Abstract
This study investigated the function of a chloride channel blocker, DIDS. Both in vitro and in vivo studies found that DIDS significantly inhibits lipopolysaccharide (LPS)-induced release of proin flammatory cytokines. Here, we show that DIDS inhibits LPS-induced inflammation, as shown by downregulation of inflammatory cytokines via inhibition of the TLR4/NF-κB pathway. Furthermore, we show that ClC-3siRNA transfection reduces LPS-induced pro-inflammation in Raw264.7 cells, indicating that ClC-3 is involved in the inhibitory effect of DIDS during LPS-induced cytokines release. In vivo, DIDS reduced LPS-induced mortality, decreased LPS-induced organic damage, and down-regulated LPS-induced expression of inflammatory cytokines. In sum, we demonstrate that ClC-3 is a pro-inflammatory factor and that inhibition of ClC-3 inhibits inflammatory induction both in vitro and in vivo, suggesting that ClC-3 is a potential anti-inflammatory target.
Highlights
Inflammation is an essential immune response that is characterized by pain, swelling, redness, heat, and impaired function[1]
Inflammatory production of IL1-α, IL1-β,IL-6 and tumor necrosis factor (TNF)-αwas induced by LPS (100 ng/mL). Both DIDS (100 μmol/L) and ClC-3 small interfering RNA (siRNA) significantly down-regulated the production of IL1-α, IL1-β, IL-6 and TNF-α(Fig. 1A)
When RAW264.7 cells were incubated with DIDS combined with a stimulatory dose of LPS, IL1-α(Fig. 1B,C), IL1-β(Fig. 1D,E), IL-6 (Fig. 1F,G), and TNF-α(Fig. 1H,I) production were significantly reduced compared to the response to LPS alone
Summary
Inflammation is an essential immune response that is characterized by pain, swelling, redness, heat, and impaired function[1]. Macrophages are activated by a variety of inflammatory stimuli, of which bacterial lipopolysaccharide plays an important role. Nuclear factor kappa B (NF-κB), a common transcription factor, regulates various genes encoding inflammatory mediators and acts as an important downstream target of MAPK signaling pathways in inflammatory and immune responses[7,8]. NF-κB signaling is a well-studied classical signaling pathway that is involved in the inflammatory response, as well as the regulation of cell proliferation and apoptosis. Chloride channels play important functional roles in diverse processes, such as blood pressure regulation, cell cycle and apoptosis, muscle tone, volume regulation, synaptic transmission and cellular excitability in mammals[9]. Chloride channels are involved in a diverse set of functions in normal physiology and acquired diseases[10], ClC-3 is closely related to the inflammatory response. ClC-3 is an essential regulator of nuclear factor NF-κB signaling, because ClC-3 deficiency significantly decreases NF-κBactivity in mouse aortic smooth muscle cells[11]
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