PUMA is invovled in ischemia/reperfusion-induced apoptosis of mouse cerebral astrocytes
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1Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu, PR China.
Abstract:
PUMA (p53-upregulated modulator of apoptosis), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and p53-independent forms of apoptosis. PUMA has been invovled in the onset and progress of several diseases, including cancer, acquired immunodeficiency syndrome, and ischemic brain disease. Although many studies have shown that ischemia and reperfusion (I/R) can induce the apoptosis of astrocytes, the role of PUMA in I/R-mediated apoptosis of cerebral astrocyte apoptosis remains unclear. To mimic in vivo I/R conditions, primary mouse cerebral astrocytes were incubated in a combinational cultural condition of oxygen, glucose, and serum deprivation (OSGD) for 1 h followed by reperfusion (OSGD/R). Cell death determination assays and cell viability assays indicated that OSGD and OSGD/R induce the apoptosis of primary cerebral astrocytes. The expression of PUMA was significantly elevated in primary cerebral astrocytes during OSGD/R. Moreover, targeted down-regulation of PUMA by siRNA transfection significantly decreased the OSGD/R-induced apoptosis of primary cerebral astrocytes. We also found that OSGD and OSGD/R triggered the release of cytochrome c in astrocytes, indicating the dependence on a mitochondrial apoptotic pathway. Reactive oxygen species (ROS) was extremely generated during OSGD and OSGD/R, and the elimination of ROS by treated with N-acetyl-L-cysteine (NAC) remarkably inhibited the expression of PUMA and the apoptosis of primary cerebral astrocytes. The activation of Caspase 3 and Caspase 9 was extremely elevated in primary cerebral astrocytes during OSGD. In addition, we found that knockdown of PUMA led to the depressed expression of Bax, cleaved caspase-9 and caspase-3 during OSGD/R. These results indicate that PUMA is invovled in the apoptosis of cerebral astrocytes upon I/R injury.
Insights
The study reveals that PUMA (p53-upregulated modulator of apoptosis) plays a key role in cerebral astrocyte apoptosis following ischemia and reperfusion (I/R) injury. Reducing PUMA levels significantly decreased I/R-induced astrocyte death, highlighting its therapeutic potential.
Area of Science:
- Molecular Biology
- Cell Biology
- Neuroscience
Background:
- PUMA (p53-upregulated modulator of apoptosis) is a critical mediator of apoptosis involved in various diseases.
- Ischemia and reperfusion (I/R) injury can induce astrocyte apoptosis, but PUMA's specific role is unclear.
- Understanding PUMA's function in cerebral I/R injury is crucial for developing neuroprotective strategies.
Purpose of the Study:
- To investigate the role of PUMA in ischemia and reperfusion (I/R)-induced apoptosis of primary mouse cerebral astrocytes.
- To elucidate the molecular mechanisms underlying PUMA's involvement in this process.
Main Methods:
- Primary mouse cerebral astrocytes were subjected to oxygen, glucose, and serum deprivation (OSGD) followed by reperfusion (OSGD/R) to mimic I/R conditions.
- Cell death and viability assays were performed.
- PUMA expression was modulated using siRNA.
- Cytochrome c release, reactive oxygen species (ROS) generation, and caspase activation were assessed.
- N-acetyl-L-cysteine (NAC) was used to scavenge ROS.
Main Results:
- OSGD/R significantly induced apoptosis in primary cerebral astrocytes.
- PUMA expression was markedly upregulated during OSGD/R.
- Down-regulation of PUMA using siRNA attenuated OSGD/R-induced astrocyte apoptosis.
- OSGD/R triggered mitochondrial-dependent apoptosis via cytochrome c release and activated Caspase 3 and Caspase 9.
- ROS generation was observed during OSGD/R, and ROS scavenging with NAC inhibited PUMA expression and apoptosis.
- Knockdown of PUMA reduced the expression of Bax, cleaved caspase-9, and cleaved caspase-3.
Conclusions:
- PUMA is a key mediator of cerebral astrocyte apoptosis induced by ischemia and reperfusion (I/R) injury.
- The PUMA-mediated apoptotic pathway involves mitochondrial signaling and is influenced by reactive oxygen species (ROS).
- Targeting PUMA may represent a potential therapeutic strategy for mitigating cerebral I/R injury.
