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Published on: August 2, 2018
GAPDH-silence preserves H9C2 cells from acute hypoxia and reoxygenation injury
Shao Liang1, Ma Aiqun2, Gemma Figtree3
1Department of Cardiology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China.
Background:
Acute hypoxia and reoxygenation injury, as a common environmental stress condition, is a basic condition of most pathophysiological processes. It has been approve that autophagy and oxidant stress could contribute to acute hypoxia and reoxygenation injury. This study is aimed to examine the effect of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silence on cell injury with acute hypoxia and reoxygenation injury by autophagy and antioxidant stress pathway.
Methods:
GAPDH expression was silenced by siRNA in H9C2 cardiomyoblasts with acute hypoxia and reoxygenation injury. Autophagy was detected by western blot for autophagy proteins and monodansylcadaverine (MDC) staining for acidic substances. Pro-apoptosis protein and flow cytometry were used to assess cell apoptosis and death and intracellular adenosine triphosphate (ATP) relative concentration was measured. Oxidant stress was assessed by measuring 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), thiobarbituric acid reactive substances (TBARS), glutathione (GSH) and super oxide dismutase (SOD).
Results:
In this study, GAPDH-silence enhanced autophagy in H9C2 cells with acute hypoxia and reoxygenation injury, decreased oxidant stress and increased antioxidant pathways; and reduced cell apoptosis and death. However, GAPDH-silence had no significant effect on cell energy.
Conclusion:
GAPDH pre-silence by siRNA reduces H9C2 cell death occurring via autophagy and anti-oxidative stress pathway in acute hypoxia and reoxygenation injury. This study enriches the understanding of GAPDH pathophysiology role, and provides potential new therapeutic targets for cardiac disease states characterized by oxidative stress.
Insights
Silencing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enhances autophagy and antioxidant pathways, reducing cell death from acute hypoxia and reoxygenation injury. This finding offers new therapeutic targets for oxidative stress-related cardiac conditions.
Area of Science:
- Cardiovascular Research
- Cellular Biology
- Biochemistry
Background:
- Acute hypoxia and reoxygenation injury are implicated in various pathophysiological processes.
- Autophagy and oxidative stress are known contributors to acute hypoxia and reoxygenation injury.
- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a role in cellular responses to stress.
Purpose of the Study:
- To investigate the effect of silencing GAPDH on H9C2 cardiomyoblasts subjected to acute hypoxia and reoxygenation injury.
- To elucidate the involvement of autophagy and antioxidant stress pathways in GAPDH-mediated cytoprotection.
- To assess the impact of GAPDH silence on cell viability, apoptosis, and energy metabolism.
Main Methods:
- GAPDH expression was silenced using siRNA in H9C2 cardiomyoblasts.
- Autophagy was assessed via western blot and monodansylcadaverine (MDC) staining.
- Cell apoptosis, death, and intracellular adenosine triphosphate (ATP) levels were measured.
- Oxidant stress markers (DCFH-DA, TBARS, GSH, SOD) were quantified.
Main Results:
- GAPDH silencing significantly enhanced autophagy and antioxidant pathways in H9C2 cells under hypoxia/reoxygenation.
- Silencing GAPDH reduced cell apoptosis and death without affecting intracellular ATP levels.
- Oxidative stress was decreased, while antioxidant capacity was increased following GAPDH silencing.
Conclusions:
- Pre-silencing of GAPDH by siRNA confers protection against cell death in acute hypoxia and reoxygenation injury.
- The protective mechanism involves the modulation of autophagy and anti-oxidative stress pathways.
- This study highlights GAPDH's pathophysiological role and suggests it as a potential therapeutic target for oxidative stress-related cardiac diseases.
