Our study provides strong evidence that hypoxic preconditioning with cobalt chloride enhances physical performance and protects muscle from exercise-induced oxidative damage via GSH, HO-1 and MT-mediated antioxidative capacity.

Shukla, Dhananjay, Saurabh Saxena, Purushotman Jayamurthy, Mustoori Sairam, Mrinalini, Singh, Swatantra Kumar Jain, Anju Bansal, and Govindaswamy Ilavazaghan. High Alt. Med. Biol. 10:57-69, 2009.-Hypoxic preco759nditioning (HPC) provides robust protection against injury from subsequent prolonged hypobaric hypoxia, which is a characteristic of high altitude and is known to induce oxidative injury in lung by increasing the generation of reactive oxygen species (ROS) and decreasing the effectiveness of the antioxidant defense system. We hypothesize that HPC with cobalt might protect the lung from subsequent hypobaric hypoxia-induced lung injury. HPC with cobalt can be achieved by oral feeding of CoCl(2) (12.5 mg kg(-1)) in rats for 7 days. Nonpreconditioned rats responded to hypobaric hypoxia (7619 m) by increased reactive oxygen species (ROS) generation and a decreased GSH/GSSG ratio. They also showed a marked increase in lipid peroxidation, heat-shock proteins (HSP32, HSP70), metallothionins (MT), levels of inflammatory cytokines (TNF-alpha, IFN-gamma, MCP-1), and SOD, GPx, and GST enzyme activity. In contrast, rats preconditioned with cobalt were far less impaired by severe hypobaric hypoxia, as observed by decreased ROS generation, lipid peroxidation, and inflammatory cytokine release and an inceased GSH/GSSG ratio. Increased expression of antioxidative proeins Nrf-1, HSP-32, and MT was also observed in cobalt- preconditioned animals. A marked increase in the protein expression and DNA binding activity of hypoxia-inducible transcriptional factor (HIF-1alpha) and its regulated genes, such as erythropoietin (EPO) and glucose transporter-1 (glut-1), was observed after HPC with cobalt. We conclude that HPC with cobalt enhances antioxidant status in the lung and protects from subsequent hypobaric hypoxia-induced oxidative stress.

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Activator protein-1 (AP-1) transcription factor plays a central role in hypoxia to modulate the expression of genes that decides the fate of the cell. The aim of the present study was to explore the role of AP-1 subunits in lung epithelial (A549) cells under hypoxia. Cell cycle studies by flow cytometry indicated that cell viability was unaffected by the initial hypoxia exposure (0.5% O at 37 °C) for 6 and 12 h. However, both transient cell cycle arrest and cell death was detected at 24 and 48 h. Flow cytometry and spectrofluorometry data confirmed the increase in ROS levels. Elevated ROS and calcium levels activated the stress-related MAPK signaling cascade. ERK and JNK were activated in early hypoxic exposure (within 6 h), whereas p38 were activated in 48 h of hypoxia. These subtypes further stimulated the subunits of AP-1 at different times of hypoxia exposure to orchestrate different genes responsible for cell proliferation (6 and 12 h) and apoptosis (24 and 48 h). Our results clearly depict the role of AP-1 heterodimer, i.e., p-c-jun/c-fos, p-c-jun/fosB, junD/c-fos, and junD/fosB in cell proliferation/survival by regulating the expression of Bcl-2 and cyclins (D1 and B1) at 6 h and 12 h of hypoxia, whereas junB/Fra-1 heterodimer have important role in apoptosis by regulating the expression of p53, Bax, and cyclin-dependent kinase inhibitors (p16, p21, p27) at 24 h and 48 h of hypoxia. Also, the cell survival signaling pathway NO-AKT interrupted at 24 h and 48 h of hypoxia indicating cell death. In conclusion, hypoxia for different time points activated different subunits of AP-1 that combined to form different heterodimers. These dimers regulated the expression of genes responsible for cell proliferation and apoptosis. Since, AP-1 plays a role in the decisive phenomenon of the cell to choose between proliferation and apoptosis; thus, its subunits or dimers could be a good therapeutic target for many diseases.

This study reports the role of MAPKs (JNK, ERK, and p38), and activator protein-1 (AP-1) transcription factor in the hypobaric hypoxia induced change in lung tissue. Healthy male Sprague-Dawley rats were exposed to hypobaric hypoxia for 6, 12, 24, 48, 72, and 120 hr. Hypoxia resulted in significant increase in reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and decreased nitric oxide (NO), these act as signaling molecules for activation of MAPK and also contribute in development of vascular leakage (an indicator of pulmonary edema) as confirmed by histological studies. Our results confirmed JNK activation as an immediate early response (peaked at 6-48 hr), activation of ERKs (peaked at 24-72 hr) and p38 (peaked at 72-120 hr) as a secondary response to hypoxia. The MAPK pathway up regulated its downstream targets phospho c-Jun (peaked at 6-120 hr), JunB (peaked at 24-120 hr) however, decreased c-Fos, and JunD levels. DNA binding activity also confirmed activation of AP-1 transcription factor in lung tissue under hypobaric hypoxia. Further, we analyzed the proliferative and inflammatory genes regulated by different subunits of AP-1 to explore its role in vascular leakage. Increased expression of cyclin D1 (peaked at 12-72 hr) and p16 level (peaked at 48-120 hr) were correlated to the activation of c-jun, c-Fos and JunB. Administration of NFκB inhibitor caffeic acid phenethyl ester (CAPE) and SP600125 (JNK inhibitor) had no effect on increased levels of Interferon-γ (IFN-γ), Interleukin-1 (IL-1), and Tumor Necrosis Factor-α (TNF-α) thereby confirming the involvement of AP-1 as well as NFκB in inflammation. Expression of c-jun, c-Fos were correlated with activation of proliferative genes and JunB, Fra-1 with pro-inflammatory cytokines. In conclusion immediate response to hypobaric hypoxia induced c-Jun:c-Fos subunits of AP-1; responsible for proliferation that might cause inhomogeneous vasoconstriction leading to vascular leakage and inflammation at increased duration of hypobaric hypoxia exposure.