Docosahexanoic acid antagonizes TNF-α-induced necroptosis by attenuating oxidative stress, ceramide production, lysosomal dysfunction, and autophagic features

Abstract

Objective It was previously reported that docosahexanoic acid (DHA) reduces TNF-α-induced necrosis in L929 cells. However, the mechanisms underlying this reduction have not been investigated. The present study was designed to investigate cellular and biochemical mechanisms underlying the attenuation of TNF-α-induced necroptosis by DHA in L929 cells. Methods L929 cells were pre-treated with DHA prior to exposure to TNF-α, zVAD, or Necrostatin-1 (Nec-1). Cell death and survival were assessed by MTT and caspase activity assays, and microscopic visualization. Reactive oxygen species (ROS) were measured by flow cytometry. C16- and C18-ceramide were measured by mass spectrometry. Lysosomal membrane permeabilization (LMP) was evaluated by fluorescence microscopy and flow cytometry using Acridine Orange. Cathepsin L activation was evaluated by immunoblotting and fluorescence microscopy. Autophagy was assessed by immunoblotting of LC3-II and Beclin. Results Exposure of L929 cells to TNF-α alone for 24 h induced necroptosis, as evidenced by inhibition of cell death by Nec-1, absence of caspase-3 activity and lamin B cleavage, and morphological analysis. DHA attenuated multiple biochemical events associated with TNF-α-induced necroptosis, including ROS generation, ceramide production, lysosomal dysfunction, cathepsin L activation, and autophagic features. DHA also attenuated zVAD-induced necroptosis but did not attenuate the enhanced apoptosis and necrosis induced by combination of TNF-α with Actinomycin D or zVAD, respectively, suggesting that its protective effects might be limited by the strength of the cell death insult induced by TNF-α. Conclusions DHA effectively attenuates TNF-α-induced necroptosis and autophagy, most likely via its ability to inhibit TNF-α-induced sphingolipid metabolism and oxidative stress. These results highlight the role of this Omega-3 fatty acid in antagonizing inflammatory cell death.

Loss of neutral ceramidase protects cells from nutrient- and energy -deprivation-induced cell death

“…For example, the Sph analogue FTY720 induced necroptotic cell death and suppressed lung tumour growth [48]. Furthermore, it has been reported that docosahexanoic acid (DHA) attenuates TNF-α-induced necroptosis in part by reducing the levels of ceramide in the cells [49]. Our data indicate that cells deficient for nCDase were protected from 2DG/AA-induced necroptosis and that this protection may be via decreased generation of sphingoid bases as nCDase −/− cells generated ceramides following 2DG/AA treatment, but not Sph and S1P.…”

Pyrethroid exposure and neurotoxicity: a mechanistic approach

“…It is regulated by NF-κB, and its production can later enhance the activation of NF-κB (86). At higher levels TNFα can cause oxidative stress via ROS accumulation (88), and several studies have demonstrated that cypermethrin can increase the levels of TNFα (19,20,89). Cypermethrin has also been demonstrated to increase IL-1 levels in brain striatum (90).…”

Programmed cell death in periodontitis: recent advances and future perspectives

“…OS as a vital pathological mechanism might shed some light on the relationship between necroptosis/pyroptosis and periodontitis. On the one hand, OS has been implicated in periodontitis (Tsai et al , ; Akalin et al , ), while on the other hand, necroptosis and pyroptosis can be initiated by OS (Pacheco et al , ; Jang et al , ). Additionally, Bullon et al () concluded that OS was the common link that connected periodontitis and metabolic syndrome.…”