Sesquiterpene lactones-enriched fractions from Xanthium mongolicum Kitag alleviate RA by regulating M1 macrophage polarization via NF-κB and MAPK signaling pathway

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by activated M1-like macrophage in the joint. Xanthium mongolicum Kitag (X. mongolicum) is a traditional medicinal plant that has long been used to treat RA and other immune diseases in China. Methods: Fractions of X. mongolicum were separated based on polarity. Anti-RA activity of the fractions were screened by LPS-stimulated RAW264.7 macrophage in vitro. The major active compounds were identified by UPLC-MS and quantified by HPLC. The anti-RA effects of the active fraction was evaluated in complete freund's adjuvant (CFA)-induced arthritis and collagen-induced arthritis (CIA) mouse models in vivo and LPS-stimulated macrophage in vitro. Results: Sesquiterpene lactones-enriched fraction from X. mongolicum (SL-XM) exhibited the strongest anti-RA activity among all components in vitro. Five major constituents i.e., Xanthinosin (1), Xanthatin (2), Mogolide D (3), Mogolide E (4), and Mogolide A (5) were identified as major compounds of SL-XM. SL-XM ameliorated symptoms of CFA and CIA induced arthritis mice model. Furthermore, SL-XM treatment inhibited LPS-induced M1 macrophages polarization. In addition, SL-XM inhibited the phosphorylation of NF-κB and MAPK signaling pathways in LPS-induced macrophage and CIA-challenged mice. Discussion: The main anti-RA active fraction of X. mongolicum may be the Sesquiterpene lactones, which includes five key compounds. SL-XM may exert its anti-RA effect by suppressing M1 macrophage polarization via the NF-κB and MAPK signaling pathway.

Keywords: Xanthium mongolicum kitag; macrophage; rheumatoid arthritis; sesquiterpene lactone; traditional Chinese medicine.

FIGURE 1

Isolation and identification of SL-XM and the major sesquiterpene lactones. (A) Photo of Xanthium mongolicum Kitag. (B,D) TLC&HPLC analysis showed that sesquiterpene lactones were enriched in E−3 and E4-fractions. ELISA assay of TNF-α in the culture supernatants of RAW264.7 cells incubated with (C) Extracts; (E) Fractions; (F) Sub-fractions of X. mongolicum for 4 h and then incubated with LPS(100 ng/mL) for additional 24 h (G) LC-MS analysis the main sesquiterpene lactones in X. mongolicum.

FIGURE 2

SL-XM dose-dependently attenuates CFA-induced RA in mice. The mice were orally administrated with SL-XM (200, 400 mg/kg) or DXM (1 mg/kg) daily after CFA injection. (A) Schematic depiction of the experimental schedule; the body weight (B), Hind paw thickness (C), Ankle thickness (D) were measured daily during experiment procedure. Serum levels of TNF-α (E), IL-6 (F), and IL-1β (G) were measured 6 days after CFA challenged. (H) Representative image of the general features, X-ray and H&E histological graphs of the ankle joint at the end of experiment. n = 6 in each group, *p < 0.05, **p < 0.01 vs. Ctrl group. ^# p < 0.05, ^## p < 0.01 vs. CFA group.

FIGURE 3

Inhibitory effects of SL-XM on M1 macrophages polarization. (A) RAW264.7 cells were stimulated with various concentration of SL-XM for 24 h, and cell viability was measured by CCK8 kit. After RAW264.7 cells were stimulated with LPS (100 ng/mL) and different concentration of SL-XM for 24h, mRNA levels of Il-6 (B), Il-1β (C), Tnf-α (D), Inos (E), Il-12b (F) were determined by qRT-PCR; protein level of iNOS (G,H) was determined by Western blot analysis; protein level of CD86 (I) was determined by immunofluorescence; percentage of CD86-positive cells (J,K) was determined by flow cytometry. *p < 0.05, **p < 0.01 vs. Ctrl group. ^# p < 0.05, ^## p < 0.01 vs. LPS group.

FIGURE 4

Promotional effect of SL-XM on the polarization of M2 macrophages. After RAW264.7 cells were stimulated with IL-4 (20 ng/mL) and different concentration of SL-XM for 24h, mRNA levels of Il-10 (A), Cd206 (B), Arg-1 (C), Pgc1-β (D), Mgl1 (E), Mgl2 (F) were determined by qRT-PCR; protein level of Arg-1 (G,H), P-STAT6 and STAT6 (G,I) was determined by Western blot analysis; protein level of CD206 (J) was determined by immunofluorescence; percentage of CD206-positive cells (K,L) was determined by flow cytometry. *p < 0.05, **p < 0.01 vs. Ctrl group. ^# p < 0.05, ^## p < 0.01 vs. LPS group.

FIGURE 5

SL-XM regulated LPS-stimulated NF-κB and MAPK signaling pathways in RAW264.7 cells. After RAW264.7 cells incubated with SL-XM for 1 h and then stimulated with LPS (100 ng/mL) for 30 min, protein levels of P-IKKα/β, IKKα, IKKβ (A), P-IκBα, IκBα (B), P-P65, P65 (C), P-P38, P38 (D), P-JNK, JNK (E), P-ERK and ERK (F) were determined by Western blot analysis. In addition, NF-κB p65 nuclear translocation was determined by immunofluorescence (G) and western blot (H). *p < 0.05, **p < 0.01 vs. Ctrl group. ^# p < 0.05, ^## p < 0.01 vs. LPS group.

FIGURE 6

Therapeutic effects of SL-XM on CIA mice. (A) Schematic depiction of the experimental schedule. From day 21 to day 45 after the initial immunization (B) Arthritis scores, (C) Hind paw thickness, (D) Onset of CIA symptoms, (E) Body weight of mice in each group were evaluated every other day. (F) Representative image of the general features, X-ray, micro-CT and H&E histological graphs of the ankle joint at the end of experiment. Mean radiological scores (G) of the hind paws of each group. Mean synovial inflammation (H), cartilage erosion (I), and bone erosion scores (J) of the hind paws of each group. n = 8 in each group, ^* p < 0.05, ^** p < 0.01 vs. Ctrl group. ^# p < 0.05, ^## p < 0.01 vs. CIA group.

FIGURE 7

SL-XM reduces M1 cytokines, while increasing M2 cytokines in serum and ankle tissue of CIA mice. At the end of experiment serum level of M1 cytokines (IL-1β, IL-6, and TNF-α) (A) and M2 cytokines (IL-10 and IL-4) (B) were measured by indicated ELISA kits. At the end of experiment mRNA level of M1 cytokines (Il-1β, Il-6, Tnf-α, and Inos) (C) and M2 cytokines (Il-10 and Arg-1) (D) in the ankle tissue of CIA mice were determined by qRT-PCR. Protein levels of NF-κB and MAPK signaling pathway components in the ankle tissue were determined by Western blot analysis (E). (F) Schematic illustration of SL-XM regulates the M1 macrophage polarization through NF-κB to attenuate experimental RA mouse model symptoms. *p < 0.05, **p < 0.01 vs. Ctrl group. ^# p < 0.05, ^## p < 0.01 vs. CIA group.