Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium.

ETHNOPHARMACOLOGICAL RELEVANCE: Di-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. AIM OF THE STUDY: Considering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. MATERIALS AND METHODS: Transcriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. RESULTS: We confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. CONCLUSIONS: Our work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

Therapeutic effects of Chinese medicine Di-Long (Pheretima vulgaris) on rheumatoid arthritis through inhibiting NF-κB activation and regulating Th1/Th2 balance.

Chinese medicine Di-Long, the dried body of Pheretima vulgaris (Chen) has been used for the treatment of joint inflammation, arthralgia and numbness of limbs for many years. This study was to investigate the anti-rheumatoid arthritis (RA) effects of Di-Long and to explore its possible mechanisms. The identification and quantification of representative components in Di-Long extracts (DL) were carried out by HPLC analysis. The anti-RA effects and mechanisms of DL were studied in CIA mice, RAW 264.7 macrophages and spleen T lymphocytes. The Th1/Th2 cell ratio in CIA mice spleens were determined by Flow cytometry. The cytokine levels were determined by ELISA method. The expressions of p-NF-κB p65 in ankle joints of CIA mice were detected by Immunohistochemistry analysis. The phosphorylation of NF-κB signaling pathway in RAW 264.7 macrophages and expressions of T-bet and GATA-3 in CIA mice spleens were determined by Western blots. The treatment with DL significantly decreased the paw thickness, arthritis scores and inflammatory cells infiltration in CIA mice. The TNF-α, IL-6 concentrations in both mice serum and macrophages secretion were markedly reduced with the treatment of DL, as well as the phosphorylation of NF-κB pathway. DL inhibited the expressions of T-bet and GATA-3 and decreased Th1/Th2 cells ratio in CIA mice spleens. DL reduced IFN-γ, IL-2 levels in mice serum and spleen T lymphocytes, and increased IL-4 levels in CIA mice serum. Chinese medicine Di-Long have significant anti-RA effects. The mechanisms might be inhibiting the activation of NF-κB signaling pathway and regulating the balance of Th1/Th2 cells.

Anti-rheumatoid arthritis effects of flavonoids from Daphne genkwa.

OBJECTIVE: This study aims to select the most effective anti-Rheumatoid Arthritis (RA) component of flavonoids from Daphne genkwa Sieb. et Zucc. by anti-inflammatory and immunomodulatory effects in vitro, and to elucidate the mechanism. METHODS: The anti-inflammatory and immunomodulatory effects of total flavonoids (TF) and four flavonoid components (genkwanin, hydroxygenkwanin, luteolin and apigenin) were determined by pharmacological approach in LPS-induced RAW 264.7 macrophages and ConA-induced T lymphocytes. Principal component analysis (PCA) was used to obtain the optimal anti-RA component in vitro. Western blot and real-time quantitative PCR (q-PCR) were used to explore the mechanisms. Finally, the in vitro anti-RA effect was verified by human rheumatoid arthritis fibroblast-like synoviocytes (FLSs). RESULTS: TF and four flavonoids significantly reduced the expressions of NO, iNOS, TNF-α, IL-6, IFN-γ and IL-2. PCA showed that genkwanin was the most effective anti-RA component in vitro. Genkwanin inhibited nuclear factor-κB (NF-κB) pathway by decreasing the phosphorylation levels of IKK, IκB and NF-κB, and down-regulated the expressions of iNOS, COX-2 and IL-6 mRNA. Genkwanin also inhibited the abnormal proliferation of FLSs and down-regulated the secretions of NO and IL-6. CONCLUSION: The most effective anti-RA component was genkwanin. Genkwanin exerts anti-RA effect through down-regulating the activation of NF-κB pathway and mRNA expressions of inflammatory mediators, and also by inhibiting the abnormal proliferation of FLSs and its NO and IL-6 secretion levels.

Genkwanin ameliorates adjuvant-induced arthritis in rats through inhibiting JAK/STAT and NF-κB signaling pathways.

