Effects of Darbepoetin alpha on TRPM2 channel and mythocondrial dysfunction in neuron cells under hypocoxic and normoxic conditions

Oxygen is essential for the proper functioning of cells and the production of energy. Two distinct mechanisms, both central and local, regulate oxygen levels within the organism. When oxygen levels decline, the body's ability to maintain homeostasis is compromised, leading to the development of a hypoxic state. While hypoxia can be tolerated in certain tissues, it can cause significant damage to others. The brain is particularly susceptible to changes in oxygen levels, and it is highly sensitive to hypoxia. Following the onset of hypoxia, the oxidant/antioxidant balance within the body is disrupted, resulting in an increase in Reactive Oxygen Species (ROS), which in turn causes oxidative stress. Oxidative stress causes functional changes in mitochondrial membrane function and Transient Receptor Potential (TRP) channels, which are widely distributed in the body. TRP are cationic channels that provide intracellular ion passage. TRPM (TRP Melastatin) is a member of the TRP family. TRPM has several sub-members and among them TRPM2 is a sub-member of TRPM. TRPM2 is a cation channel that is particularly affected by certain stimuli such as oxidative stress. TRPM2 over-activation causes cytotoxic effect in cellular structure. In hypoxic conditions, the hormone erythropoietin (EPO) is increased to enhance the oxygen level in the blood. EPO stimulates the bone marrow and increases erythrocyte production. Darbepoetin alpha (DA) is a hyperglycosylated analogue of recombinant human erythropoietin (rHuEPO) with a longer half-life. DA has been observed to exhibit decreased neurodegeneration and neuroprotective effects in cells. The objective of this study was to demonstrate the effects of DA on mitochondrial membrane and TRPM2 channel activity following hypoxia in SH-SY5Y cells differentiated into neurons in vitro. In order to achieve this objective, the SH-SY5Y cell line was differentiated into neuronal cells for a period of 14 days. The MTT assay was employed to ascertain the optimal doses of DA (10 ng and 250 ng). In the study, the expression level of HIF-1 α was quantified by RT-PCR to demonstrate that the environment was hypoxic. It was demonstrated that the HIF-1 α level increased with increasing time (24, 48, 72 hours) in the hypoxia groups. Western blot analysis was performed to assess TRPM2 channel activation following hypoxia. It was found that channel activation was statistically increased in both subject and control groups in hypoxia compared to the normoxia group (p<0.01). Mitochondrial membrane potential was evaluated by flow cytometry. The hypoxia control group showed a decrease in cell number compared to the normoxia group (p <0.05). An increase was detected in the 10 and 250 ng hypoxia DA groups compared to the control group (p <0.05). In conclusion, the effects of DA on both TRPM2 channels and mitochondrial membrane potential are observed to decrease in the SH-SY5Y cell line.