机械牵张预处理对机械通气性牵张诱导人肺泡Ⅱ型上皮细胞NF-κB和STAT3信号通路活化的影响

Effects of mechanical stretch preconditioning on pathological stretch-induced activation of NF-κB and STAT3 signaling pathways in human type Ⅱ alveolar epithelial cells

目的 探讨机械牵张预处理对机械通气性牵张诱导人肺泡Ⅱ型上皮细胞NF-κB和信号转导及转录激活因子3 （STAT3）信号通路活化的影响.方法 采用随机数字表法,将人肺泡Ⅱ型上皮细胞株（A549细胞）分为3组（n=24）：对照组（Ⅰ组）：常规培养,未进行机械通气性牵张;机械通气性牵张组（Ⅱ组）：给予20％应变率的机械牵张6h;机械牵张预处理组（Ⅲ组）：给予5％应变率的机械预牵张60 min后,将应变率调整为20％牵张6h.机械牵张频率0.3 Hz,载人波形为正弦波.处理结束后消化收集细胞,采用甲基噻唑蓝法测定细胞活力,采用比色法测定乳酸脱氢酶（LDH）释放率,采用ELISA法测定细胞培养液TNF-α、IL-8及高迁移率族蛋白1（HMGB1）浓度;采用Western blot法测定总NF-κB、磷酸化NF-κB、总STAT3和磷酸化STAT3表达水平,并以磷酸化NF-κB与总NF-κB的比值及磷酸化STAT3与总STAT3的比值分别反映其活化水平.结果 与Ⅰ组相比,Ⅱ组和Ⅲ组细胞活力降低,LDH释放率升高,细胞培养液TNF-α、IL-8及HMGB1浓度、NF-κB及STAT3活化水平升高（P＜0.05）;与Ⅱ组相比,Ⅲ组细胞活力升高,LDH释放率降低,细胞培养液TNF-α、IL-8及HMGB1浓度、NF-κB及STAT3活化水平降低（P＜0.05）.结论 机械牵张预处理减轻机械通气性牵张诱导人肺泡Ⅱ型上皮细胞损伤的机制与抑制NF-κB和STAT3信号通路活化有关.

Objective To evaluate the effects of mechanical stretch preconditioning on pathological stretch-induced activation of nuclear factor kappa B （NF-κB） and signal transducer and activator of transcription 3 （STAT3） signaling pathways in human type Ⅱ alveolar epithelial cells.Methods Human type Ⅱ alveolar epithelial cell line A549 cells cultured in vitro were randomly divided into 3 groups （n =24 each） using a random number table： control group （group Ⅰ）, pathological stretch group （group Ⅱ）, and mechanical stretch preconditioning group （group Ⅲ）.In group Ⅰ , A549 cells were cultured routinely without receiving pathological stretch.In group Ⅱ , A549 cells were exposed to 20% cyclic stretch at 0.3 Hz for 6 h.In group Ⅲ , A549 cells were exposed to 5% cyclic stretch at 0.3 Hz for 60 min, and then exposed to 20% cyclic stretch at 0.3 Hz for 6 h.After the end of the treatment, the cells were collected for determination of the cell viability （by methyl thiazolyl tetrazolium assay） and lactate dehydrogeuase （LDH）release （by colorimetric method）.The concentrations of tumor necrosis factor-alpha （TNF-α）,interleukin-8 （IL-8） and high mobility group box 1 （HMGB1） in the culture medium were detected using enzyme linked immunosorbent assay.The expression of total NF-κB, phosphorylated NF-κB, total STAT3 and phosphorylated STAT3 was detected using Western blot.The ratios of phosphorylated NF-κB to total NF-κB and phosphorylated STAT3 to total STAT3 were calculated to reflect the activation.Results Compared with group Ⅰ , the cell viability was significantly decreased, the amount of LDH released was increased, and the concentrations of TNF-α, IL-8 and HMGB1, and activation of NF-κB and STAT3 were increased in Ⅱ and Ⅲ groups.Compared with group Ⅱ , the cell viability was significantly increased, the amount of LDH released was decreased, and the concentrations of TNF-α, IL-8 and HMGB1, and activation of NF-κB and STAT3 were decreased in group Ⅲ.Conclusion The mechanism by which mechanical stretch preconditioning attenuates pathological stretch-induced damage to human type Ⅱ alveolar epithelial cells is related to inhibited activation of NF-κB and STAT3 signaling pathways.