Regulatory effect of mitoQ on the mtROS-NLRP3 inflammasome pathway in leptin-pretreated BEAS-2 cells

Abstract

Obese asthma is a phenotype of asthma whose occurrence is gradually increasing in both adults and children. The majority of studies have demonstrated that obesity is a major risk factor for asthma and the effect of obesity on the lungs is considerable. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome has been previously demonstrated to serve a role in obese asthma mediated by mitochondrial reactive oxygen species (mtROS). The aim of the present in vitro study was to investigate the effect of leptin on airway epithelial cells and the protective effect of the mitochondrial-targeted antioxidant mitoquinone (mitoQ). Human normal bronchial epithelial cell lines BEAS-2 cells were used and divided into 6 groups: Control group (negative control), DMSO group (solvent control), lipopolysaccharide (LPS) group (positive control), LPS + mitoQ group, Leptin group and Leptin + mitoQ group. CCK8 assay was used to establish the optimal concentration and incubation time of the drugs. mitoTracker probe and mitoSOX reagent were used to detect the integrity of mitochondrial membranes and the content of mtROS. mRNA expression levels were detected by reverse transcription-quantitative PCR analysis. It was revealed that the mitochondrial membrane was disrupted in the Leptin group, which recovered after treatment with mitoQ. As a result, the production of mitochondrial reactive oxygen species (mtROS) in the Leptin group was significantly increased (P<0.01), but following treatment with mitoQ, this overproduction of mtROS was significantly decreased to normal levels (P<0.01). Furthermore, the expression levels of NOD-, LRR- and pyrin domain-containing protein 3 NLRP3 and caspase-1 mRNA in the leptin-pretreated BEAS-2 cells were significantly increased compared with those in the control group (P<0.01), while they were decreased following mitoQ treatment (P<0.01). Taken together, these data suggested that leptin may promote airway inflammation partially through upregulating the mtROS-NLRP3 inflammasome signaling pathway in airway epithelial cells and mitoQ may be a potential treatment for obese asthma.

Keywords: LRR- and pyrin domain-containing protein 3 inflammasome; NOD-; leptin; mitochondrial reactive oxygen species; mitoquinone; obese asthma.

Figure 1

Effects of leptin, LPS and mitoQ on the viability of BEAS-2 cells determined by a CCK-8 assay. (A) Effect of leptin. After 24 h of treatment with leptin at the concentration of 100 ng/ml, the cell viability became significantly lower than that of the untreated cells (P<0.05). (B) Effect of LPS. After 6 h of treatment with LPS at the concentration of 10 µg/µl, the cell viability was significantly decreased compared with that of untreated cells (P<0.05). (C) Effect of mitoQ. The cell viability was significantly decreased after 1 h of treatment with mitoQ at the concentration of 500 nM compared with that of untreated cells (P<0.05). Values are expressed as the mean ± standard error of the mean of 3 samples analyzed in triplicate. ^#P<0.05 vs. untreated group incubated for 48 h; ^*P<0.05 vs. untreated group incubated for 24 h; ^∆P<0.05 vs. untreated group incubated for 6 h; ^&P<0.05 vs. untreated group incubated for 1 h. CCK-8, Cell Counting Kit-8; LPS, lipopolysaccharide; mitoQ, mitoquinone.

Figure 2

Effects of mitoQ on the mitochondrial membrane integrity in the LPS- and leptin-pretreated BEAS-2 cells. (A) Representative immunofluorescence images of samples stained with the mitoTracker probe (magnification, x100 in the upper panel and x200 in the magnified windows below). (B) Mean fluorescence intensity of the cells. Pretreatment with LPS and leptin resulted in a perforation/rupture of the mitochondrial membrane compared with the control group (P<0.01). Treatment with mitoQ significantly improved mitochondrial injury (P<0.01). Values are expressed as the mean ± standard error of the mean of 3 samples analyzed in triplicate. ^*P<0.01 vs. control group; ^∆P<0.01 vs. DMSO group; ^#P<0.01 vs. LPS group; ^&P<0.01 vs. leptin group. LPS, lipopolysaccharide; mitoQ, mitoquinone.

Figure 3

Effects of mitoQ on the production of mtROS in the LPS- and leptin-pretreated BEAS-2 cells. (A) Representative immunofluorescence images of samples stained with the mitoSOX probe and DAPI (magnification, x100). (B) Mean fluorescence intensity of the cells. LPS and leptin pretreatment resulted in an overproduction of mtROS compared with the control group (P<0.01). Treatment with mitoQ significantly attenuated this overproduction of mtROS (P<0.01). Values are expressed as the mean ± standard error of the mean of 3 samples analyzed in triplicate. ^*P<0.01 vs. control group; ^∆P<0.01 vs. DMSO group; ^#P<0.01 vs. LPS group; ^&P<0.01 vs. leptin group. mtROS, mitochondrial reactive oxygen species; LPS, lipopolysaccharide; mitoQ, mitoquinone.

Figure 4

Expression levels of NLRP3 and caspase-1 mRNA in BEAS-2 cells. The mRNA expression levels of (A) NLRP3 and (B) caspase-1 in the LPS- and leptin-pretreated BEAS-2 cells were all significantly increased compared with those in the control group (P<0.01). After mitoQ treatment, the expression levels were significantly decreased (P<0.01). Values are expressed as the mean ± standard error of the mean of 3 samples analyzed in triplicate. ^*P<0.01 vs. control group; ^∆P<0.01 vs. DMSO group; ^#P<0.01 vs. LPS group; ^&P<0.01 vs. leptin group. NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; mitoQ, mitoquinone; LPS, lipopolysaccharide.

Figure 5

Expression of IL-1β mRNA and protein in BEAS-2 cells. (A) the IL-1β mRNA expression in the LPS group and leptin group was significantly higher than that in the control group (P<0.01). Treatment with mitoQ significantly decreased the IL-1β mRNA expression induced by LPS and leptin (P<0.01). (B) The protein levels of IL-1β in the supernatant of LPS- and leptin-pretreated BEAS-2 cells were significantly increased compared with those in the control group (P<0.01). After mitoQ treatment, their expression levels were significantly decreased (P<0.01). Values are expressed as the mean ± standard error of the mean of 3 samples analyzed in triplicate. ^*P<0.01 vs. control group; ^∆P<0.01 vs. DMSO group; ^#P<0.01 vs. LPS group; ^&P<0.01 vs. leptin group. mitoQ, mitoquinone; LPS, lipopolysaccharide.