Anti‐cytokine antibodies attenuated neuroglial degeneration under hypoxic stress

Abstract

Aims/Purpose: Hypoxia is a vital cause of neurodegenerative diseases such as glaucoma and retinal ischemia. We investigated the potential neuroprotective effects of anti‐cytokine antibodies against hypoxic damage.Methods: An in vitro hypoxia model was established using cobalt chloride (CoCl2) stimulation in microglial cells (BV‐2). Immunofluorescence staining was performed to detect the expression markers of different microglial phenotypes in BV‐2. Different concentrations of CoCl2 (0–150 μM) were tested to generate dose–response curves. Following exposure to CoCl2 at an approximate IC50 of 0.1 mM, BV‐2 were treated with anti‐tumour necrosis factor‐alpha (anti‐TNF‐α) or anti‐ interleukin‐1 beta (anti‐IL‐1β) antibodies for 24 h. Cell viability was determined by MTS assay. Proteome changes in BV‐2 were analysed with the mass spectrometry‐based proteomics approach.Results: Immunocytochemistry revealed the expression of M1 marker consisting CD68 and M2 marker CD206 in the BV‐2 cells. Hypoxia induced by mimetic CoCl2 led to a concentration‐dependent viability impairment in BV‐2 and cells treated with 0.1 mM CoCl2 for 24 h showed decreased viability of ~46%‐ 50%. Interestingly, the application of anti‐TNF‐α (2ug/ml) and anti‐IL‐1β (4ug/ml) antibodies significantly improved the BV‐2 cell viability to 80% (p < 0.0001) and 76% (p < 0.0001), respectively. Proteomics and bioinformatics analyses of the differentially expressed proteins revealed that CoCl2 induced proteome alterations involved in mitochondrial dysfunction signalling pathway (p = 1.10 × 10−3), which were eliminated by the treatment with both anti‐TNF‐α and anti‐IL‐1β. Besides, CoCl2‐induced activation of endoplasmic reticulum (ER) stress response (p = 3.33 × 10−9) was alleviated by anti‐IL‐1β and anti‐TNF‐α treatment (p = 1.7 × 10−7 and p = 8.82 × 10−7, respectively). Particularly, anti‐TNF‐α antibody significantly downregulated proteins involved in apoptosis (p = 1.58 × 10−10). Anti‐IL‐1β induced Nrf2‐mediated antioxidant response and exerts protection against oxidative stress (p = 1.00 × 10−6).Conclusions: Antibodies targeting cytokines provide neuroprotective effects on microglial cells from hypoxic stress. These findings demonstrated the potential use of anti‐cytokines in the treatment of neurodegenerative diseases.