Platycodon Grandiflorus Regulated The Pulmonary Fibrosis By P2X7r-NLRP3 Signaling Pathway

Objective:Platycodon grandiflorus(PG Extract),which is a commonly used medicine for traditional Korean medicine,has a good role in protecting the lungs.Platycodin D(PD)is the main saponin component of Platycodon grandiflorus.In this study,we investigated the regulation and the potential molecular mechanisms of the extract of Platycodon grandiflorus and Platycodin D on pulmonary fibrosis based on LPS-stimulated Raw264.7 supernatant on A549 cells in vitro and cigarette smoke-induced mice in vivo.Methods:In vivo,male BALB/c mice which were given cigarette smoke for a total of 3 hours per day for 11 weeks were administrated daily with a dose of 100mg/kg,200mg/kg,300mg/kg of the PG Extract and 20mg/kg of PD to establish a pulmonary fibrosis model.Followed,lung were taken to formalin or stored at-80℃.Paraffin sections or frozen sections of lungs were subjected to H&E staining,Masson staining,Sirius red staining,immunohistochemical staining and immunofluorescence staining to observe pathological changes of lung in mice.In addition,the total protein and RNA of lung were extracted for Western blotting and RT-PCR assay.The expression levels of protein and mRNA such as α-SMA,TLR4,P2X7r,NLRP3,IL-1β were detennined.In vitro,we used LPS-stimulated Raw264.7 supernatant on A549 cells and then treated with PG Extract(100 μg/mL)and PD(5 μM)for 30 minutes.The total protein and RNA of cells were extracted for Western blotting and RT-PCR assay The expression levels of protein and mRNA such as TLR4,P2X7r,NLRP3,IL-1βwere determined.Meanwhile,immunofluorescence staining was performed to observe the expression of a-SMA,P2X7r,NLRP3 by LPS-stimulated Raw264.7 supernatant on A549 cells which was treated with PG Extract and PD.Results:In vivo,the results of H&E,Masson and other staining results confirmed that the alveolar wall of lung tissue of mice in the cigarette smoke group was significantly thickened with interstitial edema and collagen fiber deposition.However,the alveolar structures of PG Extract and PD group mice were relatively intact,and the interstitial edema and collagen fiber deposition were improved.Administration of PG Extract and PD can inhibit the expression of a-SMA and Collagen-I in cigarette model mice,and also inhibit the expressions of TLR4,P2X7r,NLRP3 and IL-1p.In vitro,stimulation of Raw264.7 cell supernatant can increase the expressions of α-SMA and Collagen-I.It can also increase the expressions of TLR4,P2X7r,NLRP3 and IL-1β.PG Extract and PD can inhibit the increase of them.And PG Extract and PD can inhibit the increase of TLR4,P2X7r,NLRP3 and IL-1β induced by the stimulation of the supernatant of RAW264.7 cells.Conclusion:PG Extract and PD can down-regulate the expressions of a-SMA and Collagen-I to improve lung injury and pulmonary fibrosis induced by cigarette smoke.And they can also improve the release of inflammatory factors in A549 cells incubated with LPS-stimulated Raw264.7 supernatant to achieve certain lung protection.P2X7r may be the core target of PG Extract and PD in the treatment of pulmonary fibrotic inflammatory response,and PG Extract and PD may also regulate the development of pulmonary fibrosis by mediating the P2X7r-NLRP3 signaling pathway and reduce inflammation caused by cigarette smoke or inflammatory supernatant by inhibiting the TLR4 signaling pathway.