Neuropeptide Substance P Attenuates Hyperoxia-Induced Oxidative Stress Injury in Type II Alveolar Epithelial Cells Via Suppressing the Activation of JNK Pathway

Exposure of mice to hyperoxia induces alveolar epithelial cell (AEC) injury, acute lung injury and death. Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lung protects against these effects, although the mechanisms are not yet clear. Hyperoxia induces cellular injury via effects on mitochondrial integrity, associated with induction of proapoptotic members of the Bcl-2 family. We hypothesized that GM-CSF protects AEC through effects on mitochondrial integrity. MLE-12 cells (a murine type II cell line) and primary murine type II AEC were subjected to oxidative stress by exposure to 80% oxygen and by exposure to H2O2. Exposure to H2O2 induced cytochrome c release and decreased mitochondrial reductase activity in MLE-12 cells. Incubation with GM-CSF significantly attenuated these effects. Protection induced by GM-CSF was associated with Akt activation. GM-CSF treatment also resulted in increased expression of the antiapoptotic Bcl-2 family member, Mcl-1. Primary murine AEC were significantly more tolerant of oxidative stress than MLE-12 cells. In contrast to MLE-12 cells, primary AEC expressed significant GM-CSF at baseline and demonstrated constitutive activation of Akt and increased baseline expression of Mcl-1. Treatment with exogenous GM-CSF further increased Akt activation and Mcl-1 expression in primary AEC. Conversely, suppression of AEC GM-CSF expression by use of GM-CSF-specific small interfering RNA resulted in decreased tolerance of oxidative stress, Furthermore, silencing of Mcl-1 prevented GM-CSF-induced protection. We conclude that GM-CSF protects alveolar epithelial cells against oxidative stress-induced mitochondrial injury via the Akt pathway and its downstream components, including Mcl-1. Epithelial cell-derived GM-CSF may contribute to intrinsic defense mechanisms limiting lung injury.

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Nrf2 Alleviates TNF-α-Induced Oxidative Stress Damage in Type II Alveolar Epithelial Cells by Down-Regulating the Expression of NOX1

10.21203/rs.3.rs-68779/v1 ◽

2020 ◽

Author(s):

Weijing Wu ◽

Jiamin Zhang ◽

Xihua Lian ◽

Xiaoping Lin ◽

Xiaoshan Su ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Binding Site ◽

Luciferase Activity ◽

A549 Cells ◽

Alveolar Epithelial Cells ◽

Type Ii ◽

Alveolar Epithelial ◽

Tnf Α ◽

Stress Damage

Abstract Objective: To study the roles of Nrf2 in acute lung injury (ALI) pathogenesis by investigating the effects of Nrf2 on regulating oxidative stress damage in TNF-α-induced type II alveolar epithelial cells (T2AECs).Methods: T2AECs were transfected with Nrf2 siRNA and overexpression vectors for six hours before being induced by TNF-α for 24 hours. Subsequently, levels of interleukins (IL-6 and IL-8), reactive oxygen species (ROS), malondialdehyde (MDA), total antioxidation capability (T-AOC), Nrf2, NOX1 and NF-kB were measured. Additionally, potential Nrf2 binding site in NOX1 promoter was predicted by AliBaba2.1 and two recombinant vectors, namely “pGL3-NOX1-1500” and “pGL3-NOX1-1489, were constructed by inserting the sequence of NOX1 promoter in full-length and that in the absence of Nrf2 binding site to pGL3 basic vector. T2AECs were transfected with these vectors prior to TNF-α induction and the luciferase activity was measured.Results: Levels of IL-6, IL-8, ROS and MDA were increased (P＜0.05) while T-AOC was decreased in TNF-α-induced A549 cells after the transfection of Nrf2 siRNA vector (P＜0.05). In contrast, concentrations of IL-6, IL-8, ROS and MDA were decreased (P＜0.05) whereas T-AOC was increased after the transfection of Nrf2 overexpression vector (P＜0.05). NOX1 promoter possesses one Nrf2 binding site. Cells transfected by “pGL3-NOX1-1500” vector had the highest luciferase activity, followed by cells transfected by “pGL3-NOX1-1489” vector and the control cells (P＜0.05).Conclusion: Nrf2 modulates NOX1 expression via binding to its promoter, by which against TNF-α-induced oxidative stress damage in T2AECs. Thus, Nrf2 might be a therapeutic target for ALI.

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