Preservation of mitochondrial homeostasis is responsible for the ameliorative effects of Suhuang antitussive capsule on non-resolving inflammation via inhibition of NF-κB signaling and NLRP3 inflammasome activation

Abstract

Ethnopharmacological relevance: Suhuang antitussive capsule (Suhuang), one of traditional antitussive Chinese patent medicines, has been used for the treatment of post-infectious cough and cough variant asthma in clinical practice. It has been demonstrated to show numerous biological actions including antitussive and anti-inflammatory effects.

Aim of the study: This study aims to investigate the effects of Suhuang on non-resolving inflammation and its underlying molecular mechanism.

Material and methods: In vitro, mitochondrial membrane potential and ROS were detected by flow cytometry analysis. mtDNA release and mPTP fluorescence were determined by Q-PCR and fluorescence microplate reader analysis. Cytochrome C release and 8-OHdG levels were evaluated by ELISA. Additionally, the effects of Suhuang on Drp1, MMP9, IκBα/NF-κB and NLRP3/ASC/Caspase-1 expression were determined by Q-PCR, gelatin zymography or immunoblot analysis. In vivo, C57/BL6 mice were orally administrated for 2 weeks with Suhuang, then lung injury was induced by LPS. Inflammatory mediators mRNA, histological assessment and NF-κB/Caspase-1/IL-1β levels were evaluated by Q-PCR, H&E staining and immunoblot analysis. Two sepsis models of mice were further used to evaluate its anti-inflammatory effects.

Results: Suhuang restored mitochondrial homeostasis by inhibiting Drp1 activation and mitochondrial fission. Besides, Suhuang reduced mPTP opening, mitochondrial membrane potential collapse, ROS overproduction and mtDNA release. Moreover, Suhuang down-regulated MMP9 expression. As a consequence of preserved mitochondrial homeostasis, Suhuang inhibited NF-κB pathway activation by prevention of NF-κB-p65 phosphorylation and IκBα degradation. Suhuang also limited NLRP3 inflammasome activation by blocking NLRP3-ASC interaction and promoting NLRP3 ubiquitination degradation. Drp1 knockdown in vitro diminished the inhibitory effects of Suhuang on inflammatory responses, indicating the essential role of Drp1 in the Suhuang's activity. Consistently, the therapeutic effects of Suhuang were confirmed in LPS-inhaled mice, which recapitulated the protective actions of Suhuang in mitochondrial homeostasis in vitro. Additionally, two sepsis models of mice confirmed the inhibitory effects of Suhuang on uncontrolled inflammation.

Conclusions: Altogether, our work reveals that Suhuang inhibits non-resolving inflammation through inhibition of NF-κB signaling and NLRP3 inflammasome activation by preserving mitochondrial homeostasis, providing new pharmacological data for the clinical use of Suhuang. Our study also suggests mitochondrial homeostasis as a potential intrinsic regulatory strategy for treating inflammatory diseases.

Keywords: 3-Methyladenine; Adenosine triphosphate; Cyclosporin A; Drp1; Lipopolysaccharide; Lung injury; Mdivi-1; Mitochondrial homeostasis; Monosodium urate; N-acetylcysteine; NF-κB signaling; NLRP3 inflammasome; Phorbol-12-myristate-13-acetate; PubChem CID: 11970143; PubChem CID: 12035; PubChem CID: 135398661; PubChem CID: 23697816; PubChem CID: 27924; PubChem CID: 3825829; PubChem CID: 5284373; PubChem CID: 5957; Suhuang antitussive capsule.