胡椒碱抑制AngⅡ诱导大鼠气道平滑肌细胞增殖和迁移

Piperine inhibits Ang Ⅱ-induced cell proliferation and migration of rat airway smooth muscle cells

目的探究胡椒碱对血管紧张素Ⅱ(AngⅡ)对大鼠气道平滑肌细胞(ASMCs)增殖和迁移的影响。方法用改良组织块和胰蛋白酶消化联合培养原代细胞ASMCs。MTT检测AngⅡ及其受体拮抗剂losartan对细胞增殖活性的影响。AngⅡ和不同浓度胡椒碱作用ASMCs后,MTT、流式细胞术和Transwell分别检测细胞增殖、细胞周期以及细胞迁移;ERK1/2抑制剂PD98059和losartan干预后,Western blot检测cyclin D1、MMP-9、p-ERK1/2、ERK1/2和β-actin等蛋白表达。结果 AngⅡ(10^(-8)、10^(-7)、10^(-6)和10^(-5)mol/L)作用24 h后,可浓度依赖性地促进ASMCs增殖(P<0.05),其中10^(-7)mol/L AngⅡ促进效果最为显著;losartan处理则抑制AngⅡ诱导的ASMCs增殖(P<0.05)。10^(-7)mol/L AngⅡ处理组ASMCs增殖活性、S期细胞分布比例、细胞迁移数目和蛋白质(cyclin D1、MMP-9和p-ERK1/2)表达均明显增加(P<0.05);而胡椒碱(10、25、50和100μmol/L)处理可浓度依赖性地减轻AngⅡ诱导的上述效应。并且,PD98059和losartan亦能阻断AngⅡ对ASMCs p-ERK1/2、cyclin D1和MMP-9的上调作用。结论胡椒碱能通过ERK1/2通路抑制AngⅡ诱导的大鼠ASMCs增殖和迁移。

Objective To explore the effects of piperine on cell proliferation and migration in angiotensin Ⅱ （Ang Ⅱ ） -treated rat airway smooth muscle cells （ASMCs）. Methods The primary ASMCs of rats were cultured by improved tissue-piece digestion inoculation and trypsin digestion. MTT assay was used to detect the effects of Ang Ⅱ and Ang Ⅱ receptor antagonist losartan on cell proliferation activity. After treatment with Ang Ⅱ and piperine, the cell proliferation activity, the cell cycle distribution and the cell migration were detected by MTT, flow cytometry and Transwell assay respectively. ERK1/2 inhibitor PD98059 and losartan were then applied to determine the expression of cyclin D1, MMP-9, p-ERK1/2, ERK1/2, and β-actin proteins by Western blot assay. Results After 24 h culture, Ang Ⅱ treatment promoted the cell proliferative activity in rat ASMCs （P〈0. 05） , and the promotive effect of 10-7 mol/L Ang Ⅱ was the most significant. Additionally, losartan blocked the Ang Ⅱ -induced cell proliferative activity in rat ASMCs （P〈0. 05）. 10^-7 mol/L Ang Ⅱtreatment resulted in the elevated cell proliferative activity, higher S phase fraction, increased migrated cell number, and enhanced expression of cyclin D1, MMP-9 and p-ERK1/2 proteins （ P 〈 0.05 ） ; these effects were dose-dependently reversed PD98059 and losartan blocked Ang Ⅱ-induced expression of p-ERK1/2, cyclin D1 and rat ASMCs. Conclusions Piperine may inhibit Ang Ⅱ-induced cell proliferation and ERK1/2 signaling pathway in rat ASMCs.