Magnolol alleviates pulmonary fibrosis inchronic obstructive pulmonary disease by targeting transient receptor potential vanilloid 4‐ankyrin repeat domain

Abstract

Transient receptor potential vanilloid 4 (TRPV4) plays a role in regulating pulmonary fibrosis (PF). While several TRPV4 antagonists including magnolol (MAG), have been discovered, the mechanism of action is not fully understood. This study aimed to investigate the effect of MAG on alleviating fibrosis in chronic obstructive pulmonary disease (COPD) based on TRPV4, and to further analyze its mechanism of action on TRPV4. COPD was induced using cigarette smoke and LPS. The therapeutic effect of MAG on COPD‐induced fibrosis was evaluated. TRPV4 was identified as the main target protein of MAG using target protein capture with MAG probe and drug affinity response target stability assay. The binding sites of MAG at TRPV4 were analyzed using molecular docking and small molecule interaction with TRPV4‐ankyrin repeat domain (ARD). The effects of MAG on TRPV4 membrane distribution and channel activity were analyzed by co‐immunoprecipitation, fluorescence co‐localization, and living cell assay of calcium levels. By targeting TRPV4‐ARD, MAG disrupted the binding between phosphatidylinositol 3 kinase γ and TRPV4, leading to hampered membrane distribution on fibroblasts. Additionally, MAG competitively impaired ATP binding to TRPV4‐ARD, inhibiting TRPV4 channel opening activity. MAG effectively blocked the fibrotic process caused by mechanical or inflammatory signals, thus alleviating PF in COPD. Targeting TRPV4‐ARD presents a novel treatment strategy for PF in COPD.

TRPV4 Activation during Guinea Pig Airway Smooth Muscle Contraction Promotes Ca2+ and Na+ Influx

“…High concentrations of KCl have been commonly used to study smooth muscle contraction elicited by membrane depolarization. We observed that incubation of tracheal tissues with 100 nM GSK219 decreased smooth muscle contraction induced by increasing KCl concentrations (10,20,40,80,120, and 160 mM) (Figure 9).…”

“…High concentrations of KCl have been commonly used to study smooth muscle contraction elicited by membrane depolarization. We observed that incubation of tracheal tissues with 100 nM GSK219 decreased smooth muscle contraction induced by increasing KCl concentrations (10,20,40,80,120, and 160 mM) (Figure 9). Since KCl-induced contraction of guinea pig ASM is entirely dependent on membrane depolarization and hence external Ca 2+ entry through L-VDCCs activation [40,61], we conducted patch-clamp experiments to rule out the possibility that GSK219 might block this channel.…”

“…Exposure to formaldehyde (a chemical broadly used in household products) and high relative humidity simultaneously intensified the activation of the TRPV4-p38 MAPK pathway, leading to worsening airway inflammation and increased mucus secretion [ 79 ]. It has also been shown that blocking TRPV4 activity and Ca 2+ influx in lung fibroblasts can effectively block the fibrotic process triggered by mechanical or inflammatory signals and alleviate pulmonary fibrosis in COPD [ 80 ]. Furthermore, in human bronchial epithelial cells, the TRPV4 channel can sense changes in volume.…”

“…D-600 was used to examine the potential involvement of L-VDCCs in ASM contraction caused by TRPV4 stimulation. In another set of experiments, cumulative concentration-response curves relating to histamine (His, 100 nM to 100 mM), carbachol (CCh, 10 nM to 32 mM), or KCl (10,20,40,80,120, and 160 mM) were generated in the absence (control) or continuous presence of 10, 100, or 1000 nM GSK219 incubated for 15 min. This procedure allowed us to examine the participation of TRPV4 during the ASM contraction (a mechanical stimulus) caused by classical bronchoconstrictor agonists and a chemical substance such as KCl.…”