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

Abstract

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-1β and TNF-α level and the expression of NLRP3, IL-1β 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-1β and TNF-α serum levels, and attenuated the upregulation of NLRP3, IL-1β and caspase-1 p20 expression in the synovial tissue of AIA rats. Network pharmacology and molecular docking results indicated that myrtenal and β-caryophyllene oxide were the main two active ingredients of PEL, and these two compounds showed significant inhibition on TNF-α, NLRP3, IL-1β and caspase-1 p20 expression in RA-FLS.

Conclusions: Myrtenal and β-caryophyllene oxide screened from PEL could suppress the activation of NLRP3 inflammasome, thereby alleviating RA symptoms.

Keywords: Liquidambaris Fructus; Myrtenal; NLRP3 inflammasome; Rheumatoid arthritis; β-Caryophyllene oxide.

Fig. 1

Petroleum ether extract of LF (PEL) alleviated the severity of adjuvant-induced arthritis (AIA), and the efficacy was superior to that of ethyl acetate extract of LF (EEL) and water extract of LF (WEL). A Mean clinical scores of AIA rats with different treatments. B Inhibition of paw inflammation of AIA rats with different treatments. Paw inhibition rate was calculated as follows: (paw volume before treatment − paw volume after treatment)/paw volume before treatment. Data were recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and two-way ANOVA, *P < 0.05 vs MC, **P < 0.01 vs MC, ***P < 0.001 vs MC, ****P < 0.0001 vs MC. (n = 6)

Fig. 2

Petroleum ether extract of LF (PEL) treatment significantly alleviated paw swelling and bone erosion in adjuvant-induced arthritis (AIA) rats, and the efficacy was superior to that of ethyl acetate extract of LF (EEL) and water extract of LF (WEL). A Macroscopic observation of paws of AIA rats with different treatments. B Micro-CT analysis of joints of AIA rats with different treatments under anesthesia using the Skyscan 1276 Micro-CT. C Bone surface area calculation of joints of AIA rats with different treatments, where higher bone surface area means more severe bone erosion. Data were recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and one-way ANOVA, *P < 0.05 vs MC, **P < 0.01 vs MC. (n = 2)

Fig. 3

Petroleum ether extract of LF (PEL) treatment significantly alleviated synovial inflammation and cartilage erosion in adjuvant-induced arthritis (AIA) rats. A Representative HE staining images and amplification of respective typical lesions (× 100) and semiquantitative score of synovial inflammation. Black arrows point to the infiltration of inflammatory cells or synovial hyperplasia. B Representative safranin O-fast green staining images (× 100) and semiquantitative score of cartilage erosion. Red arrows point to areas of cartilage loss. Blue arrows point to areas of bone erosion. Semiquantitative scores of synovial inflammation and cartilage erosion in NC were set to 0. Data were recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and one-way ANOVA, **P < 0.01 vs MC, ***P < 0.001 vs MC, ****P < 0.0001 vs MC. (n = 2)

Fig. 4

The serum levels of IL-1β and TNF-α of the adjuvant-induced arthritis (AIA) rats were quantified using ELISA. Petroleum ether extract of LF (PEL) treatment significantly reduced the production of IL-1β and TNF-α in the serum of AIA rats, and its efficacy was superior to that of ethyl acetate extract of LF (EEL) and water extract of LF (WEL). Data were recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and one-way ANOVA, ***P < 0.001 vs MC, ****P < 0.0001 vs MC. (n = 6)

Fig. 5

NLRP3 inflammasome pathway was activated in the ankle joint synovial tissue of the adjuvant-induced arthritis (AIA) rats. Treatment with petroleum ether extract of LF (PEL) significantly suppressed the expression of IL-1β, caspase-1 p20 and NLRP3, whereas treatment with ethyl acetate extract of LF (EEL) and water extract of LF (WEL) did not show significant improvement. Data were recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and one-way ANOVA, *P < 0.05 vs MC. (n = 3)

Fig. 6

Network pharmacology analysis indicated that many components’ targets were closely related to NLRP3 inflammasome activation. A Components of petroleum ether extract of LF (PEL) are represented by diamonds, and their potential targets are represented by hexagons. B Optimized PPI network between the components’ targets and the therapeutic targets of rheumatoid arthritis (RA). Components’ targets are marked in orange; therapeutic targets of RA are marked in green, with larger size indicating a more important role in the network; common targets are marked in purple; targets related to NLRP3 inflammasome activation are marked in yellow. Protein targets are represented by their gene names

Fig. 7

Binding interaction between β-caryophyllene oxide and p38 MAPK, and myrtenal and MEK1. A β-caryophyllene oxide can form hydrogen bonds with Met109 and Gly110 in the hinge region of p38 MAPK. B Myrtenal can bind with His145 and Met146 in the ATP-binding pocket of MEK1

Fig. 8

The production of IL-1β and TNF-α in human rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). ELISA was used to quantify the IL-1β and TNF-α secretion. Myrtenal and β-caryophyllene oxide could significantly reduce IL-1β production. The production of TNF-α was also significantly decreased after treatment with myrtenal and β-caryophyllene oxide. Data are recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and one-way ANOVA, *P < 0.05 vs MC, ***P < 0.001 vs MC, ****P < 0.0001 vs MC. (n = 6)

Fig. 9

The NLRP3 inflammasome pathway was activated in human rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). The expression of NLRP3, caspase-1 p20 and IL-1β in RA-FLS were significantly reduced after treating with myrtenal. β-caryophyllene oxide showed inhibitory effects on the expression of caspase-1 p20 and IL-1β, but not NLRP3. Data were recorded as mean ± SEM. Differences were assessed by Dunnett’s multiple comparisons test and one-way ANOVA, *P < 0.05 vs MC, **P < 0.01 vs MC, ***P < 0.001 vs MC. (n = 3)

Fig. 10

The predicted mechanism of the anti-RA effect of LF upon selected key targets