,2,4,6天收获细胞。观察NAC干预效果时,在PQ暴露前2 h加入10 mmol/L NAC孵育细胞。光镜下观察A549细胞的形态学改变,实时定量PCR(Real-time quantitative PCR, RT-qPCR)和Western-blotting技术检测EMT标记物E-cadherin和α-SMA的表达变化以及TGF-β1的表达水平。结果30 μmol/L PQ染毒A549细胞4 d后建立EMT模型,与对照组相比,PQ组A549细胞表现出典型的形态学改变,细胞由鹅卵石样的上皮细胞外观特征向纺锤形的间质细胞样外观特征改变。Western-blotting结果表明PQ组的A549细胞的TGF-β1、α-SMA蛋白水平表达明显高于对照组,E-cadherin蛋白水平表达低于对照组(均P<0.05)。10 mmol/L NAC预孵育可以有效逆转PQ暴露引起的上述改变,表现为EMT指标E-cadherin升高而α-SMA下降(均P<0.05),且形态学也发现在应用NAC后,纺锤形的间质细胞样的细胞减少且细胞数量有所增加。结论通过细胞实验证明,PQ暴露可以引起上皮细胞发生上皮间充质细胞转分化,NAC在此过程中具有细胞保护作用。Objective To investigate the regulatory mechanism of Paraquat (PQ)-induced epithelial-mesenchymal transition (EMT) and the protective mechanism of N-acetyl-cysteine (NAC) in PQ-induced A549 cells by EMT markers (E-cadherin and α-smooth muscle actin). Methods EMT model was established by 30 μmol/L PQ-exposed A549 cells for 4 d. The application of 10 mmol/L NAC was incubated with A549 cells for 2 h in advance. Morphological changes of A549 cells were observed under light microscope. Western blot was used to detect the expression levels of TGF-β1, E-cadherin and α-smooth muscle actin. Results In PQ-induced EMT model, the PQ group showed typical morphological changes, and the cells changed from cobblestone-like epithelial appearance to spindle-shaped mesenchymal-like appearance compared to the control group. Western blotting showed that the protein levels of TGF-β1 and α-smooth muscle actin in the PQ group were significantly increased and the protein levels of E-cadherin were decreased in the PQ group (all P<0.05). NAC preincubation can effectively reverse the above changes caused by PQ exposure: the protein levels of E-cadherin and α-smooth muscle actin were significantly increased and decreased, respectively and morphological changes showed more cobblestone-like epithelial appearance. Conclusion Cell experiments showed that PQ exposure can cause EMT in epithelial cells, and NAC has a protective effect in this process. Thus, it may provide new ideas and strategies for the treatment of pulmonary fibrosis caused by PQ poisoning in clinical practice.

关 键 词：百草枯 N-乙酰半胱氨酸 TGF-Β1 上皮间充质细胞转分化 

分 类 号：R595.4[医药卫生—内科学]