Maxing Shigan Decoction Mitigates Mycoplasma pneumonia-Induced Pyroptosis in A549 Cells via the NLRP3 Inflammasome

Fang Liu,1,2,* TianTian Liu,2,* Min Sun,2 JingMin Zhou,2 Feng Xue,2 ShuangHui Chen,2 Jia Chen,3 Lei Zhang1 1Department of Pediatrics, Pudong New Area People’s Hospital, Shanghai, 201200, People’s Republic of China; 2Department of Pediatrics, East Hospital Affiliated to Tongji University, Shanghai, 200123, People’s Republic of China; 3Department of Traditional Chinese Medicine, Shanghai Children’s Medical Center, Shanghai, 200127, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lei Zhang; Jia Chen Email cnhff3098@126.com; 304453596@qq.comBackground: Mycoplasma pneumoniae is a predominant cause of community-acquired respiratory infections. We recently discovered the clinical efficacy of Maxing shigan decoction (MXSG) in M. pneumoniae infection and designed a study to explore the mechanism of action.Methods: Serum IL-1β, IL-18, and TNF-α, and transcript expression of the NLR Family, Pyrin Domain Containing Protein 3 (NLRP3) were measured in the peripheral blood mononuclear cells (PBMCs) of 30 children with M. pneumoniae infection and 30 healthy donors. An in vitro model of M. pneumoniae infection in A549 cell culture was used to explore the curative effects and mechanisms of MXSG. Pyroptosis was measured by flow cytometry with activated caspase-1 and propidium iodide staining. IL-1β, IL-18, and TNF-α, and NLRP3 transcript expression were measured by qRT-PCR. Protein expression of NLRP3, Caspase-1, pro-caspase-1, IL-1β, pro-IL-1β, and GSDMD-N was determined by Western blotting. Experimental confirmation was performed in NLRP3-overexpressing A549 cells and in the presence of an NLRP3 inhibitor, INF39.Results: M. pneumoniae infection-induced IL-1β, IL-18, TNF-α, and mRNA expression of NLRP3 in PBMCs and promoted pyroptosis in A549 cells. It also induced IL-1β, IL-18, TNF-α, and up-regulated NLRP3, ro-IL-1β, Caspase-1, Pro-Caspase-1, and GSDMD-N in culture. Similar to the NLRP3 inhibitor INF39, MXSG (0.1, 0.2, and 0.4 mg/mL) suppressed pyroptosis induced by M. pneumoniae infection and decreased IL-1β (P < 0.001), IL-18, TNF-α in culture. MXSG down-regulated NLRP3, pro-IL-1β, Caspase-1, pro-Caspase-1, and GSDMD-N in infected cultures and mitigated NLRP3 overexpression-induced pyroptosis.Conclusion: MXSG mitigates M. pneumoniae-induced pyroptosis in A549 cells via the NLRP3 inflammasome.Keywords: Mycoplasma pneumoniae, pyroptosis, Maxing shigan decoction, NLRP3 inflammasome