Upregulation of NOX2 and NOX4 Mediated by TGF-β Signaling Pathway Exacerbates Cerebral Ischemia/Reperfusion Oxidative Stress Injury

Abstract

Background/Aims: Ischemic stroke is still one of the leading debilitating diseases with high morbidity and mortality. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) play an important role in cerebral ischemia/reperfusion (I/R) injury. However, the mechanism underlying the regulation of ROS generation is still not fully elucidated. This study aims to explore the role of transforming growth beta (TGF-β) signals in ROS generation. Methods: Sprague–Dawley rats were subjected to I/R injury, and PC-12 cells were challenged by hypoxia/reoxygenation (H/R) and/or treated with activin receptor-like kinase (ALK5) inhibitor Sb505124 or siRNA against ALK5. Brain damage was evaluated using neurological scoring, triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, infarct volume measurement, TUNEL staining, and caspase-3 activity measurement. Expression of TGF-β and oxidative stress-related genes was analyzed by real-time polymerase chain reaction and Western blot; NOX activity and ROS level were measured using spectrophotometry and fluorescence microscopy, respectively. Results: I/R contributed to severe brain damage (impaired neurological function, brain infarction, tissue edema, apoptosis), TGF-β signaling activation (upregulation of ALK5, phosphorylation of SMAD2/3) and oxidative stress (upregulation of NOX2/4, rapid release of ROS [oxidative burst]). However, Sb505124 significantly reversed these alterations and protected rats against I/R injury. As in the animal results, H/R also contributed to TGF-β signaling activation and oxidative stress. Likewise, the inhibition of ALK5 or ALK5 knockdown significantly reversed these alterations in PC-12 cells. Other than ALK5 knockdown, ALK5 inhibition had no effect on the expression of ALK5 in PC-12 cells. Conclusions: Our studies demonstrated that TGF-β signaling activation is involved in the regulation of NOX2/NOX4 expression and exacerbates cerebral I/R injury.

Targeting Oxidative Stress and Inflammation to Prevent Ischemia-Reperfusion Injury

“…In theory, blocking pathways or targeting the key molecules of ROS generation can reduce the oxidative damage caused by CIRI. Some progress has been made in antioxidant treatments for CIRI (Kim et al, 2009;Hu et al, 2013;Watanabe et al, 2016;Mahmood et al, 2017;Lou et al, 2018;Song et al, 2018;Xing et al, 2018;Zhu et al, 2018;Chu et al, 2019;Han et al, 2019;Li S. et al, 2019;Liu and Zhang, 2019).…”

“…NAPDH oxidase is a specific ROS-producing enzyme, particularly of NOX4, which is mainly expressed in neurons. Drugs that inhibit NOX4 may have protective effects for brain tissue after ischemia/reperfusion injury (Lou et al, 2018). Casein kinase 2 (CK2) is an important regulator of NADPH oxidase, and inactivation of CK2 in brain tissue can increase NOX2 activity, and increase ROS production and neuronal death (Kim et al, 2009).…”

The interrelationship between cerebral ischemic stroke and glioma: a comprehensive study of recent reports

“…57,58 However, Duox1/2 has an EF-hand domain for calcium binding instead of the heme group. 59 Specific signals (viz., TGF-β, 60 AKT, 61 PKC, 62 MAPK, ERK, etc.) induce conformational changes in the NOX complex in a phosphorylation-dependent manner and allow production of a notably large amount of ROS.…”

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants

“…Expressed highly in brain astrocytes [47], NOX4 has been associated with subarachnoid hemorrhage-induced oxidative stress [48]. Furthermore, NOX4 is upregulated in response to transforming growth factor β1 signaling in numerous cell types [49][50][51][52]. Given the effects of Mn-TM-2-PyP on SOD2 expression, we also quantified catalase expression after treatment with antioxidants in control optic nerve head astrocytes.…”