丹参酮ⅡA磺酸钠对海水浸泡人肺上皮A549细胞水通道蛋白5的影响

Effect of sulfortanshinone sodium injection on the expression and activity of aquaporin-5 of human alveolar epithelial cells after seawater exposure

目的 探讨水通道蛋白5（AQP5）在海水浸泡致细胞损伤中的变化,同时了解丹参酮Ⅱ A可能的作用机制.方法 体外传代培养肺腺癌细胞株A549细胞,接种于培养皿中,按不同海水含量分为空白对照组及15%、25%、50%、75%、100%海水组;以25%海水浸泡不同时间分为空白对照组及海水1、4、8 h组;按给予不同剂量丹参酮ⅡA干预分为空白对照组、25%海水组及25、50、75、100μg/ml丹参酮ⅡA干预4 h组.用蛋白质免疫印迹法（Western blotting）检测AQP5蛋白表达;用免疫组化法检测AQP5阳性表达.结果 Western blotting结果显示,25%与50%海水组8 h时A549细胞AQP5蛋白表达均较空白对照组明显增高（1.053±0.231、1.116±0.316比0.101±0.081,均P＜0.05）;海水1 h组AQP5表达较空白对照组稍有增加（0.306±0.125比0.288±0.098,P＞0.05）,4 h组（1.423±0.377）明显增加（P＜0.01）,8 h组AQP5表达（1.507±0.461）较4 h组略有增加,但差异无统计学意义;25μg/ml与50 μg/ml丹参酮Ⅱ A组4 h时AQP5蛋白表达较25%海水组明显减少（0.580±0.186、0.499±0.172比1.013±0.287,均P＜0.05）.免疫组化显示,25%海水4 h组AQP5阳性表达较空白对照组明显增多（7.21±0.78比0.41±0.07,P＜0.01）,染色变深;25μg/ml丹参酮ⅡA干预4 h组AQP5阳性表达（3.02±0.23）较25%海水4 h组明显减少（P＜0.05）.结论 丹参酮Ⅱ A在25μg/ml浓度时毒副作用最小,对海水浸泡A549细胞的保护作用最佳,其机制可能与抑制AQP5的过度表达有关.

Objective To explore the effects of tanshinone Ⅱ A on the activity of aquaporin-5 （AQP5）in human alveolar epithelial cells （A549） after seawater exposure and its possible mechanism. Methods Routinely cultured A549 cells were divided into different groups according to different content of seawater：blank control group, 15%, 25%, 50%, 75%, 100% seawater groups; they were divided into different groups according to the duration of exposure to 25 % seawater ： blank control group, 1, 4, 8 hours groups ;they were also divided into different groups according to concentration of tanshinone ⅡA and exposed to seawater for 4 hours： blank control group, 25% seawater group, 25, 50, 75, 100 μg/ml tanshinone ⅡA intervention groups. The expressions of AQP5 were respectively assayed by Western blotting and immunohistochemistry. Results The results of Western blotting showed that the expressions of AQP5 were remarkably higher at 8 hours of exposure to seawater in 25% and 50% seawater groups than those in blank control group （1. 053±0. 231, 1. 116±0. 316 vs. 0. 101±0. 081, both P〈0. 05）; the expression of AQP5 in 1-hour group showed a slight increase compared with blank control group （0. 306±0. 125 vs. 0. 288±0. 098,P〉0. 05）, that in 4-hour group was increased significantly （1. 423±0. 377, P〈0. 01）, and in 8-hour group （1. 507± 0. 461 ） it was slightly higher than that in 4-hour group without statistical significance. The AQP5 expression was significantly lower in tanshinone ⅡA 25 μg/ml and 50μg/ml intervention groups than that in 25% seawater group （0. 580 ± 0. 186, 0. 499 ± 0. 172 vs. 1.013 ± 0. 287, both P 〈 0. 05）. Immunohistochemistry showed that the expression of AQP5 was markedly up-regulated after A549 cells were stimulated with 25% seawater for 4 hours as compared with blank control group （7.21±0. 78 vs. 0. 41 ±0.07, P〈0.01）, but intervention of tanshinone ⅡA significantly inhibited the up-regulation of AQP5 expression （3.02±0.23） induced by 25% seawater （P〈0.05）. Conclusion The experimental results showed that tanshinone ⅡA is innocuous to A549 at a dosage of 25 μg/ml, and it can decrease the overexpression of AQP5 induced by seawater.