Schisandrin treatment suppresses the proliferation, migration, invasion, and inflammatory responses of fibroblast-like synoviocytes from rheumatoid arthritis patients and attenuates synovial inflammation and joint destruction in CIA mice.
Rheumatoid arthritis (RA) is a systemic autoimmune disease causing joint dysfunction. As disease-modifying anti-rheumatic drugs (DMARDs) have poor efficacy in 20% to 25% of RA patients, additional novel RA medications are urgently needed. Schisandrin (SCH) has multiple therapeutic effects. However, whether SCH is effective against RA remains unknown.
To investigate how SCH affects the abnormal behaviours of RA fibroblast-like synoviocytes (FLSs) and further elucidate the underlying mechanism of SCH in RA FLSs and collagen-induced arthritis (CIA) mice.
Cell Counting Kit-8 (CCK8) assays were used to characterize cell viability. EdU assays were performed to assess cell proliferation. Annexin V-APC/PI assays were used to determine apoptosis. Transwell chamber assays were used to measure cell migration and invasion in vitro. RT-qPCR was used to assess proinflammatory cytokine and MMP mRNA expression. Western blotting was used to detect protein expression. RNA sequencing was performed to explore the potential downstream targets of SCH. CIA model mice were used to assess the treatment efficacy of SCH in vivo.
Treatments with SCH (50, 100, and 200 μΜ) inhibited RA FLSs proliferation, migration, invasion, and TNF-α-induced IL-6, IL-8, and CCL2 expression in a dose-dependent manner but did not affect RA FLSs viability or apoptosis. RNA sequencing and Reactome enrichment analysis indicated that SREBF1 might be the downstream target in SCH treatment. Furthermore, knockdown of SREBF1 exerted effects similar to those of SCH in inhibiting RA FLSs proliferation, migration, invasion, and TNF-α-induced expression of IL-6, IL-8, and CCL2. Both SCH treatment and SREBF1 knockdown decreased activation of the PI3K/AKT and NF-κB signalling pathways. Moreover, SCH ameliorated joint inflammation and cartilage and bone destruction in CIA model mice.
SCH controls the pathogenic behaviours of RA FLSs by targeting SREBF1-mediated activation of the PI3K/AKT and NF-κB signalling pathways. Our data suggest that SCH inhibits FLS-mediated synovial inflammation and joint damage and that SCH might have therapeutic potential for RA.
Lin W
，Liu Y
，Zhang S
，Xu S
，Qiu Q
，Wang C
，Liu D
，Shen C
，Xu M
，Shi M
，Xiao Y
，Chen G
，Xu H
，Liang L
... -
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Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/β-catenin pathway.
Shikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification.
This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms.
SKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-α-induced migration and invasion in RA FLS. The involvement of Wnt/β-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for β-catenin and western blot for pathway-related proteins.
SKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1β, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/β-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3β (Ser9) and β-catenin, the raised protein level of GSK-3β and the decreased nuclear translocation of β-catenin. Interestingly, the combination of LiCl (Wnt/β-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/β-catenin inhibitor) enhanced the therapeutic roles of SKN.
SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/β-catenin pathway.
Liu FY
，Wang MQ
，Liu MM
，Li T
，Wang XH
，Jiang F
，Wu XJ
，Cheng J
，Cai L
，Li R
... -
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PPAR-γ alleviates the inflammatory response in TNF-α-induced fibroblast-like synoviocytes by binding to p53 in rheumatoid arthritis.
