Knocking down TRPM2 expression reduces cell injury and NLRP3 inflammasome activation in PC12 cells subjected to oxygen-glucose deprivation
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Published on: May 3, 2024
Tao Pan1, Qiu-Jiao Zhu2, Li-Xiao Xu3
1Department of Neonatology, Children's Hospital Affiliated to Suzhou University, Suzhou, Jiangsu Province, China.
Abstract:
Transient receptor potential melastatin 2 (TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mechanism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma (PC12) cells injured by oxygen-glucose deprivation (OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reoxygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2 (CXCL2), NACHT, LRR, and PYD domain-containing protein 3 (NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.
Insights
Reducing TRPM2 expression protects against cerebral ischemia injury. This study shows TRPM2 knockdown inhibits apoptosis and oxidative stress, offering a potential therapeutic strategy for brain injury.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Transient receptor potential melastatin 2 (TRPM2) is an ion channel implicated in cerebral ischemia.
- The precise role of TRPM2 reduction in repairing cerebral injury and its underlying mechanisms remain unclear.
Purpose of the Study:
- To investigate the protective effects of reducing TRPM2 expression on oxygen-glucose deprivation (OGD)-induced injury in pheochromocytoma (PC12) cells.
- To elucidate the molecular mechanisms by which TRPM2 knockdown mitigates neuronal damage.
Main Methods:
- PC12 cells were transfected with TRPM2 shRNAs and subjected to OGD.
- Apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS), and intracellular calcium levels were measured via flow cytometry.
- Expression of CXCL2, NALP3 (NLRP3 inflammasome), and caspase-1 was assessed using RT-qPCR and Western blotting.
Main Results:
- TRPM2 knockdown significantly reduced apoptosis rates, mitochondrial membrane potential loss, ROS production, and intracellular calcium accumulation in OGD-treated PC12 cells.
- TRPM2 knockdown suppressed the activation of the NLRP3 inflammasome pathway, indicated by reduced NALP3 and caspase-1 expression.
Conclusions:
- TRPM2 knockdown confers neuroprotection against OGD-induced injury in PC12 cells.
- This protective effect is mediated by the inhibition of apoptosis, reduction of oxidative stress, preservation of mitochondrial function, and attenuation of NLRP3 inflammasome activation.
