Opening of microglial K(ATP) channels inhibits rotenone-induced neuroinflammation

Abstract

As activated microglia (MG) is an early sign that often precedes and triggers neuronal death, inhibition of microglial activation and reduction of subsequent neurotoxicity may offer therapeutic benefit. The present study demonstrates that rat primary cultured MG expressed Kir6.1 and SUR2 subunits of K(ATP) channel, which was identical to that expressed in BV-2 microglial cell line. The classic K(ATP) channel opener pinacidil and selective mitochondrial K(ATP) (mito-K(ATP)) channel opener diazoxide prevented rotenone-induced microglial activation and production of pro-inflammatory factors (tumour necrosis factor[TNF]-alpha and prostaglandin E(2)[PGE(2)]). And the effects of pinacidil and diazoxide were reversed by mito-K(ATP) blocker 5-hydroxydecanoate (5-HD), indicating that mito-K(ATP) channels participate in the regulation of microglial activation. Moreover, the underlying mechanisms involved the stabilization of mitochodrial membrane potential and inhibition of p38/c-Jun-N-terminal kinase (JNK) activation in microglia. Furthermore, the in vivo study confirmed that diazoxide exhibited neuroprotective effects against rotenone along with the inhibition of microglial activation and neuroinflammation. Thus, microglial mito-K(ATP) channel might be a novel prospective target for the treatment of neuroinflammation-related degenerative disorders such as Parkinson's disease.

1

Primary cultured microglia expressed Kir6.1 and SUR2 subunits of K_ATP channels but not Kir6.2 and SUR1. (A) RT-PCR analysis of the Kir6.1 and SUR2 subunits in microglia. M, DNA marker; BT, whole brain tissue; MG, microglia. (B) Western blotting analysis of the Kir6.1 and SUR2 sub-units in microglia. (C) Immunolocalization of Kir6.1 and SUR2 subunits in microglia was examined using antibodies for Kir6.1 (green), SUR2 (green), colocalized with the microglial marker OX-42 (red). Scale bar: 25 μm.

2

Effects of K_ATP channel openers on rotenone-induced microglial activation and production of pro-inflammatory and neurotoxic factors. (A) Rotenone induced a decrease in the viability of microglia in a concentration-dependent manner. (B) K_ATP channel openers attenuated rotenone-induced morphological change of microglia. Rot, rotenone; Pin, pinacidil; Dia, diazoxide; Gli, glibenclamide; 5-HD, 5-hydroxydecanoate. (C) K_ATP channel openers reduced fluorescence intensity of rotenone-induced ED1-positive microglia (red). (D) K_ATP channel openers inhibited rotenone-induced production of TNF-α and PGE2 from microglia. (E) K_ATP channel openers inhibited rotenone-induced production of intracellular reactive oxygen species (ROS) in microglia. Scale bar: 50 μm (B and C); 100 μm (E). **P<0.01, ***P <0.001 versus control group; ###P <0.001 versus Rot group; ††P <0.01, †††P <0.001 versus Rot + Pin group; ‡P <0.05, ‡‡‡P <0.001 versus Rot + Dia group. Data are presented as the mean ± S.E.M. of four independent experiments.

3

Effects of mito-K_ATP channel opener diazoxide on rotenone-induced mitochon-drial depolarization and p38/JNK phos-phorylation in microglia. (A) JC-1 fluorescence imaging of mitochondria. Quantification of mitochondrial membrane potential expressed as a ratio of J-aggregate to JC-1 monomer (red: green) fluorescence. Scale bar: 25 μm. (B) Rotenone induced p38/JNK phosphoryla-tion in microglia, indicative of p38/JNK activation. Microglia were treated with 10 nm rotenone for indicated times. (C) Pre-treatment with diazoxide suppressed rotenone-induced p38/JNK phosphoryla-tion in microglia. 30 min after treatment, microglia were harvested and phosphory-lated p38/JNK were analysed. Lower: Representative blots are shown. Upper: Densitometric analysis of the phosphory-lated forms of p38/JNK. *P <0.05, ***P <0.001 versus control group; ###P <0.001 versus Rot group; †††P <0.001 versus Rot + Dia group. Data are presented as the mean ± S.E.M. of four independent experiments.

4

Effects of mito-K_ATP channel opener diazoxide on rotenone-induced rat behavioural symptoms. (A) Catalepsy test. (B) Rotarod test. ***P <0.001 versus control group; ###P <0.001 versus rotenone (2.5 mg/kg/day) group. Data are presented as the mean ± S.E.M., n= 12 per group.

5

Effects of mito-K_ATP channel opener diazoxide on rotenone-induced dopaminer-gic neuronal death and microglial activation in rat substantia nigra. (A) Images of immunohistochemistry. TH-positive cells (brown) represent dopaminergic neurons. Anti-GFAP was employed for staining astro-cytes (brown). OX-42 and ED1 immunos-taining (brown) were carried out to study microglial activation. Scale bar: 100μm (black), 20 μm (white). (B) TH-positive cell numbers in substantia nigra. ***P<0.001 versus control group; ###P <0.001 versus rotenone (2.5 mg/kg/day) group. (C) GFAP-positive cell numbers in substantia nigra. Data are expressed as mean ± S.E.M., n = 6 per group.

6

Effects of mito-K_ATP channel opener diazoxide on rotenone-induced production of pro-inflammatory factors in rat brain and peripheral blood. (A) Diazoxide decreased the content of TNF-α in subtantia nigra and peripheral blood compared with that of rotenone-treated rats. (B) Diazoxide down-regulated the mRNA levels of TNF-α and COX-2 in substantia nigra and striatum. ***P <0.001 versus vehicle group; ##P <0.01, ###P <0.001 versus Rot group. Data are expressed as mean ± S.E.M., n= 6 per group.