Department of Anesthesiology, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, Hubei, China; Institute of Anesthesiology, Hubei University of Medicine, Shiyan, 442000, Hubei, China.
Department of Anesthesiology, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, Hubei, China.
Biomed Pharmacother. 2020 May;125:109995. doi: 10.1016/j.biopha.2020.109995. Epub 2020 Feb 27.
We previously observed that amphiregulin (Areg), a ligand of epithelial growth factor receptor (EGFR), was highly expressed in lipopolysaccharide (LPS)-induced acute lung injury (ALI) lung tissues mainly by the classically activated (M1) alveolar macrophages (AMs). Areg also plays a protective role in LPS-induced injury in lung tissues and alveolar epithelial cells (AECs). However, whether Areg is co-expressed with tumor necrosis factor (TNF)-α in ALI lung tissues, and can directly inhibit TNF-α-induced AEC injury remains unclear.
We first detected the kinetic expressions of Areg and TNF-α in LPS-stimulated lung tissues and M1 AMs and then identified the role of exogenous recombinant Areg (rmAreg) in the injured lung tissues. The effect of Areg on TNF-α-induced apoptosis in MLE-12 cells, a kind of AECs, was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The activation of the EGFR-AKT pathway and caspase-3, -8, and -9 were detected by Western blotting. The EGFR knockdown by small interfering RNA was used to assess the role of EGFR in Areg functions.
Areg production occurred in close parallel with TNF-α expression in M1 AMs and ALI lung tissues, and rmAreg attenuated LPS-induced ALI in mice. TNF-α stimulation induced significant apoptosis in MLE-12 cells, but this apoptosis was inhibited under rmAreg treatment. Moreover, rmAreg enhanced the activation of EGFR and AKT, and reduced the expressions of cleaved caspase-3, -8, and -9 in ALI lung tissues and TNF-α-challenged MLE-12 cells. However, the EGFR knockdown significantly inhibited the Areg-induced improvement in apoptosis, enhancement of EGFR and AKT activation, and reduction of cleaved caspase-3, -8, and -9 expressions.
Areg and TNF-α were synchronously produced by ALI lung tissues and M1 AMs, and Areg directly inhibited the TNF-induced apoptosis and transduction of caspase death signals in AECs via the EGFR pathway.
我们之前观察到，表皮生长因子受体（EGFR）配体 Amphiregulin（Areg）在脂多糖（LPS）诱导的急性肺损伤（ALI）肺组织中主要由经典激活（M1）肺泡巨噬细胞（AMs）高度表达。Areg 在 LPS 诱导的肺组织和肺泡上皮细胞（AEC）损伤中也发挥保护作用。然而，Areg 是否与肿瘤坏死因子（TNF）-α在 ALI 肺组织中共同表达，以及是否能直接抑制 TNF-α诱导的 AEC 损伤尚不清楚。
我们首先检测了 LPS 刺激的肺组织和 M1 AMs 中 Areg 和 TNF-α的动态表达，然后鉴定了外源性重组 Areg（rmAreg）在损伤肺组织中的作用。通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记染色检测 Areg 对 MLE-12 细胞（一种 AEC）中 TNF-α诱导的细胞凋亡的影响。通过 Western blot 检测 EGFR-AKT 通路和 caspase-3、-8、-9 的激活。用小干扰 RNA 敲低 EGFR 来评估 EGFR 在 Areg 功能中的作用。
Areg 的产生与 M1 AMs 和 ALI 肺组织中 TNF-α的表达密切平行，rmAreg 减轻了 LPS 诱导的小鼠 ALI。TNF-α刺激诱导 MLE-12 细胞发生明显凋亡，但在 rmAreg 处理下，这种凋亡受到抑制。此外，rmAreg 增强了 EGFR 和 AKT 的激活，并降低了 ALI 肺组织和 TNF-α刺激的 MLE-12 细胞中 cleaved caspase-3、-8 和 -9 的表达。然而，EGFR 敲低显著抑制了 Areg 诱导的凋亡改善、EGFR 和 AKT 激活增强以及 cleaved caspase-3、-8 和 -9 表达降低。
Areg 和 TNF-α是由 ALI 肺组织和 M1 AMs 同步产生的，Areg 通过 EGFR 途径直接抑制 TNF 诱导的 AEC 凋亡和半胱天冬酶死亡信号转导。
