Dai Shaohua, Xu Qirong, Liu Sheng, Yu Bentong, Liu Jichun, Tang Jian
Department of Thoracic Surgery, The First Affiliated Hospital of Nanchang UniversityJiangxi, China.
Am J Transl Res. 2017 Oct 15;9(10):4470-4480. eCollection 2017.
This study was conducted to investigate the mechanism of autophagy and its signaling pathways in ischemia/reperfusion injury (IRI). Pulmonary microvascular endothelial cells (PMVECs) were used to construct I/R models. The cells were then treated with autophagy inhibitor 3-MA and infected with adenovirus expressing Beclin 1-shRNA. The expression of CD31, LC3-II, Bcl-2, Bax, LC3-II, Beclin 1, AKT, p-AKT, AMPK and p-AMPK, apoptosis, cell viability and migration ability were determined. Over 95% isolated PMVECs were positive for CD31. The expression of LC3-II and Beclin 1 was up-regulated in I/R cells. 3-MA and Beclin 1 knockdown inhibited the expression of LC3-II and Beclin 1 and autophagosome formation. Autophagy induced by hypoxia was antagonistic against apoptosis, which increased after treatment with 3-MA and knockdown of Beclin 1. 3-MA and Beclin 1 knockdown downregulated and upregulated the expression of Bcl-2 and Bax, respectively. Apoptosis mediated by hypoxia and reperfusion-induced autophagy was reduced by 3-MA and Beclin-1 knockdown, which increased and reduced the expression of Bcl-2 and Bax, respectively, leading to significant decreased Bax/Bcl-2 ratio. In these cells, expression of p-AKT, p-AMPK and p-mTOR was up-regulated. After treatment with 3-MA and Beclin 1 knockdown, expression of p-AKT and p-AMPK was significantly reduced.
本研究旨在探讨自噬及其信号通路在缺血/再灌注损伤（IRI）中的作用机制。采用肺微血管内皮细胞（PMVECs）构建I/R模型。然后用自噬抑制剂3-MA处理细胞，并用表达Beclin 1-shRNA的腺病毒感染细胞。检测CD31、LC3-II、Bcl-2、Bax、LC3-II、Beclin 1、AKT、p-AKT、AMPK和p-AMPK的表达、细胞凋亡、细胞活力及迁移能力。分离的PMVECs中CD31阳性率超过95%。I/R细胞中LC3-II和Beclin 1的表达上调。3-MA和Beclin 1基因敲低抑制了LC3-II和Beclin 1的表达及自噬体形成。缺氧诱导的自噬对细胞凋亡具有拮抗作用，3-MA处理和Beclin 1基因敲低后细胞凋亡增加。3-MA和Beclin 1基因敲低分别下调和上调Bcl-2和Bax的表达。3-MA和Beclin-1基因敲低减少了缺氧和再灌注诱导的自噬介导的细胞凋亡，分别增加和降低了Bcl-2和Bax的表达，导致Bax/Bcl-2比值显著降低。在这些细胞中，p-AKT、p-AMPK和p-mTOR的表达上调。3-MA处理和Beclin 1基因敲低后，p-AKT和p-AMPK的表达显著降低。
