Cardamonin Inhibited IL-1β Induced Injury by Inhibition of NLRP3 Inflammasome via Activating Nrf2/NQO-1 Signaling Pathway in Chondrocyte

Abstract

In this study we investigated the role and mechanism of cardamonin on IL-1β induced injury in OA. CHON-001 cells were treated with cardamonin and IL-1β and transfected with silencing nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability was detected by Cell Counting Kit-8 assay and flow cytometer assay was utilized for cell apoptosis assessment. IL-6, IL-8, TNF-α and Nrf2 mRNA expression was tested by qRT-PCR. Western blot was employed to evaluate MMP-3, MMP-13, Collagen II, Nrf2, NQO-1, NLRP3, Caspase 1 and apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) protein levels. In CHON-001 cells, IL-1β suppressed cell viability and Collagen II level while promoting cell apoptosis and expression of pro-inflammatory cytokines (IL-6, IL-8, TNF-α), MMPs (MMP-3, MMP-13), NQO-1, and NLRP3 inflammasome (NLRP3, Caspase 1 and ASC), with no significant influence on Nrf2. Cardamonin reversed the effect of IL-1β on cell viability, cell apoptosis, pro-inflammatory cytokines, MMPs, Collagen II, and NLRP3 inflammasome levels. In addition, cardamonin advanced Nrf2 and NQO-1 expression of CHON-001 cells. SiNrf2 reversed the function of cardamonin on IL-1β-induced cell apoptosis and expression of pro-inflammatory cytokines, Nrf2, NQO-1, and NLRP3 inflammasome in chondrocytes. Taken together Cardamonin inhibited IL-1β induced injury by inhibition of NLRP3 inflammasome via activating Nrf2/NQO1 signaling pathway in chondrocyte.

Keywords: IL-1β; NLRP3 inflammasome; Nrf2/NQO-1; Osteoarthritis; cardamonin.

Fig. 1. Cardamonin reversed the effect of interleukin (IL)-1β on repressing viability and inducing apoptosis as well as inflammatory factor levels in chondrocytes.

(A) Chemical structure of cardamonin. (B and C) Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay after treatment of different cardamonin concentrations (B) as well as treatment of IL-1β in combination with cardamonin (C). (D) Cell apoptosis was tested through flow cytometer assay after treatment of IL-1β and cardamonin. (E) Representative images of cell apoptosis tested by flow cytometer assay after treatment of IL-1β and cardamonin. (F) IL-6, IL-8 and tumor necrosis factor (TNF)-α mRNA levels were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) after treatment of IL-1β and cardamonin. ^^^p < 0.001 vs. Control group; ^p < 0.05 vs. Control group; ***p < 0.001 vs. IL-1β group; **p < 0.01 vs. IL-1β group; *p < 0.05 vs. IL-1β group. All experiments were repeated independently at least three times. Data were performed as the means ± SD.

Fig. 2. Cardamonin reversed the interleukin (IL)-1β-induced expression of collagen-related factors and activation of nucleotide binding oligomerization domain-like receptor 3 (NLRP3) inflammasome in chondrocytes.

(A and B) Representative images of matrix metalloproteinase (MMP)-3, MMP-13 and Collagen II (A) as well as nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (NQO-1), NLRP3, Caspase 1 and ASC (B) protein expression detected by western blot after treatment of IL-1β and cardamonin. β-Actin was used as a loading control. (C and D) MMP-3, MMP-13 and Collagen II (C) as well as Nrf2, NQO-1, NLRP3, Caspase 1 and ASC (D) protein expression was detected by western blot after treatment of IL-1β and cardamonin. β-Actin was used as a loading control. ^^^p < 0.001 vs. Control group; ***p < 0.001 vs. IL-1β group; **p < 0.01 vs. IL-1β group; *p < 0.05 vs. IL-1β group. All experiments were repeated independently at least three times. Data were performed as the means ± SD.

Fig. 3. Silencing nuclear factor erythroid 2-related factor 2 (siNrf2) reversed the effect of cardamonin on Nrf2 expression and cell apoptosis in interleukin (IL)-1β-mediated chondrocytes.

(A and B) Relative Nrf2 mRNA expression was tested through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) after transfection of siNrf2 without (A) or with (B) treatment of IL-1β and cardamonin. (C) Relative Nrf2 protein expression was assessed by western blot after treatment of IL-1β and cardamonin as well as transfection of siNrf2. β-Actin was used as a loading control. (D) Cell apoptosis was tested through flow cytometer assay after treatment of IL-1β and cardamonin as well as transfection of siNrf2. (E) Representative images of cell apoptosis tested by flow cytometer assay after treatment of IL-1β and cardamonin as well as transfection of siNrf2. ^†††p < 0.001 vs. silencing negative control (siNC) group; ***p < 0.001 vs. Control group; ^^^p < 0.001 vs. IL-1β group; ###p < 0.001 vs. IL-1β+Cardamonin+siNC group. All experiments were repeated independently at least three times. Data were performed as the means ± SD.

Fig. 4. Silencing nuclear factor erythroid 2-related factor 2 (siNrf2) reversed the effect of cardamonin on interleukin (IL)-1β-induced expression of inflammatory factors and activation of nucleotide binding oligomerization domain-like receptor 3 (NLRP3 ) inflammasome in chondrocytes.

(A) IL-6, IL-8 and tumor necrosis factor (TNF)-α mRNA expression levels were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) after treatment of IL-1β and cardamonin as well as transfection of siNrf2. (B and C) Representative images of NAD(P)H quinone dehydrogenase 1 (NQO-1), NLRP3, Caspase 1 and ASC protein bands (B) and NQO-1, NLRP3, Caspase 1 and ASC protein expression levels (C) were evaluated through western blot after treatment of IL-1β and cardamonin as well as transfection of siNrf2. β-Actin was used as a loading control. ***p < 0.001 vs. Control group; ^^^p < 0.001 vs. IL-1β group; ^###p < 0.001 vs. IL-1β+Cardamonin+silencing negative control (siNC) group; ^##p < 0.01 vs. IL-1β+Cardamonin+siNC group; ^#p < 0.05 vs. IL-1β+Cardamonin+siNC group. All experiments were repeated independently at least three times. Data were performed as the means ± SD.