KMS99220 Exerts Anti-Inflammatory Effects, Activates the Nrf2 Signaling and Interferes with IKK, JNK and p38 MAPK via HO-1

Abstract

Neuroinflammation is an important contributor to the pathogenesis of neurodegenerative disorders including Parkinson's disease (PD). We previously reported that our novel synthetic compound KMS99220 has a good pharmacokinetic profile, enters the brain, exerts neuroprotective effect, and inhibits NFκB activation. To further assess the utility of KMS99220 as a potential therapeutic agent for PD, we tested whether KMS99220 exerts an anti-inflammatory effect in vivo and examined the molecular mechanism mediating this phenomenon. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, oral administration of KMS99220 attenuated microglial activation and decreased the levels of inducible nitric oxide synthase and interleukin 1 beta (IL-1b) in the nigrostriatal system. In lipopolysaccharide (LPS)-challenged BV-2 microglial cells, KMS99220 suppressed the production and expression of IL-1b. In the activated microglia, KMS99220 reduced the phosphorylation of IκB kinase, c-Jun N-terminal kinase, and p38 MAP kinase; this effect was mediated by heme oxygenase-1 (HO-1), as both gene silencing and pharmacological inhibition of HO-1 abolished the effect of KMS99220. KMS99220 induced nuclear translocation of the transcription factor Nrf2 and expression of the Nrf2 target genes including HO-1. Together with our earlier findings, our current results show that KMS99220 may be a potential therapeutic agent for neuroinflammation-related neurodegenerative diseases such as PD.

Keywords: IκB kinase; Nrf2; heme oxygenase-1; mitogen-activated protein kinases; neuroinflammation.

Fig. 1. KMS99220 possesses anti-inflammatory properties

(A and B) Mice were administered with MPTP only or co-treated with 10 or 30 mg/kg KMS99220. The nigral sections were subjected to Iba-1 immunohistochemistry (A; scale bar = 100 μm), and the immunodensity of Iba-1-positive microglia was determined (B). The data are expressed as % of vehicle-treated group ± SEM (n = 10); *P < 0.05 vs vehicle-treated group; ^#P < 0.05 vs MPTP-treated group. (C and D) Mice were administered with MPTP only or co-treated with 30 mg/kg KMS99220. (C) Western blot analysis in striatal tissues for iNOS (130 kDa). β-actin (43 kDa) was used as an internal control. (D) ELISA results for IL-1β in nigral tissues. The data are expressed as % of vehicle-treated group ± SEM (n = 3); **P < 0.01 vs vehicle-treated group; ^##P < 0.01 vs MPTP-treated group. (E and F) BV-2 cells were exposed to various concentrations of KMS99220 with 0.2 μg/ml LPS. (E) RT-PCR results for IL-1β. GAPDH was used as an internal control. (F) ELISA for IL-1β at 24 h exposure to KMS99220. The data are expressed as % of LPS-treated control ± SEM (n = 3); ^##P < 0.01 vs LPS-treated control.

Fig. 2. KMS99220 suppresses the activation of IKK and MAPKs in activated microglia

BV-2 cells were pretreated with various concentrations of KMS99220 for 1 h and then exposed to 0.2 μg/ml LPS for 0.5 h. The total and phosphorylated levels of IKK (85/87 kDa), p38 MAPK (43 kDa), JNK (46/54 kDa), and ERK (42/44 kDa) were analyzed by Western blot analysis.

Fig. 3. HO-1 mediates the KMS99220-induced inhibition of IKK, p38 and JNK

(A and B) BV-2 cells were transfected with control or HO-1 siRNA for 24 h. (A) Western blot results of HO-1. (B) Western blot results of total and phosphorylated levels of IKK, JNK, p38 MAPK, and ERK in BV-2 cells after treatment with 10 μM KMS99220 for 1 h and incubation with 0.2 μg/ml LPS for 0.5 h. (C) Western blot results of total and phosphorylated levels of IKK, JNK, p38 MAPK, and ERK after pretreatment with SnPP for 1 h and subsequent treatment with 10 μM KMS99220 for 1 h followed by 0.2 μg/ml LPS for 0.5 h.

Fig. 4. KMS99220 activates the Nrf2 signaling pathway and induces HO-1 production in microglia

(A–I) BV-2 cells were treated with various concentrations of KMS99220. (A) Nuclear extracts from BV-2 cells were obtained after incubation with KMS99220 for 3 h, and the amount of nuclear Nrf2 (57 kDa) was analyzed by Western blot analysis, using lamin B (67 kDa) as an internal control. After incubation with KMS99220 for 24 h, Western blot analysis was performed for HO-1 (32 kDa) (B), NQO1 (31 kDa) (C), GCLC (73 kDa) (D), and GCLM (31 kDa) (E) using β-actin as an internal control. After incubation for 6 h, RT-PCR was performed for HO-1 (F), NQO1 (G), GCLC (H), and GCLM (I) using GAPDH as an internal control. The data are expressed as fold changes compared with untreated control ± SEM (n = 3); ^#P < 0.05, ^##P < 0.01 vs untreated control. (J) BV-2 cells were exposed to various concentrations of KMS99220 with 0.2 μg/ml LPS. After 6 h, RT-PCR was performed against HO-1, NQO1, GCLC, and GCLM using GAPDH as an internal control.

Fig. 5. A proposed mechanism of the anti-inflammatory effect of KMS99220 in microglia and dopaminergic (DA) neurons