Myrtenal and β-caryophyllene oxide screened from Liquidambaris Fructus suppress NLRP3 inflammasome components in rheumatoid arthritis

Background: Liquidambaris Fructus (LF) is the infructescence of Liquidambar formosana. In Traditional Chinese Medicine, LF has been used to treat joint pain, a common symptom of arthritis and rheumatism; however, a lack of pharmacological evidence has limited its applications in modern clinics. Therefore, this study aims to explore the protective effect of LF on rheumatoid arthritis (RA) and to identify its active ingredients. Methods: Rats with adjuvant-induced arthritis (AIA) were divided into 4 groups and administered petroleum ether extract of LF (PEL), ethyl acetate extract of LF (EEL), water extract of LF (WEL), or piroxicam (PIR) respectively for 3 weeks. Two additional groups were used as normal control (NC) and model control (MC) and administered distilled water as a placebo. The clinical scores for arthritis, bone surface, synovial inflammation and cartilage erosion were used to evaluate the therapeutic efficacy of each treatment. The serum IL-1 beta and TNF-alpha level and the expression of NLRP3, IL-1 beta and caspase-1 p20 in the synovial tissue of AIA rats were evaluated by ELISA and Western blot. The active ingredients of LF were investigated using network pharmacology and molecular docking methods, and their inhibition of NLRP3 inflammasome activation was verified in the human rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) model. Results: PEL could alleviate paw swelling, bone and joint destruction, synovial inflammation and cartilage erosion in the AIA rats, with significantly superior efficacy to that of EEL and WEL. PEL reduced IL-1 beta and TNF-alpha serum levels, and attenuated the upregulation of NLRP3, IL-1 beta and caspase-1 p20 expression in the synovial tissue of AIA rats. Network pharmacology and molecular docking results indicated that myrtenal and beta-caryophyllene oxide were the main two active ingredients of PEL, and these two compounds showed significant inhibition on TNF-alpha, NLRP3, IL-1 beta and caspase-1 p20 expression in RA-FLS. Conclusions: Myrtenal and beta-caryophyllene oxide screened from PEL could suppress the activation of NLRP3 inflammasome, thereby alleviating RA symptoms.