Paeoniflorin inhibits PRAS40 interaction with Raptor to activate mTORC1 to reverse excessive autophagy in airway epithelial cells for asthma.

ABSTRACT

BACKGROUND: Bronchial asthma is a chronic condition characterized by airway inflammation and remodeling, which pose complex pathophysiological challenges. Autophagy has been identified as a practical strategy to regulate inflammation and remodeling processes in chronic inflammatory diseases with pathological characteristics, such as asthma. PF (Paeoniflorin) is a potential new autophagy regulatory compound. Previous studies have reported that PF can inhibit airway inflammation to alleviate allergic asthma, but whether this is mediated through the regulation of autophagy and the molecular mechanism of action remains unclear. PURPOSE: The aim of this study was to evaluate the inhibitory effect of natural small molecule PF on asthma by regulating epithelial autophagy. METHODS: The rat asthma model was established through intraperitoneal injection of OVA and aluminum hydroxide suspension, followed by atomized inhalation of OVA for a period of two weeks. Following treatment with PF, histopathology was observed using Masson and H&E staining, while airway Max Rrs was evaluated using a pulmonary function apparatus. Levels of inflammatory cells in BALF were detected using a blood cell analyzer, and levels of inflammatory factors in BALF were detected through Elisa. Expressions of p-PRAS40 and p-Raptor were observed through immunohistochemistry, and levels of Beclin1 and LC3B were observed through immunofluorescence. The structure and quantity of autophagosomes and autophagolysosomal were observed through TEM. An autophagy model of 16HBE cells was established after treatment with 10ng/mL IL13 for 30 minutes. PRAS40 (AKT1S1) overexpression and mutation of PF and Raptor binding site (K207M& L302I& Q417H) were introduced in 16HBE cells. Autophagy in cells was measured by mFRP-GFP-LC3 ADV fluorescent tracer. The binding sites of PF and Raptor were analyzed using the Autodock Tool. The p-mTOR, p-Raptor, p-PRAS40, LC3II/LC3I were detected through Western blot, and interaction between PRAS40-Raptor and Raptor-mTOR was detected through Co-IP. RESULTS: The results showed that PF effectively reduced airway inflammation, improved airway pathological changes and remodeling, and maintained lung function. Additionally, PF was found to reverse excessive autophagy in airway epithelial cells. Interestingly, PF activated the mTORC1 subunit PRAS40 and Raptor in airway epithelial cells by regulating their phosphorylation. PRAS40 is an endogenous mTOR inhibitor that promotes autophagy. PF competitively binds Raptor to PRAS40, promoting Raptor-mTOR interactions to activate mTORC1, an outcome that can be reversed by PRAS40 overexpression and site-specific amino acid codon mutations in Raptor. CONCLUSION: These findings suggest that PF intervention and inhibition of PRAS40-Raptor interaction are effective treatments for bronchial asthma. By activating mTORC1, PF effectively reverses excessive autophagy in airway epithelial cells, leading to improved airway function and reduced inflammation.