Changes In Expression Of S100A6/RAGE Induced By Cigarette Smoke And Radon Inhalation

Objective:To observe changes in expression of receptor for advanced glycation endproducts(RAGE) and calcium binding protein S100A6 in lung tissue of rats and bronchial epithelial cells BEAS-2B after long term exposure to cigarette smoke and radon gas.Methods:1. In vivo experiment: (1) Exposure to cigarette smoke: 27 healthy male Wistar rats were divided into 9 groups in random with 3 rats in each group, 5% smoke for 5 weeks (5%Sm-5W), 15 weeks (5%Sm-15W), 30 weeks(5%Sm-30W), 60% smoke for 5 weeks (60%Sm-5W), 15 weeks (60%Sm-15W), 30 weeks (60%Sm-30W) and 3 control groups in 3 time points (CSm). Rats were exposed to cigarette smoke in a gas inhalation box for twice a day, 30 min each and 5 days a week. (2) Exposure to radon: 12 healthy male Wistar rats were divided into the control group and 3 exposure groups of low, middle and high dose with 3 rats in each group. Rats were exposed to radon and its progeny with a multifunction ecological radon room at a constant level of 100000Bq/KL to a cumulative dose of 64, 121 and 236WLM respectively. (3) Combined exposure to cigarette smoke and radon: 12 healthy male rats were divided into control group and 3 exposure groups of low, middle and high dose, with 3 rats in each group. Cigarette smoke concentration was 60% and radon accumulation dose was up to 17, 34 and 68 WLM. (4) All the rats were weighed every two weeks. After exposure rats were sacrificed, the right lung was fixed for histological observation and immunohistology of RAGE and S100A6, the left lung was used for protein extraction and RAGE/S100A6 protein expression with western blot.2. In vitro experiment: Human bronchial epithelial cells BEAS-2B were divided into the control group, 5% cigarette smoke group(5%T) and 20% cigarette smoke group(20%T), with 3 parallels in each group. Cells in exponential growth phase were planted onto transwell membrane in 1×10~5/ml for 1.5ml per well before exposure. Cells were exposed to cigarette smoke for 10 min once every two days in a gas inhalation box with oxygen concentration of 21% and flow rate of 10ml/min. After the exposure cells were trypsinized into dishes for further growth and then exposed to smoke gas, which was noted as the first exposure passage (5%T-1/20%T-1) and continued until to the eight passage (5%T-8/20%T-8). Plating efficiency, serum resistance, and soft agar colony formation were detected in every passage and protein expression of RAGE and S100A6 in each group was measured by western blot.Results:(1) In rats exposed to radon, inflammatory cell infiltration, thickening of lung interval, hyperaemia, hydropsia of the interstitium were observed. Epithelial cells shedding into bronchial intracavitary and part pulmonary emphysema were observed in 60% cigarette smoking group. Macrophagocyte and pulmonary emphysema were found in the combined exposure groups. Body weight increase slowed down in cigarette smoking groups especially the 60% group. Compared with the control and smoking groups, the body weight in combined exposure groups decreased significantly but not in radon exposure groups. Organ coefficient of testis decreased in all groups but the liver coefficient increased only in high dose radon group. (2) S100A6 expression increased in 5%Sm-5W, 5%Sm-15W, and 5%Sm-30W groups compared with the control group, with a dose related increase in the 60%Sm-15W and 60%Sm-30W groups. The expression of S100A6 increased in all radon exposure groups (64 WLM, 121WLM, 236 WLM) with a highest amount in the 121WLM group. In the combined exposure groups, S100A6 also increased especially in the L-15W group. In in vitro experiment, the expression of S100A6 in human bronchial epithelial cells of BEAS-2B was revealed inhanced gradually along with the passages in a dose-response manner after exposure to smoke gas. (3) The expression of RAGE in lung tissue of rats changed in an independent way, with an increase in 5% cigarette smoking group and all radon groups, and first increase and then decrease in the combined exposure group. (4) Human bronchial epithelial cells were malignantly transformed after exposure to cigarette smoke, as identified by cell morphology, increased serum resistance and plating efficiency, and acquired ability of growth in soft agar. Conclusions:1. Pathology of lung tissue in rats changed significantly after exposure to cigarette smoke and radon, and pulmonary emphysema was observed in the combined wxposure group. Decrease in body weight gain and organ coefficient of testis and liver reflected general toxic effect of cigarette smoke and radon inhalation. 2. S100A6 expression in lung tissue of rats increased in a dose-response manner after exposure to cigarette smoke, radon and combined inhalation, which might be used as an early biomarker in lung carcinogenesis induced by cigarette smoke and radon. 3. Human bronchial epithelial cells could be malignantly transformed by smoke gas, which could be used as a cell model in future study on lung carcinogenesis induced by cigarette smoke.