Thymoquinone Regulated The Pulmonary Fibrosis By P2X7r-NLRP3 Signaling Pathway

Objective:Thymoquinone(TQ)is a kind of quinones with pharmacological activity,which has a good anti-inflammatory effect.In this study,A549 cells were stimulated by LPS-stimulated THP-1 supernatant in vitro and C57BL/6 mice were exposed to cigarette smoke in vivo to establish a pulmonary fibrosis model,and investigate the regulatory effect of thymoquinone on pulmonary fibrosis and its potential molecular biological mechanism.Methods:In vitro,we selected human lung adenocarcinoma cells A549 and mononuclear macrophage THP-1 cells for cell experiments.A549 cells were treated with LPS-stimulated THP-1 cells and then treated with TQ of 1～5μM for 6 hours After 6 hours,the total protein of A549 cells were extracted for Western blotting to detect the expression levels of proteins such as α-SMA,P2X7r,NLRP3,P-STAT3 and other proteins,or extracted RNA for quantitative analysis by RT-PCR.Meanwhile,the P2X7r gene was silenced to evaluate its role in the P2X7r-NLRP3 signaling pathway In order to further verify the effect of TQ on α-SMA and P2X7r,immunofluorescence staining experiments were also carried out in this study.In vivo,male C57BL/6 mice were exposed to cigarette smoke for a total of 3 hours per day and kept for 8 weeks to establish a pulmonary fibrosis model.Mice in the thymoquinone group were given 20mg/kg or 40mg/kg TQ once a day by gavagmg,while the dexamethasone group was admimstered 0.5mg/kg DEX.Followed,we took lung tissue from mice for follow-up experiments,and observed pathological changes in lung tissues by H&E staining,Sirius red staining and IHC staining.In addition,we were extracted total protein and RNA from lung tissues,and determined the expression of α-SMA,P2X7r,NLRP3,P-STAT3 etc.Results:In vitro experiments showed that TQ could significantly inhibit the abnormal expression of α-SMA,P2X7r,NLRP3,P-STAT3 in A549 cells induced by THP-1 superlative stimulation,it also can inhibited α-SMA and P2X7r’s red positive fluorescence expression significantly.In vivo experiments,to observed the H&E staining,Sirius red staining and IHC staining results,it was found that TQ could significantly improved the damage of alveolar cavity structure induced by cigarette smoke in mice,and reduced the excessive deposition of collagen lipid.And fluorescence staining results of immune tissues also showed that TQ could significantly inhibited the red positive fluorescence expression of P2X7r.In addition,TQ could successfully down-regulated the expression of protein and mRNA such as α-SMA,P2X7r,NLRP3 and P-STAT3 etc.Conclusion:Thymoquinone can significant improvement the sustained occurrence of cell damage and inflammatory response,which induced by LPS-stimulated THP-1 cells supernatant and cigarette smoke.Its mechanism of action may be to prevent the release of pro-inflammatory factors by regulating the P2X7r-NLRP3 and STAT3 signaling pathways,thus playing a protective role on the lung.Moreover,inhibition of P2X7r-NLRP3 signaling pathway may be a key and effective way to improve the development mechanism of pulmonary fibrosis.