Autophagy participates in the development of cerebral ischemia stroke. Autophagy-related 3 (ATG3), an important autophagy regulator, was reported to be upregulated in a rat model of cerebral ischemia/reperfusion (CI/R) injury and an oxygen–glucose deprivation/reoxygenation (OGD/R) cell model. However, the detailed role of ATG3 in CI/R injury remains elusive. An in vitro cellular model was established to mimic CI/R injury by exposing hBMECs and bEnd.3 cells to OGD/R. OGD/R-induced injury were evaluated by cell counting kit-8 (CCK-8), LDH release assay, caspase-3 activity assay and TUNEL assay. Inflammation was assessed by detecting mRNA expression and concentrations of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) using qRT-PCR and ELISA, respectively. The protein levels of ATG3, light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were determined by western blot analysis. We successfully established an in vitro OGD/R injury model using hBMECs and bEnd.3 cells. ATG3 was time-dependently upregulated and ATG3 knockdown inhibited autophagy in OGD/R-challenged brain microvascular endothelial cells. Moreover, autophagy inhibition by ATG3 interference attenuated OGD/R-induced viability inhibition and increase of LDH release, caspase-3 activity, programmed cell death, and production of IL-1β, IL-6 and TNF-α. Inhibition of autophagy by ATG3 silencing activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in OGD/R-challenged brain microvascular endothelial cells. Furthermore, inhibition of the PI3K/Akt pathway reversed the protective effects of ATG3 silencing on OGD/R-induced injury and inflammation. In conclusion, autophagy inhibition by ATG3 knockdown remitted OGD/R-induced injury and inflammation in brain microvascular endothelial cells via activation of the PI3K/Akt pathway.

自噬参与脑缺血性脑卒中的发生发展。据报道，自噬相关 3 (ATG3) 是一种重要的自噬调节因子，在脑缺血/再灌注 (CI/R) 损伤大鼠模型和氧糖剥夺/复氧 (OGD/R) 细胞模型中表达上调。然而，ATG3 在 CI/R 损伤中的详细作用仍不清楚。通过将 hBMEC 和 bEnd.3 细胞暴露于 OGD/R 来建立体外细胞模型来模拟 CI/R 损伤。通过细胞计数试剂盒8（CCK-8）、LDH释放测定、caspase-3活性测定和TUNEL测定评估OGD/R诱导的损伤。通过分别使用 qRT-PCR 和 ELISA 检测白细胞介素-1β (IL-1β)、IL-6 和肿瘤坏死因子-α (TNF-α) 的 mRNA 表达和浓度来评估炎症。ATG3、轻链 3 (LC3)-I、LC3-II、p62、通过蛋白质印迹分析测定蛋白激酶 B (Akt) 和磷酸化 Akt (p-Akt)。我们使用 hBMEC 和 bEnd.3 细胞成功建立了体外 OGD/R 损伤模型。在 OGD/R 攻击的脑微血管内皮细胞中，ATG3 呈时间依赖性上调，并且 ATG3 敲低会抑制自噬。此外，ATG3 干扰引起的自噬抑制减弱了 OGD/R 诱导的活力抑制，并增加了 LDH 释放、caspase-3 活性、程序性细胞死亡以及 IL-1β、IL-6 和 TNF-α 的产生。ATG3 沉默对自噬的抑制激活了 OGD/R 攻击的脑微血管内皮细胞中的磷酸肌醇 3-激酶 (PI3K)/Akt 通路。此外，PI3K/Akt 通路的抑制逆转了 ATG3 沉默对 OGD/R 诱导的损伤和炎症的保护作用。综上所述，