Apelin/APJ系统在脂多糖诱导大鼠肺微血管内皮细胞损伤中的变化及其作用研究

摘要

目的探讨Apelin/APJ系统在脂多糖诱导大鼠肺微血管内皮细胞( PMVECs)损伤中的变化,并研究Apelin的作用及机制。方法采用植块法培养大鼠PMVECs, VIII因子相关抗原免疫细胞化学染色进行鉴定。噻唑蓝( MTT)比色法测定PMVECs活力；RT-PCR法检测Apelin、APJ mRNA表达的变化；Western blot法检测大鼠PMVECs中PCNA蛋白表达及Akt 的磷酸化水平。结果 LPS 在短时间内能够使Apelin、APJ mRNA表达水平呈代偿性上升( P<0．01),但随着作用时间延长,基因表达受到明显抑制,低于对照组(P<0．05或P<0．01),提示Apelin/APJ系统可能参与了LPS诱导的大鼠PMVECs损伤。 MTT结果表明,10-9~10-6 mol · L-1的Apelin明显促进了大鼠PMVECs增殖( P<0．05或P<0．01),且具有一定的浓度和时间依赖性。而且,Apelin还不同程度地改善了LPS诱导的PMVECs细胞损伤( P<0．05或P<0．01)。另外,Western blot结果显示,Apelin还明显逆转了LPS诱导的PCNA蛋白表达和Akt磷酸化水平的降低(P<0．05或P<0．01)。结论 Apelin/APJ系统参与了LPS诱导的大鼠PMVECs损伤。 Apelin对于维护肺微血管内皮细胞功能,干预LPS诱导的PMVECs损伤起着重要作用,可能与其激活Akt磷酸化通路有关。%Aim To explore the changes of Apelin/APJ system in LPS-induced injury of rat pulmonary mi-crovascular endothelial cells( PMVECs) , and the effect and mechanism of Apelin. Methods PMVECs were cultured with the explant technique, and the identifica-tion of rat PMVECs was carried out by immunocyto-chemical staining of factorⅧrelated antigen. MTT as-say was used to evaluate the viability of PMVECs. The mRNA expression of Apelin and APJ was detected by RT-PCR. The protein expression of PCNA and the phosphorylation of Akt was analyzed by Western blot. Results The mRNA expression of Apelin and APJ showed a compensatory increase after LPS treatment for a short period of time ( P<0. 01 ) , but with the exten-sion of time, which was significantly inhibited, even lower than the control group ( P<0. 05 or P<0. 01 ) , suggesting that Apelin/ APJ system might be involved in LPS-induced PMVECs injury. MTT results showed that 10 -6 ~10 -9 mol · L-1 Apelin obviously promoted the proliferation of rat PMVECs ( P <0. 05 or P <0. 01 ) , and with certain concentration and time de-pendence. Moreover, Apelin also improved the LPS-induced PMVECs injury in different degrees ( P<0. 05 or P < 0. 01 ) . In addition, Western blot analysis showed that Apelin significantly reversed the decrease of the protein expression of PCNA and the Akt phos-phorylation level induced by LPS ( P <0. 05 or P <0. 01 ) . Conclusions The Apelin/APJ system is in-volved in LPS-induced PMVECs injury. Apelin plays an important role in protecting the pulmonary microvas-cular endothelial function and reversing the LPS-in-duced PMVECs injury, which might be related to the activation of Akt phosphorylation pathway.