Apelin通过Akt/eNOS机制促进肺血管内皮细胞一氧化氮释放

Apelin promotes NO release involving the mechanism of Akt/eNOS in pulmonary endothelial cells

目的探讨Apelin对肺血管内皮细胞释放一氧化氮(NO)的影响及其机制。方法培养新生鼠肺微血管内皮细胞(PMVECs)。(1)以1×105个/孔密度接种至24孔板,80%融合后,无血清培养24 h。分为2组:Apelin组,加入Apelin至终浓度为10-8mol/L;对照组,加入等量无血清DMEM培养液。每组设立4个复孔。继续培养,并分别在0 min、2 min、5 min、15 min、30 min时间点取培养液测NO浓度。(2)以5×105个/孔密度接种至6孔板,80%融合后,无血清培养24 h。分为3组:Apelin组,加入Apelin至终浓度为10-8mol/L。Apelin+Akt抑制剂(Akt inhibitor)组,加入Apelin前用5μmol/L浓度的Akt抑制剂预处理30 min。对照组加入等量DMEM培液。每组设立3复孔。继续培养5 min后立即提取总蛋白,用于Western blot检测磷酸化Akt和磷酸化内皮型一氧化氮合酶(eNOS)蛋白表达。结果与0 min时间点比较,培养液中NO浓度分别在2 min、5 min、15 min时间点增加(P<0.01或P<0.05),其中在5 min时间点达到高峰。与对照组比较,Apelin组磷酸化Akt和磷酸化eNOS表达增加(P<0.01),与Apelin组比较,Apelin+Akt inhibitor组磷酸化eNOS表达降低(P<0.01)。结论Apelin促进PMVECs释放NO,Akt/eNOS磷酸化参与Apelin促进NO释放机制。

Objective To investigate the effects of apelin on release of nitric oxide （NO） and its mechanism in pulmonary microvaseular endothelial cells （PMVECs）. Methods PMVECs between passage 2 and 3 cultured from neonatal rat of 24 hours after birth were starved serum-free for 24 hours, and then used to following studies. Part Ⅰ ： The effects of apelin on release of NO in PMVECs at different time： 1 × 10^5 cells per well grown in 24-well plates were divided randomly into 2 groups： apelin group was added with 10^-8 mol/L apelin; control goup with equal volume of serum-free DMEM. Then the concentrations of NO in cultural medium were detected in both groups at five different time points of 0 min, 2 min, 5 min, 15 min and 30 min. Part Ⅱ ： The effects of apelin on phosphorylations of endothelial nitric-oxide synthase（eNOS） and Akt： 5 × 10^5 cells per well grown in 6-well plates were divided into 3 groups： apelin group was added with 10^-8 mol/L of apelin; apelin ＋Akt inhibitor group was pretreated with 5 μmol/L of Akt inhibitor 30 rnin prior to incubated with apetin; control group with equal volume of serum-free DMEM. Five rain later Western blot was used to assay protein expressions of phosphorylations of endothelial nitric oxide synthase and Akt. Results Compared with the control group, the concentration of NO in cultural medium increased at the three time points of 2 min, 5 min,and 15 main （P〈0.01 or P〈0.05）, with the peak at the point of 5 min（P〈0.01）. Compared with the control group, apelin induced an increase in protein expressions of phosphorylation of both Akt and eNOS significantly in apelin group（P〈0. 01）. Compared with the apelin group,protein expressions of phosphorylation of eNOS significantly decreased in Apelin ＋ Akt inhibitor group （P〈0.01）. Conclusions Apelin can induce NO release. Apelin promoting phosphorylation of Akt/ eNOS is possibly one of the mechanisms to induce NO release.