血管紧张素转换酶2对人肺微血管内皮细胞炎性损伤的调控作用

Regulation of angiotensin converting enzyme 2 on inflammatory injury of human pulmonary microvascular endothelial cells

目的探讨血管紧张素转换酶2(ACE2)对人肺微血管内皮细胞(HPMECs)炎性损伤的调控作用。方法利用原代培养HPMECs,构建脂多糖(LPS)损伤模型,(1)量效实验:分别用终浓度为50、100、500、1000 ng/ml的LPS刺激HPMECs 24 h,采用细胞计数试剂盒(CCK-8)法检测细胞活性,采用乳酸脱氢酶(LDH)法检测细胞损伤程度,采用蛋白质印迹实验(Western Blot)检测ACE2的表达情况;(2)以ACE2激动剂DIZE(或ACE2抑制剂MLN-4760)预培养HPMECs后加入LPS再培养24 h,同时设空白对照组(CTRL,正常内皮细胞培养基)及LPS、激动剂(或抑制剂)单独处理组,收集细胞培养上清液,采用LDH法检测细胞损伤程度,采用酶联免疫吸附测定法(ELISA)测定上清液中白介素(IL)-1β、IL-6水平,采用Western Blot法测定ACE2及MasR蛋白表达情况。结果在LPS刺激24 h后,与CTRL组比较,HPMECs细胞活力下降,且随着LPS浓度的升高,细胞活力下降明显(P<0.001);ACE2蛋白表达增加,以100 ng/ml组ACE2增加最为显著(2.24±0.57),差异均有统计学意义。与LPS组比较,LPS+DIZE组LDH相对释放活性降低(P<0.0001),炎症因子IL-1β、IL-6释放减少(P<0.01),MasR蛋白表达升高(P<0.01),差异均有统计学意义。与LPS组比较,LPS+MLN-4760组LDH相对释放活性增加(P<0.01),炎症因子IL-1β、IL-6释放增加(P<0.05),ACE2、MasR蛋白表达均降低(P<0.0001),差异均有统计学意义。结论LPS诱导HPMECs细胞损伤模型中ACE2表达上调,ACE2功能活化有利于增强MasR表达,减少炎症因子释放,在体外表现为对LPS所诱导的细胞损伤的保护作用。

Objective To investigate the regulatory effect of angiotensin converting enzyme 2(ACE2)on inflammatory injury of human pulmonary microvascular endothelial cells(HPMECs).Methods The primary cultured HPMECs were treated with lipopolysaccharide(LPS)as an injury model.LPS dose dependent response and ACE2 agonist/antagonist intervention experiments were carried out:(1)Dose dependent response experiment:HPMECs were treated with LPS at different concentrations of 50,100,500,and 1000 ng/ml for 24 h.Cell counting kit-8(CCK-8)method was used to detect the cell viability.Lactate dehydrogenase(LDH)activity was tested to reflect the degree of cell injury and ACE2 expression was tested by Western blot.(2)HPMECs were pre-incubated with the ACE2 agonist DIZE(or ACE2 inhibitor MLN-4760),followd by adding LPS,culture for another 24 hours.Meanwhile a blank control group(normal endothelial cell medium)and LPS,agonist(or inhibitor)were used.Interleukin 1β(IL-1β)and IL-6 levels in the cell culture supernatant were determined by enzyme-linked immunosorbent assay.The expression of ACE2 and MasR were testes by Western Blot.Results After 24 h of LPS stimulation,the cell viability of HPMECs decreased compared with that of CTRL group,and the cell viability decreased significantly with the increase of LPS concentration(P<0.001).The ACE2 expression increased,and reach the highest level in 100 ng/ml group(2.24±0.57).Compared with LPS group,LDH relative release activity,IL-1βand IL-6 release were significantly decreased(all P<0.01)while MasR protein expression was significantly increased(P<0.01)in LPS+DIZE group.Compared with LPS group,the relative release activity of LDH and IL-1βand IL-6 level were increased while ACE2 and MasR expression decreased significantly in LPS+MLN-4760 group(P<0.0001).Conclusion LPS-induced HPMECs cell injury model up-regulates ACE2 expression.Activating ACE2 function can enhance the expression of MasR and reduce the release of inflammatory factors,thereby alleviating the cell injury induced by LPS.