白藜芦醇苷对内毒素诱导BEAS-2B细胞中Clara细胞分泌蛋白表达和Ca^(2+)浓度的影响

Effects of polydatin on Clara cell secretory protein and Ca^(2+) in LPS-induced activation of BEAS-2B cells

目的探讨白藜芦醇苷(PD)对内毒素(LPS)诱导的人支气管上皮细胞BEAS-2B中Clara细胞分泌蛋白(CCSP)和Ca2+的影响。方法 MTT法筛选PD和LPS最佳浓度;复制LPS诱导的人支气管上皮细胞炎症反应模型,实验分为正常对照组、LPS模型组、LPS+PD组、PD+LPS组及PD对照组。real-time PCR法检测CCSP mRNA的表达量,Western blotting检测CCSP蛋白含量,流式细胞仪检测细胞内Ca2+浓度。结果经过MTT法筛选,PD的最适浓度为0.5 mmol·L-1,LPS的最适浓度为100 mg·L-1。各组CCSP mRNA和蛋白表达情况为:与正常对照组相比,LPS模型组的表达量显著减少(P<0.01);与LPS模型组相比,LPS+PD组和PD+LPS组的表达量均显著增多(P<0.01),且PD+LPS组的表达量增加更多(P<0.01)。各组Ca2+浓度检测结果:LPS+PD组和PD+LPS组的荧光强度显著低于LPS模型组(P<0.01),PD+LPS组的荧光强度更低(P<0.05)。结论在LPS诱导的BEAS-2B细胞中,PD可促进CCSP mRNA和蛋白的表达,降低胞内Ca2+浓度,预防性应用的效果明显优于治疗组。

AIM To explore the effects of polydatin （PD） on Clara cell secretory protein and Ca2＋ in LPS-induced activation of bronchial epithelial cell BEAS-2B. METHODS The optimal concentrations of PD and LPS were assayed by MTF method. The model of LPS-induced inflammation in BEAS-2B was copied. The cells were divided into five groups： normal control group, LPS group, LPS ＋ PD group, PD ＋ LPS group, and PD control group. The expression of CCSP mRNA was determined by real-time PCR, the expression of CCSP protein was determined by Western blotting, the concentration of intracellular Ca2＋ was detected by flow cytometry. RESULTS The appropriate concentration of PD was 0.5 mmol·L-1 and the optimal concentration of LPS was 100 mg·L-1. CCSP mRNA and protein expressions of the LPS group were less （P 〈 0.01） than the normal control group, while CCSP mRNA and protein expressions of the LPS ＋ PD group and the PD ＋ LPS group were more than the LPS group （P 〈 0.01）, those of the PD ＋ LPS group were the most （P 〈 0.01）. The results of Ca2＋ concentration were： fluorescence intensity of the LPS ＋ PD group and the PD ＋ LPS group was lower than the LPS group （P 〈 0.01）, while the PD ＋ LPS group was the lowest （P 〈 0.05）. CONCLUSION PD plays a crucial role in lung protection by contributing to CCSP gene and protein expression and reducing intracellular Ca2＋ concentration. A more pronounced effect was shown in prophylactic pre-treatment group.