Zafirlukast ameliorates Docetaxel-induced activation of NOD-like receptor protein 3 (NLRP3) inflammasome, mediated by sirtuin1 (SIRT1) in hepatocytes

Abstract

Docetaxel-associated liver injury has become a serious public health problem, resulting in therapy discontinuation, liver failure, and death. Zafirlukast is a typical leukotriene receptor antagonist used for prophylaxis and chronic treatment of asthma. In this study, we investigate whether treatment with Zafirlukast could alleviate Docetaxel-induced cytotoxicity in hepatocytes. Our results indicate that Zafirlukast mitigated Docetaxel-induced toxicity in LO-2 hepatocytes. Firstly, Zafirlukast reduced the production of 8-hydroxy-2p-deoxyguanosine (8-OHdG) and increased the levels of reduced glutathione (GSH) against Docetaxel. Secondly, Zafirlukast elevated the levels of mitochondrial membrane potential (ΔΨm) and adenosine triphosphate (ATP). Thirdly, Zafirlukast prevented Docetaxel-induced release of lactate dehydrogenase (LDH) and increased cell viability of LO-2 hepatocytes against Docetaxel. We also found that Zafirlukast ameliorated Docetaxel-induced apoptosis by reducing Caspase-3 and Caspase-9 activity. Mechanistically, our results demonstrate that Zafirlukast inhibited the activation of NOD-like receptor protein 3 (NLRP3), mediated by SIRT1. Based on these findings, we conclude that the administration of Zafirlukast might have a protective effect against Docetaxel-induced cytotoxicity in hepatocytes.

Keywords: Docetaxel; LDH; SIRT1; Zafirlukast; hepatocytes.

Figure 1.

Zafirlukast ameliorated Docetaxel-induced oxidative stress in LO-2 hepatocytes. Cells were stimulated with Docetaxel (20 μM) in the presence or absence of Zafirlukast (5, 10 μM) for 24 hours. (a). The levels of 8-OHdG; (b). The levels of reduced GSH; (c). The levels of ROS generation (####, P < 0.0001 vs. control group; **, ***, P < 0.01, 0.005 vs. Docetaxel group)

Figure 2.

Zafirlukast mitigated Docetaxel-induced mitochondrial dysfunction in LO-2 hepatocytes. (a). Mitochondrial membrane potential (ΔΨm) was measured using RH123 staining; (b). The levels of intracellular ATP (####, P < 0.0001 vs. control group; **, ***, P < 0.01, 0.005 vs. Docetaxel group)

Figure 3.

Zafirlukast attenuated the cytotoxicity of Docetaxel in LO-2 hepatocytes. The release of LDH (####, P < 0.0001 vs. control group; **, ***, P < 0.01, 0.005 vs. Docetaxel group)

Figure 4.

Zafirlukast prevented Docetaxel-induced apoptosis in LO-2 hepatocytes. The activity of Caspase-3 and Caspase-9 (####, P < 0.0001 vs. control group; **, ***, P < 0.01, 0.005 vs. Docetaxel group) was measured using commercial kits

Figure 5.

Zafirlukast prevented Docetaxel-induced activation of NLRP3 inflammasome in LO-2 hepatocytes. (a). mRNA of NLRP3; (b). Protein levels of NLRP3; (c). The levels of IL-1β and IL-18 (####, P < 0.0001 vs. control group; **, ***, P < 0.01, 0.005 vs. Docetaxel group)

Figure 6.

Zafirlukast restored Docetaxel-induced reduction of SIRT1 in LO-2 hepatocytes. (a). mRNA of SIRT1; (b). Protein levels of SIRT1 (####, P < 0.0001 vs. control group; **, ***, P < 0.01, 0.005 vs. Docetaxel group)

Figure 7.

Silencing of SIRT1 abolished the protective effects of Zafirlukast against Docetaxel-induced damages in LO-2 hepatocytes. Cells were transduced with lentiviral SIRT1 shRNA, followed by stimulation with Docetaxel (20 μM) in the presence or absence of Zafirlukast (10 μM) for 24 hours. (a). Real time PCR revealed successful knockdown of SIRT1; (b). mRNA of NLRP3; (c). The levels of IL-1β and IL-18; (d). The activity of Caspase-3 (####, P < 0.0001 vs. control group; ***, P < 0.005 vs. Docetaxel group; $$, P < 0.01 vs. Docetaxel+ Zafirlukast group)