Yadav Subodh K, Sharma Sanjeev K, Farooque Abdullah, Kaushik Gaurav, Kaur Balwinder, Pathak Chander M, Dwarakanath Bilikere S, Khanduja Krishan L
Department of Biophysics, PGIMER Chandigarh, Chandigarh, 160012, India.
Present address: Department of CSIC, PGIMER Chandigarh, Chandigarh, 160012, India.
Lipids Health Dis. 2016 Aug 15;15(1):129. doi: 10.1186/s12944-016-0300-x.
Smoking is one of the leading causes of millions of deaths worldwide. During cigarette smoking, most affected and highly exposed cells are the alveolar epithelium and generated oxidative stress in these cells leads to death and damage. Several studies suggested that oxidative stress causes membrane remodeling via Phospholipase A2s but in the case of cigarette smokers, mechanistically study is not yet fully defined. In view of present perspective, we evaluated the involvement of cytosolic phospholipase A2 (cPLA2) IVA as therapeutic target in cigarette smoke induced pathologies in transformed type I and type II alveolar epithelial cells.
Transformed type I (WI26) and type II (A549) alveolar epithelial cells were used for the present study. Cigarette smoke condensate (CSC) was prepared from most commonly used cigarette (Gold Flake with filter) by the Indian population. CSC-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichlorodihydrofluorescin diacetate (DCFH-DA), cell membrane integrity using fluorescein diacetate (FDA) and ethidium bromide (EtBr) staining, super oxide dismutase (SOD) levels, cPLA2 activity and molecular involvement of specific cPLA2s at selected 24 h time period.
CSC-induced response on both type of epithelial cells shown significantly reduction in cell viability, declined membrane integrity, with differential escalation of ROS levels in the range of 1.5-15 folds and pointedly increased cPLA2 activity (p < 0.05). Likewise, we observed distinction antioxidant potential in these two types of lineages as type I cells had considerably higher SOD levels when compared to type II cells (p < 0.05). Further molecular expression of all cPLA2s increased significantly in a dose dependent manner, specifically cytosolic phospholipase A2 IVA with maximum manifestation of 3.8 folds. Interestingly, CSC-induced ROS levels and cPLA2s expression were relatively higher in A549 cells as compared to WI26 cells.
The present study indicates that among all cPLA2s, specific cPLA2 IVA are the main enzymes involved in cigarette smoke induced anomalies in type I and type II lung epithelial cells and targeting them holds tremendous possibilities in cigarette smoke induced lung pathologies.
吸烟是全球数百万人死亡的主要原因之一。在吸烟过程中，受影响最大且暴露程度最高的细胞是肺泡上皮细胞，这些细胞中产生的氧化应激会导致细胞死亡和损伤。多项研究表明，氧化应激通过磷脂酶A2导致膜重塑，但对于吸烟者而言，其作用机制尚未完全明确。基于当前的研究视角，我们评估了胞质磷脂酶A2（cPLA2）IVA作为治疗靶点在香烟烟雾诱导的I型和II型肺泡上皮细胞病变中的作用。
本研究使用了转化的I型（WI26）和II型（A549）肺泡上皮细胞。香烟烟雾冷凝物（CSC）由印度人群最常用的香烟（带过滤嘴的金叶烟）制备而成。通过MTT法检测细胞活力、使用2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）测量活性氧（ROS）、使用荧光素二乙酸酯（FDA）和溴化乙锭（EtBr）染色检测细胞膜完整性、检测超氧化物歧化酶（SOD）水平、cPLA2活性以及在选定的24小时时间段内特定cPLA2的分子参与情况，评估CSC诱导的分子变化。
CSC对两种类型上皮细胞的诱导反应均显示细胞活力显著降低、膜完整性下降，ROS水平在1.5至15倍范围内有不同程度的升高，且cPLA2活性明显增加（p < 0.05）。同样，我们观察到这两种细胞系的抗氧化潜力存在差异，I型细胞的SOD水平明显高于II型细胞（p < 0.05）。所有cPLA2的进一步分子表达均呈剂量依赖性显著增加，特别是胞质磷脂酶A2 IVA，最大表现为3.8倍。有趣的是，与WI26细胞相比，CSC诱导的A549细胞中的ROS水平和cPLA2表达相对更高。
本研究表明，在所有cPLA2中，特定的cPLA2 IVA是香烟烟雾诱导I型和II型肺上皮细胞异常所涉及的主要酶，针对它们在香烟烟雾诱导的肺部病变中具有巨大的治疗潜力。
