Study on the mechanism of action of Penehyclidine hydrochloride on LPS-induced acute lung injury by regulating autophagy through the mTOR/Keap1/Nrf2 signaling pathway

Acute lung injury (ALI) is a clinical syndrome characterized by pulmonary inflammation and edema, leading to impaired oxygenation and respiratory failure. Penehyclidine hydrochloride (PHC) has anticholinergic, anti-inflammatory, and antioxidant properties. In this paper, we investigated the protective role of PHC in ALI and explored its mechanism of action. Both in vivo and in vitro experiments were performed using LPS induction to establish an ALI model. Following PHC intervention, the assessment of lung injury was conducted using pathological section examination, mouse lung injury scoring, and ELISA to measure oxidative stress markers including myeloperoxidase (MPO), malondialdehyde (MDA), Super Oxide Dismutase (SOD), and Glutathione Peroxidase (GSH-Px), as well as inflammatory cytokine levels of TNF-α, IL-1β, and IL-18. Immunoblotting and immunofluorescence assays were employed to detect Autophagy markers and the mTOR/Keap1/Nrf2 signaling pathway. To confirm the role of Autophagy in the protective effects of PHC against ALI, we administered PHC in combination with Rapamycin (RAPA) or 3-Methyladenine (3-MA) to the model groups and evaluated the aforementioned parameters. Our findings revealed that in the LPS-induced ALI model, there was significant pulmonary histopathological damage and increased levels of MPO, MDA, TNF-α, IL-1β, and IL-18, along with decreased levels of SOD and GSH-Px in lung tissue or serum. These alterations were all reversed following PHC treatment. Additionally, compared to the ALI group, PHC administration reversed the expression of mTOR/Keap1/Nrf2 and Autophagy proteins LC3, Beclin-1 and p62 induced by LPS. Treatment with the mTOR Inhibitor (Autophagy Inducer RAPA) blocked the protective effects of PHC on lung injury, the mTOR/Keap1/Nrf2 signaling pathway, and Autophagy, while co-treatment with the Autophagy inhibitor 3-MA showed a significant protective effect on ALI. The results suggest that PHC has a notable protective effect on ALI, which may be achieved by modulating the mTOR/Keap1/Nrf2 signaling pathway to inhibit Autophagy.

Acute lung injury; Autophagy; MTOR/Keap1/Nrf2 signaling pathway; Penehyclidine hydrochloride.