Tibetan medicine Ershiwuwei Lvxue Pill attenuates collagen-induced arthritis via inhibition of JAK2/STAT3 signaling pathway.

Ershiwuwei Lvxue Pill (ELP, མགྲིན་མཚལ་ཉེར་ལྔ།), a traditional Tibetan medicine preparation, has been used hundreds of years for the clinical treatment of rheumatoid arthritis (RA) in the highland region of Tibet, China. However, the underlying mechanism of its therapeutic effect remains unclear. The present study aimed to investigate the potential pharmacological mechanisms of anti-arthritic effect of ELP. The main chemical constituents of ELP were analyzed by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF-MS). Forty-eight male Wistar rats (220 ± 20 g) were randomly divided into six groups: normal group, collagen-induced arthritis (CIA) group, methotrexate group (1.05 mg/kg), ELP groups (115, 230 and 460 mg/kg). CIA rat models were assigned to evaluate the anti-RA activity of ELP by determining the paws swelling, arthritis score, organ coefficients of spleen and thymus, and histopathological analysis of knee joints of synovial tissues. The levels of TNF-α, IL-10, IL-6 and IL-17 in serum were measured by ELISA. In addition, mRNA and protein expression levels associated with JAK2/STAT3 signaling pathway in synovial tissues of CIA rats were detected by qRT-PCR, immunohistochemistry and Western blot analyses. Fourteen main chemical constituents of ELP were quantitatively determined by UPLC-Q-TOF-MS analysis. Treatment with ELP reduced the paw swelling, arthritis score and organ coefficients of spleen and thymus. Histopathological examination revealed the protective effects of ELP on CIA rats with knee joint injury. The levels of serum pro-inflammatory cytokines (TNF-α, IL-6 and IL-17) were markedly reduced while the anti-inflammatory cytokine IL-10 was significantly increased with the treatment of ELP. Further investigations showed ELP down-regulated the mRNA and protein expression levels of Bcl-2, whereas up-regulated Bax, SOCS1 and SOCS3. Meanwhile, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 proteins from synovial tissues were dramatically decreased with the treatment of ELP, whereas no changes of the mRNA and protein expression levels of JAK2 and STAT3 were observed. These results indicated that ELP reduced the severity of arthritis and joint swelling, suggesting an antirheumatic effect on CIA rats. The possible mechanism is related to inhibiting inflammatory response and inducing apoptosis in synovial tissues by regulating JAK2/STAT3 signaling pathway. However, further in vivo and in vitro investigations are still needed to clarify the underlying mechanism of ELP in treating RA.

Liu C ，Zhao Q ，Zhong L ，Li Q ，Li R ，Li S ，Li Y ，Li N ，Su J ，Dhondrup W ，Meng X ，Zhang Y ，Tu Y ，Wang X ... -  《-》

Ethyl acetate extract of Tibetan medicine Rhamnella gilgitica ameliorated type II collagen-induced arthritis in rats via regulating JAK-STAT signaling pathway.

Rhamnella gilgitica Mansf. et Melch. (སེང་ལྡེང་།, RG) is a traditional Tibetan medicinal plant that is currently grown throughout Tibet. According to the theory of Tibetan medicine, RG is efficient for removing rheumatism, reducing swelling, and relieving pain. Hence, it has been used for the treatment of rheumatoid arthritis (RA) in Tibet for many years. However, there are no previous reports on the anti-RA activities of ethyl acetate extract of RG (RGEA). This study aimed to explore the anti-RA effect and mechanism of RGEA on collagen-induced arthritis (CIA) in rats. The CIA model was established in male Wister rats by intradermal injection of bovine type II collagen and Complete Freund's Adjuvant at the base of the tail and left sole, respectively. The rats were orally administered with RGEA (9.71, 19.43, or 38.85 mg/kg) for 23 days. The body weight, swelling volume, arthritis index score, thymus and spleen indices, and pathological changes were observed to evaluate the effect of RGEA on RA. Furthermore, the inflammatory cytokines in serum, such as interleukin1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin6 (IL-6), interleukin17 (IL-17), interferon-γ (INF-γ), interleukin4 (IL-4), and interleukin10 (IL-10) were measured by enzyme linked immunosorbent assay (ELISA) to explore the anti-inflammatory effects of RGEA. The terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining was used to examine apoptosis. Finally, the protein and gene expression of B-cell lymphoma-2-associated X (Bax), B-cell lymphoma 2 (Bcl-2), Caspase3, janus-activated kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), suppressor of cytokine signaling1 (SOCS1), and 3 (SOCS3) in synovial tissue were detected using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR). After the treatment with RGEA, the body weight of rats was restored, both the arthritis index and paw swelling were suppressed, and spleen and thymus indices were decreased. RGEA reduced the inflammatory cells and synovial hyperplasia in the synovial tissue of the knee joint, and suppressed bone erosion. Meanwhile, RGEA decreased the levels of IL-1β, IL-6, IL-17, TNF-α, and INF-γ, while increased the levels of IL-4 and IL-10. TUNEL fluorescence apoptosis results confirmed that RGEA obviously promoted the apoptosis of synovial cells. Further studies showed that RGEA inhibited the proteins and mRNAs expression of JAK2 and STAT3 as well as increased the proteins and mRNAs expression of SOCS1 and SOCS3. In addition, RGEA upregulated the expression of Bax and Caspase3, and downregulated the expression of Bcl-2. The anti-RA effectof RGEA might be related to the promotion of apoptosis and inhibition of inflammation, which regulated the JAK-STAT pathway.

Su J ，Li Q ，Liu J ，Wang H ，Li X ，Wüntrang D ，Liu C ，Zhao Q ，RuyuYao ，Meng X ，Zhang Y ... -  《-》

Abelmoschus manihot (L.) medik. seeds alleviate rheumatoid arthritis by modulating JAK2/STAT3 signaling pathway.

