目的:检测自噬成分的表达,p38MAPK(p38)和磷酸化叉头盒转录因子O-1(pFoxO1)在慢性血栓栓塞性肺动脉高压(CTEPH)大鼠肺血管内皮细胞中的表达,探讨组织因子(TF)调控自噬的可能机制。
方法:从CTEPH(CTEPH组)和健康大鼠(对照组(ctrl组))中分离肺动脉内皮细胞(PAECs),并在不同时间点与TF共培养12h,24h,48小时,剂量包括0nM,10nM,100nM,1µM,10µM,100µM,并与TFPI共培养48小时,包括0nM,2.5nM,5nM。叉头盒转录因子O-1(FoxO1)的表达,测量PAEC中的pFoxO1、p38、Beclin-1和LC3B。共免疫沉淀(co-IP)测定用于检测FoxO1和LC3之间的相互作用。
结果:在12小时时,CTEPH组(与TF从0nM到100µM共培养)中p-FoxO1/FoxO1的蛋白表达明显低于ctrl组,24h,和48h(P<0.05),CTEPH组(与TFPI从0nM到5nM共培养)在48h时显着低于ctrl组(P<0.05)。0nM处理的CTEPH组中p38的蛋白表达,10nM,100nM或1µMTF持续48小时比ctrl组显著增加(P<0.05),CTEPH组(与TFPI浓度从0nM到5nM共培养)在48小时比ctrl组显著增加(P<0.05)。在24h和48h后,CTEPH组相同浓度(与TF从0nM到100µM共培养)的Beclin1蛋白表达显着低于ctrl组(P<0.05),而CTEPH组(与TFPI浓度从2.5nM到5nM共培养)在48h时显着降低(P<0.05)。相同浓度的LC3-II/LC3-I蛋白表达(与TF0nM共培养,1µM,10µM,和100µM)在12小时后,CTEPH组明显低于ctrl组(P<0.05),在CTEPH组(与TFPI浓度从0nM至5nM共培养)中明显低于ctrl组48小时(P<0.05)。在不同剂量和时间点,对照组和CTEPH组的FoxO1和LC3之间存在密切的相互作用。
结论:来自CTEPH大鼠的PAECs自噬活性被破坏。TF,FoxO1和p38MAPK在PAECs的自噬活性中起关键作用。TF可能通过p38MAPK-FoxO1通路调节自噬活性。
OBJECTIVE: To detect the expression of autophagy components, p38 MAPK (p38) and phosphorylated forkhead box transcription factor O-1 (pFoxO1) in pulmonary vascular endothelial cells of chronic thromboembolic pulmonary hypertension (CTEPH) rats and to investigate the possible mechanism through which tissue factor (TF) regulates autophagy.
METHODS: Pulmonary artery endothelial cells (PAECs) were isolated from CTEPH (CTEPH group) and healthy rats (control group (ctrl group)) which were cocultured with TF at different time points including 12 h, 24 h, 48 h and doses including 0 nM,10 nM, 100 nM, 1µM, 10µM, 100µM and cocultured with TFPI at 48 h including 0 nM, 2.5 nM, 5 nM. The expression of forkhead box transcription factor O-1 (FoxO1), pFoxO1, p38, Beclin-1 and LC3B in PAECs was measured. Coimmunoprecipitation (co-IP) assays were used to detect the interaction between FoxO1 and LC3.
RESULTS: The protein expression of p-FoxO1/FoxO1 was significantly lower in the CTEPH groups (cocultured with TF from 0 nM to 100 µM) than in the ctrl group at 12 h, 24 h, and 48 h (P < 0.05) and was significantly lower in the CTEPH groups (cocultured with TFPI from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of p38 in the CTEPH groups treated with 0 nM, 10 nM, 100 nM or 1 µM TF for 48 h significantly increased than ctrl groups (P < 0.05) and was significantly increased in the CTEPH groups (cocultured with TFPI concentration from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of Beclin1 at the same concentration (cocultured with TF from 0 nM to 100 µM) was significantly lower in the CTEPH groups than ctrl groups after 24 h and 48 h (P < 0.05) and was significantly decreased in the CTEPH groups (cocultured with TFPI concentration from 2.5 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of LC3-II/LC3-I at the same concentration (cocultured with TF 0 nM, 1 µM, 10 µM, and 100 µM) was significantly lower in the CTEPH than in the ctrl groups after 12 h (P < 0.05) and was significantly lower in the CTEPH groups (cocultured with TFPI concentration from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). There were close interactions between FoxO1 and LC3 in the control and CTEPH groups at different doses and time points.
CONCLUSIONS: The autophagic activity of PAECs from CTEPH rats was disrupted. TF, FoxO1 and p38 MAPK play key roles in the autophagic activity of PAECs. TF may regulate autophagic activity through the p38 MAPK-FoxO1 pathway.