Effect and Mechanism of Yuxuebi Tablet Against Collagen-Induced Arthritis of Rats / 中国实验方剂学杂志

Objective:To explore the intervention effect of Yuxuebi tablet （YXB） on collagen-induced arthritis （CIA） in rats and its anti-inflammatory mechanism. Method:Following CIA modeling， the rats in the drug administration groups were separately treated with intragastric administration of YXB （0.1， 0.2， and 0.4 g·kg^-1） and methotrexate （MTX， 0.4 mg·kg^-1）， once a day. The incidence of CIA， mechanical pain threshold （MPT） and cold pain threshold （CPT） were evaluated once every three days. After continuous administration for 30 days， the peripheral blood of rats was collected for the determination of platelet （PLT） count and fibrinogen （FIB） content. The hematoxylin-eosin （HE） staining was conducted to analyze the pathological changes in joint tissues. The protein expression levels of interleukin （IL）-1<italic>β</italic>， IL-8， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） p65， phosphorylated NF-<italic>κ</italic>B （p-NF-<italic>κ</italic>B） p65， Ras， and Raf-1 in joint tissues of CIA rats were detected by immunohistochemistry （IHC） and Western blot. The rheumatoid arthritis fibroblast-like synoviocytes （RA-FLS） were induced by tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>， 10 μg·L^-1） <italic>in vitro</italic> and then subjected to transwell migration/invasion assay， followed by the detection of protein expression levels of Ras， Raf-1， and p-NF-<italic>κ</italic>B p65 in RA-FLS by Western blot. Result:Compared with the control group， the model group exhibited an increased incidence of CIA， significantly decreased MPT （<italic>P</italic><0.05，<italic>P</italic><0.01）， elevated CPT （<italic>P</italic><0.01） and PLT and FIB in the peripheral blood， worsened histopathological score of joints， enhanced RA-FLS migration and invasion， and up-regulated inflammatory factors （<italic>P</italic><0.01）. The comparison with the model group revealed that YXB at different doses obviously reduced the incidence of CIA， increased MPT， down-regulated CPT and PLT and FIB in the peripheral blood （<italic>P</italic><0.05，<italic>P</italic><0.01）， ameliorated the pathological changes like synovial hyperplasia and bone and cartilage destruction （<italic>P</italic><0.05，<italic>P</italic><0.01）， and inhibited RA-FLS migration and invasion. Besides， the low-， medium-， and high-dose YXB reversed the IL-1<italic>β</italic>， IL-8， Ras， Raf-1， and p-NF-<italic>κ</italic>B p65 expression in joint tissues of CIA rats to different extents， as well as the protein expression of Ras， Raf-1 and p-NF-<italic>κ</italic>B p65 in RA-FLS （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:YXB reduces the incidence of CIA， ameliorates the clinical symptoms of RA and the pathological changes in joint tissues， and inhibits the formation of synovium， which may be attributed to its inhibition against Ras/Raf-1/NF-<italic>κ</italic>B signaling pathway.

Brusatol inhibits the invasion behavior of rheumatoid arthritis fibroblast-like synoviocytes through regulating mechanical properties of cytoskeleton / 医用生物力学

Objective To investigate the effects of brusatol on mechanical properties of the cytoskeleton as well as the invasion behavior of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Methods Cytoskeleton staining method was used to determine the regulatory effects of brusatol on mechanical properties of the RA FLS cytoskeleton. Transwell chamber assay was used to detect the effects of brusatol on the cytoskeleton and invasion behavior of RA FLS. The effects of brusatol on the expression of matrix metalloproteinase-2/3 (MMP-2/3) of RA FLS were measured by zymography and Western blotting methods. Results Cytoskeleton staining and microscope observation showed that brusatol could significantly reduce the formation number of RA FLS pseudopodia, thus inhibited cell movement ability via regulating mechanical properties of cytoskeleton. The invasion behavior of RA FLS was inhibited by brusatol, and brusatol could down-regulate the expression of MMP-2/3. Conclusions Brusatol plays an important role in regulating mechanical properties of cytoskeleton and inhibiting the invasion behavior of RA FLS. Meanwhile, brusatol can inhibit the invasion behavior of RA FLS through down-regulating the expression of MMP-2/3. The research findings provide the corresponding experimental basis for further development of new drugs for RA treatment.

Effects of cyclic mechanical stretch on BMP-2 expression in RA and OA fibroblast-like synoviocytes / 医用生物力学

Objective To explore the role of mechanical stimulation in synovium under different pathological conditions through studying the effects of cyclic mechanical stretch on the expression of bone morphogenetic protein 2 (BMP-2) in rheumatoid arthritis (RA) and osteoarthritis (OA) fibroblast-like synoviocytes (FLS). Method 6% and 0.5 Hz stretch generated by Flex cell 4000 tension systems was applied on normal, RA and OA FLS of human knee joint source under normal and inflammatory conditions for 2 h or 6 h, respectively. Results Cyclic mechanical stretch of 6%, 0.5 Hz had no significant effects on the expression of BMP-2 in normal, RA and OA FLS at 2 h, while in RA FLS it increased significantly at 6 h. Inflammatory cytokines (IL-1β) didn’t influence normal FLS at 2 h, but made BMP-2 mRNA significantly increased at 6 h. IL-1β increased BMP-2 mRNA of RA FLS significantly both at 2 h and 6 h. IL-1β increased BMP-2 mRNA of OA FLS significantly only at 2 h, but had no significant effect at 6 h. The co-effect of IL-1β and cyclic mechanical stretch induced the ascension of BMP-2 expression significantly in normal and RA FLS at 2 h, and in normal, RA and OA FLS at 6 h. Conclusions Response of BMP-2 mRNA to mechanic stimulation and IL-1β in normal, RA and OA FLS were different. Inflammation may play a more important role than mechanical stimulation in the pathogenesis of RA and OA. Synergetic effect in inflammation and mechanical stimulation were found in OA FLS at 6 h, which reveals that they may co-act in the occurrence and development of OA.

Role of Synovial Fibroblasts in Rheumatoid Arthritis / 한양의대학술지

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial tissue proliferation with progressive joint destruction. The etiology of RA remains unknown, but many factors, including autoimmunity, cytokines and genetic factors, participate in its pathogenesis. There is growing evidence that activated fibroblast like synoviocytes (FLS), as part of a complex cellular network, play an important role in the pathogenesis of RA. It has been understood that proinflammatory cytokines secreted from macrophages and lymphocytes may influence the activation of FLS, but invasive and aggressive behaviour of RA-FLS maintained even in the absence of inflammatory stimuli. This kind of partial transformation is characterized by alterations in the expression of regulatory genes such as p53 and signaling cascade, as well as changes in pathway leading to apoptosis. Under the influences of proinflammatory cytokines in rheumatoid joints, RA-FLS is actively involved in the matrix degradation through the production of matrix metalloproteinases (MMP) and cathepsin. In addition, activated RA-FLS exert specific effects on other cell types such as macrophages and lymphocytes. While careful mapping of cytokine networks a decade ago led to the successful development of anti-cytokine therapy, the elucidation of gene mutations and detailed signaling transduction pathways that are specific to RA as well as mechanisms of action of MMP may provide the new targets for novel therapeutic interventions for RA.