Identification of the target protein and molecular mechanism of honokiol in anti-inflammatory action

Searching the targets of natural products is very important for drug discovery and elucidating the mechanism of drug action and disease. Honokiol (HK), as the major active component of Magnolia officinalis Rehder & E.H.Wilson, has been widely used in medicine and cosmetics. Among its bioactivities, its anti-inflammatory activity is particularly impressive. However, the target protein of HK in anti-inflammatory action and its regulatory mechanism are unclear.Here, we identified the target protein and molecular mechanism of the anti- inflammatory action of HK.First, an LPS-induced septic shock model and DSS-induced ulcerative colitis model were used to assess the anti-inflammatory efficacy of HK. Second, the drug affinity responsive target stability, proteomics analysis, thermal shift assays and cellular thermal shift assays were used to identify and validate the target of HK. Finally, western blot, ELISA, LDH immunofluorescence staining, shRNA and LC/MS for L-leucine analysis were performed to determine the mechanism of the anti-inflammatory action of HK.This study revealed that HK significantly alleviated LPS-induced septic shock and DSS-induced ulcerative colitis in vivo, suggesting that HK has significant anti-inflammatory activity. HK treatment dramatically reduced IL-1β release and caspase-1 activation at different time points, showing that HK could inhibit both NLRP3 inflammasome priming and activation processes in cells. HK also suppressed adaptor apoptosis speck-like protein oligomerization. Mechanistically, SLC3A2 was identified as a direct target of HK in THP-1 cells. HK downregulated SLC3A2 expression by promoting its degradation via proteasome-mediated proteolysis. Further study demonstrated that HK triggered SLC3A2 to suppress NLRP3 inflammasome activation by significantly reducing the content of L-leucine transported into cells and lysosomes to block the mTORC1 pathway.Our work identified HK as a promising anti-inflammatory drug candidate through the SLC3A2/L-leucine/mTORC1/NLRP3 pathways.