Cayratia albifolia C.L.Li exerts anti-rheumatoid arthritis effect by inhibiting macrophage activation and neutrophil extracellular traps (NETs)

Abstract

Background: Cayratia albifolia C.L.Li (CAC), commonly known as "Jiao-Mei-Gu" in China, has been extensively utilized by the Dong minority for several millennia to effectively alleviate symptoms associated with autoimmune diseases. CAC extract is believed to possess significant anti-inflammatory properties within the context of Dong medicine. However, an in-depth understanding of the specific pharmaceutical effects and underlying mechanisms through which CAC extract acts against rheumatoid arthritis (RA) has yet to be established.

Methods: Twenty-four Sprague-Dawley rats were divided into four groups, with six rats in each group. To induce the collagen-induced arthritis (CIA) model, the rats underwent a process of double immunization with collagen and adjuvant. CAC extract (100 mg/kg) was orally administered to rats. The anti-RA effects were evaluated in CIA rats by arthritis score, hind paw volume and histopathology analysis. Pull-down assay was conducted to identify the potential targets of CAC extract from RAW264.7 macrophage lysates. Moreover, mechanism studies of CAC extract were performed by immunofluorescence assays, real-time PCR and Western blot.

Results: CAC extract was found to obviously down-regulate hind paw volume of CIA rats, with diminished inflammation response and damage. 177 targets were identified from CAC extract by MS-based pull-down assay. Bioinformatics analysis found that these targets were mainly enriched in macrophage activation and neutrophils extracellular traps (NETs). Additionally, we reported that CAC extract owned significant anti-inflammatory activity by regulating PI3K-Akt-mTOR signal pathway, and inhibited NETosis in response to PMA.

Conclusions: We clarified that CAC extract significantly attenuated RA by inactivating macrophage and reducing NETosis via a multi-targets regulation.

Keywords: Cayratia albifolia C.L.Li; Macrophage; NETosis; PI3K-Akt pathway; Rheumatoid arthritis.

Fig. 1

The main chemical components of CAC extract

Fig. 2

CAC extract prevented arthritis progression in CIA rats. A, B The hind paw volume (A) and arthritis index (AI) score (B) of hind paws in rats from: CON group (n = 6), CIA group (n = 6), CAC group (100 mg/kg/day, n = 6), MTX group (1 mg/kg/3 days, n = 6). C H&E staining of each group (scale bars: 50 μm). D–E IHC analysis of the expression of TNF-α (D) and IL-6 (E) in each group (scale bars: 25 μm). ^*P < 0.05 vs. CIA group; ^**P < 0.01 vs. CIA group; ^***P < 0.001 vs. CIA group; ^## P < 0.01 vs. control group; ^### P < 0.001 vs. control group

Fig. 3

Identification of pharmacological targets of CAC extract via ‘Target-fishing’ strategy. A Representative transmission election microscopy (TEM) image of Fe₃O₄ beads (scale bar, 500 nm). B Workflow of target identification of CAC extract based on pull-down/MS. C, D The pathway-gene network analysis of 177 potential target proteins (C) and proteins associated with RA (D) on Cytoscape

Fig. 4

CAC extract inhibited neutrophils extracellular traps (NETs) formation. A Representative image of purified neutrophils after Wright-Giemsa’s staining (scale bar, 100 μm). B CAC extract inhibited PMA-induced cfDNA release in neutrophils. C–E Representative immunofluorescence image of cfDNA (C), MPO (D) and CitH3 (E) staining of neutrophils (scale bar, 100 μm). PMA-induced (1 μM) cfDNA release in neutrophils was inhibited by CAC extract (50, 100, 200 μg/ml). The expressions of MPO and CitH3 induced by PMA (1 μM) in neutrophils were suppressed by CAC extract (50, 100, 200 μg/ml). *P < 0.05 vs. PMA group; **P < 0.01 vs. PMA group; ***P < 0.001 vs. PMA group; ^#P < 0.05 vs. control group

Fig. 5

CAC extract inhibited macrophage-mediated inflammation response via PI3K-Akt pathway. A LPS (1 μg/ml) with or without CAC (50, 100 and 200 μg/ml) did not affect cell viability. B CAC extract attenuated NO production. C–E CAC extract decreased mRNA expression levels of IL-6 (B), iNOS (C) and MCP-1 (D). F CAC extract inhibited PI3K-Akt-mTOR pathway in LPS-activated RAW264.7 cells. G Quantitative analysis for relative phosphorylation levels of PI3K (p-PI3K), Akt (p-Akt) and mTOR (p-mTOR) were performed by normalizing to model group. *P < 0.01 vs. LPS group; **P < 0.01 vs. LPS group; ***P < 0.001 vs. LPS group; ^## P < 0.001 vs. control group; ^### P < 0.001 vs. control group; N.S., not significant by ANOVA with Dunnett’s post-hoc test