Studies have found that neutrophil extracellular traps (NETs) which are the specific dying form of neutrophil upon activation have fundamental role in the rheumatoid arthritis onset and progression. The purpose of this study was to explore the therapeutic effect of Sinomenine on adjuvant-induced arthritis in mice, and the neutrophil activities regulated by Sinomenine. The rheumatoid arthritis model was established by local injection of adjuvant and the Sinomenine treatment was administered orally for 30 days, during which, arthritic scores were evaluated and the joint diameter was measured to determine disease progression. The joint tissues and serum were acquired for further tests after sacrifice. Cytometric beads assay was performed to measure the concentration of cytokines. For paraffin-embedded ankle tissues, hematoxylin and erosin staining and Safranin O-fast staining were adopted to monitor the tissue changes of joint. In order to analyze the inflammation, NETs and autophagy of neutrophils in vivo, immunohistochemistry assays were applied to detect the protein expression levels in the local joints. To describe the effect brought by Sinomenine on inflammation, autophagy and NETs in vitro, the western blotting and the immunofluorescence assays were performed. The joint symptoms of the adjuvant induced arthritis were alleviated by the Sinomenine treatment significantly in terms of the ankle diameter and scores. The improvement of local histopathology changes and decrease of inflammatory cytokines in the serum also confirmed the efficacy. The expression levels of interleukin-6, P65 and p-P65 in the ankle areas of mice were remarkably reduced by Sinomenine. Compared with the model group, the decreased expression levels of lymphocyte antigen 6 complex and myeloperoxidase in the Sinomenine treating group showed the inhibitory effect of Sinomenine on the neutrophil migration. The expression of protein arginine deiminase type 4 (PAD4), ctrullinated histone H3 (CitH3) and microtubule-associated protein 1 light chain 3B (LC3B) had the similar tendency. Upon activation of lipopolysaccharide (LPS) in vitro, Sinomenine suppressed the phosphorylation of P65, extracellular signal-regulated kinase (ERK) and P38 of neutrophil. Meanwhile, Sinomenine inhibited NETs formation induced by phorbol 12-myristate 13-acetate (PMA), which were demonstrated by the decreased expression of neutrophil elastase (NE), PAD4 and CitH3. Sinomenine also inhibited PMA-induced autophagy in vitro based on the changes of Beclin-1 and LC3B. Sinomenine has good efficacy in treating adjuvant induced arthritis via regulating neutrophil activities. Apart from inhibiting activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, the mechanism includes suppression of NETs formation via autophagy inhibition.
Tibetan medicine is an important part of traditional Chinese medicine and a significant representative of ethnic medicine in China. Tibetan medicine is gradually recognized by the world for its unique curative effects. Wuwei Shexiang pills (WPW) has been widely used to treat “Zhenbu” disease (Also known as rheumatoid arthritis) in Tibetan medicine, however, its potential bioactive ingredients and mechanism for RA treatment remain unclear. In this study, we used a combination of gas chromatography-mass spectrometry (GC-MS), ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC-Q-TOF/MS), network analysis and experimental validation to elucidate the potential pharmacodynamic substances and mechanisms of WPW in the treatment of rheumatoid arthritis (RA). The results showed that songoramine, cheilanthifoline, saussureanine C, acoric acid, arjunolic acid, peraksine, ellagic acid, arjungenin and other 11 components may be the main activities of WPW in the treatment of RA. PIK3CA, AKT, MAPK, IL-6, TNF, MMP1, MMP3, and CDK1 are considered as core targets. PI3K-AKT, MAPK, apoptosis, cell cycle, and other signaling pathways may be the key pathways for WPW to play a role in the treatment of RA. Furthermore, we validated the underlying molecular mechanism of WPW predicted by network analysis and demonstrated its possible mechanism through in vivo animal experiments. It was found that WPW could significantly improve the degree of paw swelling, and reduce ankle joint diameter and arthritis index. Further histomorphological analysis showed that WPW could reduce the degree of synovial tissue inflammation and ankle joint cartilage damage. Meanwhile, WPW could down-regulate the levels of IL-6, IL-1β, and IL-17, and increase the levels of IL-10 and IL-4 in the serum of AA rats. TUNEL staining confirmed that WPW could significantly promote the apoptosis of synovial cells. Moreover, the immunohistochemical results showed that WPW decreased the expression of PI3K, AKT, MAPK, MMP1, MMP3, CDK1, and Bcl-2, as well as increased the expression of Bax protein. In conclusion, we successfully combined GC-MS, UPLC-Q-TOF/MS, network analysis, and experimental validation strategies to elucidate the inhibition of inflammation by WPW in AA model rats via PI3K/AKT, MAPK, cell cycle and apoptotic pathways process. This not only provides new evidence for the study of potential pharmacodynamic substances and the mechanism of WPW in the treatment of RA, but also provides ideas for the study of other Tibetan medicine compound preparations.
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