Combined effect of tumor necrosis factor (TNF)-α and heat shock protein (HSP)-70 in reducing apoptotic injury in hypoxia: A cell culture study

Studies have demonstrated neuroprotective effects of either TNF-α or HSP-70 in ischemia/reperfusion injury following exercise. However, the protective mechanisms involving combined effect of the two proteins, particularly in neuronal apoptosis, remain unclear. This study aims to elucidate the beneficial role of TNF-α and HSP-70 in the regulation of apoptotic proteins and ERK signaling in hypoxic injury. Cortical neurons from 20 Sprague–Dawley rat embryos were isolated and cultured in five groups with or without pretreatment with recombinant TNF-α, HSP-70 protein or both prior to hypoxic conditions: (1) control; (2) control/hypoxia; (3) TNF-α/hypoxia; (4) HSP-70/hypoxia and (5) TNF-α/HSP-70/hypoxia. Western blotting was used to detect pro- and anti-apoptotic proteins, including Bax, AIF, Bcl-xL, Bcl-2, and pERK1/2 protein. TNF-α and HSP-70 significantly (p < 0.05) reduced the levels of pro-apoptotic proteins, Bax and AIF. Also, pretreatment of hypoxic brain tissue with TNF-α and HSP-70 significantly (p < 0.05) enhanced the levels of anti-apoptotic protein, Bcl-xL. TNF-α and HSP-70 together increased Bcl-2 levels by 70%. Hypoxia caused a significant (p < 0.05) increase in ERK1/2 phosphorylation levels by 224%. The most effective inhibition of ERK levels was obtained by the combined administration of TNF-α and HSP-70. This study suggested that TNF-α and HSP-70 together enhance the decrease in pro-apoptotic protein levels and the increase in anti-apoptotic protein levels in the event of neuronal hypoxia through ERK1/2 signal transduction.