腹水草对人胃上皮细胞GES-1的保护作用及其对PKA、CREB、AQP1的调控研究

Protective Effect of Veronicastrum Axillare on Human Gastric Epithelial Cells(GES-1) and Its Modulation on the PKA, CREB and AQP1

[目的]研究腹水草(Veronicastrum axillare,V.axillare)含药血清对乙醇损伤的人胃上皮细胞(gastric epithelial cells-1,GES-1)的保护作用及其对环腺苷酸依赖性激酶(protein kinase A,PKA)、环磷腺苷效应元件结合蛋白(c AMP-response element binding protein,CREB)、水通道蛋白1(aquaporin 1,AQP1)表达水平的调控作用。[方法]20只雄性SD大鼠随机分为5组(n=4),连续灌胃给药14d,制备正常组(生理盐水20 m L·kg-1)、雷尼替丁组(0.027g·kg-1)以及V.axillare低、中、高剂量组(0.7、1.4、2.8 g·kg-1)含药血清。MTT法检测体外培养的GES-1细胞的活性,研究V.axillare含药血清及PKA抑制剂H-89(25μmol·m L-1)预处理对5%乙醇诱导GES-1细胞活力的影响。荧光定量RT-PCR检测PKA、CREB及AQP1 m RNA的表达水平,Western blot检测AQP1蛋白表达水平。[结果]模型组和H-89组GES-1细胞活性低于正常组,差异有统计学意义(P<0.01);乙醇造模处理显著上调PKA、CREB及下调AQP1的表达水平,差异有统计学意义(P<0.01);H-89处理显著抑制PKA和CREB m RNA表达水平(P<0.01);加入各剂量V.axillare含药血清不能显著改善H-89对PKA、CREB和AQP1 m RNA表达水平的抑制作用。V.axillare中、高剂量含药血清细胞活性高于模型组,PKA、CREB m RNA表达水平低于模型组,差异有统计学意义(P<0.01),而V.axillare中、低剂量含药血清AQP1表达水平高于模型组,差异有统计学意义(P<0.01)。[结论]V.axillare含药血清显著减轻乙醇对GES-1细胞的损伤,对GES-1细胞具有保护作用,其保护机制与下调PKA、CREB及上调AQP1的表达有关。

Objective] To study the protective effect of Veronicastrum axillare on human gastric epithelial cells（GES-1） against ethanol damage, and its modulation on the expression of PKA, CREB and AQP1. [Methods] 20 male SD rats were divided into five groups and prepared by 14 consecutive daily intragastric administration of 0.9% saline, Ranitidine（0.18%, 0.027g·kg-1）, and V. axillare decoction（0.7, 1.4, 2.8g·kg-1 respectively for low/mid/high dose groups）. The drug-loaded serums from different groups were prepared and used to culture GES-1 cells in vitro. Cell damage was induced by 5% ethanol, and the effect of drug-loaded serums and PKA inhibitor H-89（25μmol·mL-1） pretreatment on cell viabilities were measured with MTT assays. The mRNA expressions for PKA, CREB and AQP1 were measured with RT-PCR, and the AQP1 protein expression was measured with Western blot. [Results] Compared with the normal group, 5% ethanol and H-89 significantly reduced cell viability（P〈00.01）. Ethanol treatment could significantly up-regulate PKA, CREB, and down-regulate AQP1（P〈0.01）. H-89 treatment greatly inhibited the expression of PKA and CREB（P〈0.01）, and addition of V. axillare could not effectively reverse this inhibition. Compared with the model group, V. axillare medium and high dose groups significantly increased cell viability and down-regulated PKA, CREB（P〈0.01）, V. axillare medium and low dose groups significantly up-regulated AQP1（P〈0.05, P〈0.01）. [Conclusion] V. axillare-loaded serum significantly protected GES-1 cells against ethanol damage. The protection was related to the down-regulation of PKA, CREB, and the up-regulation of AQP1.