BACKGROUND: Genkwanin is a flavone isolated from the traditional Chinese herb Daphne genkwa. Our previous work proved that four flavonoids (including genkwanin) isolated from D. genkwa (FFD) significantly improved the symptoms of arthritis in rat models. Recent studies have revealed that genkwanin exhibited anti-inflammatory and immunomodulatory activities, both of which were closely related to the pathology of rheumatoid arthritis (RA). Therefore, studying the anti-RA effects and mechanisms of genkwanin may give us insight into FFD's therapeutic effects on RA. PURPOSE: This study aimed to investigate the anti-rheumatoid arthritis activity of genkwanin on adjuvant-induced arthritis (AIA) model in rats and explore the underlying mechanisms. METHODS: The anti-rheumatoid arthritis activity of genkwanin was evaluated on AIA rat model by determining the paw swelling degrees and arthritis index scores, along with histopathological analysis of joint tissues. The serum cytokine levels were measured by ELISA method, and serum NO levels were measured by Griess method. The expression and phosphorylation levels of proteins in JAK/STAT and NF-κB signaling pathways were determined by western blot analysis and immunohistochemistry analysis. RESULTS: Genkwanin significantly decreased the paw swelling and arthritis index in AIA rats and also decreased the inflammation and bone destruction in joint tissues. The serum TNF-α, IL-6, and NO concentrations were markedly reduced while the IL-10 concentration was markedly increased with the treatment of genkwanin. Genkwanin inhibited the activation of JAK/STAT and NF-κB signaling pathways in synovial tissues of AIA rats. CONCLUSION: Genkwanin exerted anti-rheumatoid arthritis effects on AIA rats through inhibiting the activation of JAK/STAT and NF-κB signaling pathways. The results obtained in this work lead us to suggest that Genkwanin could play a crucial role on the previously demonstrated anti-rheumatoid arthritis activity of flavonoid extract of D. genkwa (namely FFD).

Therapeutic effects of Smilax glabra and Bolbostemma paniculatum on rheumatoid arthritis using a rat paw edema model.

Smilax glabra Roxb. (Tufuling) and Bolbostemma paniculatum (Maxim.) Franquet (Tubeimu) are used as couplet medicine in traditional Chinese medicine for the treatment of arthritis. This study is conducted to provide evidence on their therapeutic effects on rheumatoid arthritis (RA) and to explore its possible mechanisms of action. The identification and quantification of representative components (Astilbin and Tubeimoside I) in the n-butyl alcohol fraction of this couplet medicine (BFCM) were carried out by HPLC-UV assays. The contents of Astilbin and Tubeimoside I in BFCM were 13.13% (15.434 min) and 3.4% (18.619 min) respectively. For the assessment of anti-RA and anti-inflammatory activities, a carrageenan-induced paw edema model in rats was used. The swelling rates of paws and levels of IL-1ß, IL-6 and TNF-α in the swelling tissue were determined. We observed that the BFCM exhibited significant inhibitory activity on carrageenan-induced paw edema model (p<0.01). The down regulated levels of IL-1ß, IL-6 and TNF-α (all p<0.05) were reported. The results indicate that BFCM possesses significant anti-RA and anti-inflammatory effects, and it has a potential to be developed as a new therapeutic agent against RA.

Analgesia effect of baicalein against NTG-induced migraine in rats.

BACKGROUND: Migraine is a complex nervous system disease characterized by typical throbbing and unilateral headache, which causes severe healthy and social issues worldwide. The purpose of this study was to investigate the effect of baicalein (BAI) on the treatment of migraine. MATERIAL AND METHODS: Twenty-four rats were randomly divided equally into four groups, including a blank group, model group, positive group (ibuprofen tablets 82mg/kg), and BAI group (60mg/kg). All rats were intragastrically treated with the corresponding treatment for 10 consecutive days, and they were subcutaneously injected with NTG (10mg/kg) 1h after the last treatment, except in the blank group. After model establishment, the behaviors of all rats, including scratching head and shaking body were observed continuously for 100min. Four hours after NTG treatment, all rats were anaesthetized and the blood was collected. Thereafter, nitric oxide (NO) in plasma was determined by colorimetric method, the level of calcitonin gene-related peptide (CGRP) and endothelin (ET) were detected by radioimmunoassay method. In addition, immunohistochemistry was applied to detect c-Fos neuronal activity in trigeminal nucleus caudalis (TNC). RESULTS: Behavioral research showed that BAI administration alleviated the hyperalgesia in migraine rats. Compared with the model group, the levels of NO and CGRP in BAI administration groups were markedly decreased (p<0.01), and the levels of ET was significantly increased (p<0.01). Meanwhile, immunohistochemistry results showed that NTG treatment significantly activated c-Fos neurons while BAI treatment inhibited the expression of c-Fos. CONCLUSIONS: BAI could alleviate the migraine-like headache induced by NTG, which is related to the regulation of vasoactive substances. These findings may contribute to the further study of BAI as a potential drug for migraine pharmacotherapy.