Nicorandil inhibits inflammasome activation and Toll-like receptor-4 signal transduction to protect against oxygen-glucose deprivation-induced inflammation in BV-2 cells

Abstract

Background and purpose: Our previous studies have demonstrated adenosine triphosphate-sensitive potassium channel (KATP channel) openers could protect against inflammatory response in brain disease, but little is known about the mechanisms involved in KATP channel openers inhibiting neuroinflammation.

Methods and results: In the present study, we found that oxygen-glucose deprivation (OGD) resulted in BV-2 cells activation, significantly increased tumor necrosis factor-alpha and interleukin-1beta (IL-1β) levels, accompanied by downregulating Kir6.1 subunit. Pretreatment with nicorandil, a KATP channel opener, could attenuate OGD-induced BV-2 cells activation and inhibit pro-inflammatory factors release. Further study demonstrated that OGD activated Toll-like receptor-4 (TLR4) signaling pathway and NOD-like receptor pyrin domain containing three inflammasome, thereby increased IL-1β production. Pretreatment with nicorandil could reverse the two pathways involved in IL-1β production.

Conclusions: Our findings reveal that KATP channel openers could protect against OGD-induced neuroinflammation via inhibiting inflammasome activation and TLR4 signal transduction.

Keywords: ATP-sensitive potassium channel; Inflammasome; Neuroinflammation; Nicorandil; Oxygen-glucose deprivation.

Figure 1

Oxygen–glucose deprivation (OGD) induces downregulation of Kir6.1 subunit in activated BV‐2 cells. The timecourse of lactate dehydrogenase (LDH) release (A) and MTT activity (B) of BV‐2 cells after OGD/reoxygenation. (C) The representative morphological alteration of BV‐2 cells after OGD 1 h and reoxygenation 24 h. Scale bar: 40 μM. (D) Western blotting analyses and represented immunoblots of K_ATP channels subunits in BV‐2 cells. *P < 0.05, **P < 0.01 versus control group. Results are shown as mean ± SEM of every three individual experiments.

Figure 2

Opening K_ATP channels decreases production of pro‐inflammatory cytokines from activated BV‐2 cells. (A) The represented appearance of BV‐2 cells under microscope. Scale bar: 40 μM. ELISA assays of tumor necrosis factor‐alpha (TNF‐α) (B), Interleukin‐1beta (IL‐1β) (C), and IL‐10 (D) in the medium of BV‐2 cells. *P < 0.05, **P < 0.01 versus lane 1; #P < 0.05 versus lane 4. Results are shown as mean ± SEM of every seven individual experiments.

Figure 3

Opening K_ATP channels downregulates Toll‐like receptor‐4 (TLR4) and phosphorylated IkappaB kinase (IKK) complex in activated BV‐2 cells. (A) Represented immunoblots of TLR4, IKKβ, and high mobility group box 1 (HMGB1). Western blotting analyses of TLR4 (B), pIKK (C), and HMGB1 (D) in BV‐2 cells. **P < 0.01 versus lane 1; #P < 0.05 versus lane 4. Results are shown as mean ± SEM of every three individual experiments.

Figure 4

Opening K_ATP channels suppresses inflammasome activation and production of interleukin‐1beta (IL‐1β) induced by oxygen–glucose deprivation (OGD). (A) Represented immunoblots of NLRP3, cleaved Caspase‐1, and IL‐1β. Western blotting analyses of NLRP3 (B), cleaved Caspase‐1 (C), and maturation of IL‐1β (D). *P < 0.05, **P < 0.01 versus lane 1; #P < 0.05, ##P < 0.01 versus lane 4. Results are shown as mean ± SEM in every four individual experiments.