Tao Y ，Liu J ，Li M ，Wang H ，Fan G ，Xie X ，Fu X ，Su J ... -  《-》

Ershiwuwei Lvxue Pill alleviates rheumatoid arthritis by different pathways and produces changes in the gut microbiota.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that often results in joint destruction. Ershiwuwei Lvxue Pill (ELP), a prescription of Tibetan medicine, has been used for centuries for the clinical treatment of RA in Tibet, China. In a previous study, we reported that ELP could ameliorate RA symptoms in CIA rats by inhibiting the inflammatory response and inducing apoptosis in synovial tissues. It is still needed further to clarify the mechanisms of action of ELP in mitigating RA. In this study, we aim to elucidate the mechanism of action of ELP to improve RA joint damage and explore the changes in the intestinal flora and host metabolites. Firstly, we analyzed the main absorbed constituents of ELP in the serum of rats by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF/MS). Then, we verified the alleviating effects of ELP on cartilage injury and bone erosion as well as the inflammatory response in CIA rats by microCT, H&E staining, safranin-O staining, and ELISA. Moreover, we investigated the main factors that mediate joint damage, including the production of matrix metalloproteinases (MMPs) and osteoclast activity in the ankle of rats by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Further, we explored the molecular mechanisms of the MMPs production and osteoclast activity in CIA rats treated with ELP through various experiments such as ELISA, qRT-PCR, western blotting, and immunofluorescence assay. Besides, we investigated gut microbiota composition by 16S rDNA sequencing and serum metabolites through untargeted metabolomics. In addition, we analyzed the correlation between gut microbiota and metabolites by Spearman correlation analysis. In this study, we identified 20 compounds from rat serum samples, which could be the ELP components that improve RA. Moreover, we found that ELP could alleviate cartilage and bone injury by reducing MMP-1, MMP-3, and MMP-13 expression and osteoclast activity in CIA rats. Further studies demonstrated that ELP could reduce joint damage by inhibiting osteoprotegerin (OPG)/receptor activator for nuclear factor-κB ligand (RANKL) /nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinases (JNK) signal pathways. The 16S rDNA sequencing analysis indicated that there was a significant difference in the gut microbiota composition between the normal and CIA rats, and these differences were changed after ELP administration. ELP could alter the gut microbiota by increasing the abundance of the genus Lactobacillus and decreasing the abundance of Dorea, [Eubacterium]_ventriosum_group, Anaerostipes, Collinsella, Coprococcus_1, Ruminiclostridium_5, Ruminococcus_1, Family_XIII_UCG-001, Butyricicoccus, Erysipelotrichaceae_UCG-003, Lachnoclostridium, Faecalibacterium, Lachnospiraceae_UCG-010, Roseburia, Rs-E47_termite_group_norank, Treponema_2 genera. Non-targeted metabolomics analysis showed that ELP reduced arachidonic acid levels. The serum arachidonic acid level was significantly correlated with the abundance of 41 genera, particularly Collinsella and Lactobacillus. Our study shows that ELP can improve RA joint damage by inhibiting MMPs production and osteoclast activity, and regulating intestinal flora and host metabolites, which provides a novel insight into the ELP in alleviating RA.

Li Y ，Liu C ，Luo J ，Zeng Y ，Meng X ，Wang S ，Zhang Y ... -  《-》

Triterpene saponins from Guo-gang-long attenuate collagen-induced arthritis via regulating A20 and inhibiting MAPK pathway.

The stems of Entada phaseoloides (L.) Merr commonly named "Guo-gang-long", is a traditional Chinese folk medicine that has been used clinically in China for the treatment of arthritis. Our previous study described that triterpene saponins isolated from "Guo-gang-long" could inhibit the inflammatory response. However, the potential mechanism of "Guo-gang-long" on treatment of arthritis, and whether the triterpene saponins responsible for its anti-arthritic effect are unclear. To investigate the function and mechanisms of the triterpene saponins from E. phaseoloides (ES) in collagen-induced arthritis (CIA) rats. The chemical components of ES were analyzed by HPLC. Anti-arthritic activity of ES was investigated in CIA rats, which was established by immunization with bovine type II collagen. Three doses of ES (25, 50 and 100 mg/kg) were administrated using oral gavage to CIA rats daily for 3 weeks. The anti-arthritic activity of ES was evaluated by clinical arthritis scoring, paw swelling and mechanical sensitivity, as well as histological changes in CIA rats. The impacts of ES on the regulation of the ubiquintin-editing enzyme A20 and MAPK signaling pathway, and production of pro-inflammatory cytokines in CIA rats were examined by Western blot, quantitative real-time PCR, ELISA, and immunohistochemical staining, respectively. ES treatment relieved the paw swelling, hyperalgesia and joint destruction, and prevented the progression of arthritis in CIA rats. Meanwhile, ES suppressed the excessive mRNA levels and protein expression of TNF-α and IL-17 in synovial tissues and hind paw joints, and reduced the production of IL-1β, TNF-α and IL-17 in serum. Furthermore, ES up-regulated A20 and suppressed the phosphorylation of p38 and ERK1/2 in hind paw joints, as well as inhibiting the activation of spinal p38 in CIA rats. ES could relieve rheumatic symptoms and prevent the development of rheumatoid arthritis. The effects of ES may be mediated by reducing proinflammatory cytokine levels, up-regulating A20 expression, reducing p38 and ERK1/2 activation in periphery, and inhibiting of phospho-p38 in spinal cord.

Xiong H ，Luo M ，Ju Y ，Zhao Z ，Zhang M ，Xu R ，Ren Y ，Yang G ，Mei Z ... -  《-》