Rheumatoid arthritis (RA) is characterized by synovial inflammation, synoviocyte expansion and damage to cartilage and bone. We recently reported that peroxisome proliferator-activated receptor (PPAR)-γ inhibited the proliferation and activation of fibroblast-like synoviocytes (FLS), and was downregulated in RA synovial. In this study we investigated the role of PPAR-γ in RA and the underlying mechanisms. Adjuvant-induced arthritis (AIA) was induced in rats; from D15, AIA rats were orally administered pioglitazone (30 mg·kg-1·d-1) or rosiglitazone (4 mg·kg-1·d-1) for 14 days. Collagen-induced arthritis (CIA) was induced in wild-type and Ppar-γ+/- mice. We showed that the expression of PPAR-γ was significantly reduced, whereas that of TNF-α was markedly increased in human RA FLS. In CIA mice, knockdown of PPAR-γ expression (Ppar-γ+/-) aggravated the ankle inflammation. Similarly, T0070907 (a PPAR-γ antagonist) or si-PPAR-γ promoted the activation and inflammation of TNF-α-induced FLS in vitro. On the contrary, administration of PPAR-γ agonist pioglitazone or rosiglitazone, or injection of ad-Ppar-γ into the ankle of AIA rat in vivo induced overexpression of PPAR-γ, reduced the paw swelling and inflammation, and downregulated activation and inflammation of FLS in RA. Interesting, injection of ad-Ppar-γ into the ankle also reversed the ankle inflammation in Ppar-γ+/- CIA mice. We conducted RNA-sequencing and KEGG pathway analysis, and revealed that PPAR-γ overexpression was closely related to p53 signaling pathway in TNF-α-induced FLS. Co-IP study confirmed that p53 protein was bound to PPAR-γ in RA FLS. Taken together, PPAR-γ alleviates the inflammatory response of TNF-α-induced FLS by binding p53 in RA.
Li XF
，Yin SQ
，Li H
，Yang YL
，Chen X
，Song B
，Wu S
，Wu YY
，Wang H
，Li J
... -
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Exploring the pharmacological mechanisms and key active ingredients of total flavonoids from Lamiophlomis rotata (Benth.) Kudo against rheumatoid arthritis based on multi-technology integrated network pharmacology.
Lamiophlomis rotata (Benth.) Kudo (LR, Lamiaceae) is a traditional Tibetan medicinal material in China. Tibetan medicine classic and research report suggested that LR could be used to cure rheumatoid arthritis (RA). However, the anti-RA active ingredients and pharmacological mechanisms of LR have not been elucidated.
To explore the mechanisms and key active ingredients of total flavonoids from LR (TFLR) against RA.
First, the mechanisms of TFLR against RA were investigated on collagen-induced arthritis (CIA) rat model by analyzing paw appearance, paw swelling, arthritis score, spleen index, thymus index, inflammatory cytokine (TNF-α, IL-1β, IL-6 and IL-17) levels in serum, histopathology of ankle joint and synovium from knee joint (hematoxylin-eosin, safranin O-fast green and DAB-TUNEL staining), and apoptosis-related protein (PI3K, Akt1, p-Akt, Bad, p-Bad, Bcl-xL and Bcl-2) levels in the synovium of ankle joints (Western blot). Then, the crucially active ingredients of TFLR against RA were explored by network pharmacology, ingredient analysis, in vitro metabolism and TNF-α-induced human RA synovial fibroblast MH7A proliferation assays. Network pharmacology was applied to predict the key active ingredients of TFLR against RA. The ingredient analysis and in vitro metabolism of TFLR were performed on HPLC, and MH7A proliferation assay were applied to evaluate the predicted results of network pharmacology.
TFLR shown excellently anti-RA effect by reducing paw swelling, arthritis score, spleen index, thymus index and inflammatory cytokine (IL-1β, IL-6 and IL-17) levels, and improving the histopathological changes of ankle joint and synovium from knee joint in CIA rats. Results of Western blot indicated that TFLR reversed the changes of PI3K, p-Akt, p-Bad, Bcl-xL and Bcl-2 levels in the ankle joint synovium of CIA rats. Results of network pharmacology exhibited that luteolin was identified as the pivotal active ingredient of TFLR against RA. The ingredient analysis of TFLR indicated that the main ingredient in TFLR was luteoloside. The in vitro metabolism study of TFLR suggested that luteoloside could be converted to luteolin in artificial gastric juice and intestinal juice. Results of MH7A proliferation assay showed that there was no significant difference between TFLR and equal luteoloside on the viability of MH7A cells, indicating that luteoloside was the key active ingredient of TFLR against RA. Additionally, the luteolin (same mol as luteoloside) showed better inhibitory effect on the viability of MH7A cells than luteoloside.
TFLR showed anti-RA effect, and the mechanism was related to promoting synovial cell apoptosis mediated by PI3K/Akt/Bad pathway. Meanwhile, this work indicated that luteoloside was the key active ingredient of TFLR against RA. This work lays a foundation for providing TFLR product with clear mechanism and stable quality to treat RA.
Zhan H
，Chen R
，Zhong M
，Wang G
，Jiang G
，Tao X
，Chen M
，Jiang Y
... -
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ResearchGate
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钛学术
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