Bufei formula attenuates airway mucus hypersecretion in COPD through inhibition of TRIM56-mediated ITGB4 ubiquitination.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Bufei Formula (BFF), a traditional Chinese medicine, has been widely utilized in the clinical treatment of chronic obstructive pulmonary disease (COPD), though its underlying mechanisms remain unclear and warrant further investigation. AIM OF THE STUDY: To investigate the mechanisms by which BFF alleviates COPD airway mucus hypersecretion through the TRIM56-mediated ubiquitination. MATERIALS AND METHODS: A cigarette smoke-induced COPD mouse and cigarette smoke extract (CSE)-induced airway epithelial cells were established to evaluate the effect of BFF on airway mucus hypersecretion. The target protein of BFF was identified using DARTS technology. Co-immunoprecipitation (Co-IP) combined with mass spectrometry (MS) was used to identify the downstream proteins of TRIM56. RESULTS: BFF can effectively improve lung function and pathological changes, and inhibit inflammation in COPD mice, as well as downregulate the levels of mucin and MUC5AC in the lung. BFF-4, an active fraction derived from BFF, directly inhibits the expression of MUC5AC and TNF-α induced by CSE in epithelial cells. DARTS analysis and single-cell sequencing analysis revealed that TRIM56 is a target of BFF-4, and its mediated ubiquitination contributes to the effects of BFF-4. Moreover, high levels of TRIM56 and ubiquitinated proteins are expressed both in vivo and in vitro. BFF-4 significantly downregulates these expressions. Furthermore, overexpression of TRIM56 and ubiquitinated proteins markedly diminishes the inhibitory effect of BFF-4 on mucin expression. Overexpression of TRIM56 also counteracts the inhibition of ubiquitination by BFF-4. Co-IP-MS analysis revealed that TRIM56 binds to ITGB4, which in turn regulates the expression of MUC5AC. Furthermore, molecular docking identified the top five small molecules with the highest affinity for TRIM56 were identified, suggesting their potential for direct interaction with TRIM56. CONCLUSION: BFF-4 effectively ameliorates airway mucus hypersecretion in COPD by inhibiting TRIM56-mediated downregulation of protein ubiquitination, including ITGB4.