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.

Fatigue Analysis Of High Dump Truck Operators In Indonesia’s Coal Mining Industry: A Case Study

@#A coal mining industry typically applies a 24-hours working time, which enforces some workers to stay conscious during night shift, opposing human body's biological clock. This study aims to analyse the level of fatigue experienced by high dump truck operators (HD operators) in a coal mining site in East Kalimantan, Indonesia. This study utilizes primary data which obtained from distributing Industrial Fatigue Research Committee (IFRC) survey to all HD operators and secondary data (for Fatigue Likelihood Scoring -FLS) which consists of HD operators’ working schedule that currently applied in the company. Results obtained is analyzed using Fatigue Risk Management System (FRMS) framework which combines FLS classification and Dawson-McCulloch’s model of fatigue risk trajectory. This study reveals that based on IFRC survey, HD operators experienced low/mild fatigue due to insignificant influence of fatigue-related factors contained in the survey. However, consideration for improvement is in need since the resultof fatigue for night shift operators is close to moderate level. In addition, based on FLS, the level of fatigue indicates that HD operators experienced excessive working hours, in which in FRMS graph classified as fatigue-related errors. Thus, this studyproposes several strategies as the hazard control mechanism: (1) providing optimum resting time, (2) equipping operators with audio music that lead to positive energy and increasing work focus, and (3) adding afternoon shift to balance the working hours.

An Iranian Version of the Fukushima Lumbar Spinal Stenosis Scale (FLS-25): A Validation Study

STUDY DESIGN: Cross-sectional study. PURPOSE: To translate and validate the Fukushima lumbar spinal stenosis (LSS) scale 25 (FLS-25) for use in Iran. OVERVIEW OF LITERATURE: Tools measuring patient-reported outcomes should satisfy certain psychometric properties. METHODS: FLS-25 is a self-administered scale for evaluating symptoms of LSS. A forward-backward procedure was applied to translate the questionnaire from English into Persian. A sample of patients with LSS completed the questionnaire at two points in time: once before surgery and once 6 months after the surgery. The Neurogenic Claudication Outcome Score (NCOS) was also used for assessment. The psychometric properties of FLS-25 were evaluated for internal consistency, test-retest and interobserver reliabilities, responsiveness to change, known-group comparison, and convergent validity. RESULTS: In all, 131 patients were included in the study. The mean age of the patients was 61.4 (standard deviation, 11.1) years. The Cronbach's alpha coefficient for FLS-25 was 0.89. Test-retest reliability as carried out by the intraclass correlation coefficient was 0.94 (95% confidence interval, 0.95). Interobserver agreement as measured by the kappa statistics also was found to be acceptable (kappa value, 0.88), and validity was found to be satisfactory. The instrument was able to discriminate between the subgroups of patients who differed in symptom severity. The correlation between FLS-25 and NCOS scores was excellent, indicating good convergent validity (r=0.82, p<0.001). The results also indicated that the instrument was responsive to change (p<0.001). CONCLUSIONS: The Iranian version of FLS-25 performed well, and the findings suggest that it is a valid measure of symptom severity in LSS patients.

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.

IL-17 and Toll-like Receptor 2 or Toll-like Receptor 4 Combined Engagement Upregulates RANKL and IL-6 in Human Rheumatoid Synovial Fibroblasts / 대한류마티스학회지

OBJECTIVE: The aim of this study was to clarify whether stimulation of recombinant IL-17, TLR2 and TLR4 by their specific ligands induces the production of RANKL and IL-6 in the fibroblast-like synoviocytes (FLSs) from RA patients. METHODS: FLSs were isolated from RA synovial tissues and they were stimulated with the IL-17, TLR2 ligand bacterial peptidoglycan (PGN) and TLR4 ligand lipopolysaccharide (LPS). The RANKL levels were assessed by RT-PCR and western blotting. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 in the RA synovium were quantified by immunohistochemistry and these values were compared with the values obtained in the osteoarthritis synovium. The increased IL-6 production in the culture supernatants of the RA FLSs was quantified by sandwich ELISA. RESULTS: The mRNA and protein levels of RANKL and IL-6 increased in the RA FLSs stimulated with PGN, LPS and IL-17, or PGN plus IL-17 or LPS plus IL-17. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 were much higher in the RA synovium than those in the osteoarthritis (OA) synovium. CONCLUSION: We observed synergistic effects of TLR-2, TLR-4 and IL-17 upon the induction of RANKL. In conclusion, our data supports the previous evidence of an important role of TLR-2, TLR-4 and IL-17 in the pathogenesis of RA.

Macrophage Migration Inhibitory Factor (MIF) Induced Stromal Cell-derived Factor 1 (SDF-1) Production Via Nuclear Factor KappaB (NF-kappaB) Signaling in Rheumatoid Arthritis Fibroblast Like Synoviocytes (RA-FLS)

BACKGROUND: Stromal cell-derived factor (SDF)-1 is a potent chemoattractant for activated T cells into the inflamed Rheumatoid arthritis (RA) synovium. To determine the effect of macrophage migration inhibitory factor (MIF) on the production of SDF-1 in the inflamed RA synovium. METHODS: The expression of SDF-1 and MIF in RA and Osteoarthritis (OA) synovium was examined by immunohistochemical staining. The SDF-1 was quantified by RT-PCR and ELISA after RA fibroblast like synoviocyte (FLS) were treated with MIF in the presence and absence of inhibitors of intracellular signal molecules. The synovial fluid (SF) and serum levels of MIF and SDF-1 in RA, OA and healthy control were measured by ELISA. RESULTS: Expression of SDF-1 and MIF in synovium was higher in RA patients than in OA patients. The production of SDF-1 was enhanced in RA FLS by MIF stimulation. Such effect of MIF was blocked by the inhibitors of NF-kappaB. Concentrations of SDF-1 in the serum and SF were higher in RA patients than in OA patients and healthy control. SDF-1 and MIF was overexpressed in RA FLS, and MIF could up-regulate the production of SDF-1 in RA FLS via NF-kappaB- mediated pathways. CONCLUSION: These results suggest that an inhibition of interaction between MIF from T cells and SDF-1 of FLS may provide a new therapeutic approach in the treatment of RA.

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.

Role of Immune Response to Type II Collagen in the Pathogenesis of Rheumatoid Arthritis

Type II collagen (CII), major component of hyaline cartilage, has been considered as an auto-antigen in rheumatoid arthritis (RA). However, the clinical and biological significances with regard to the CII autoimmunity need to be clarified in human RA. The presence of antibodies to CII has been identified in sera, synovial fluid, and cartilage of patients with RA. In our study, the increased titer of IgG anti-CII in sera was well correlated with C-reactive protein, suggesting that this antibody may reflect the inflammatory status of RA. The titer of anti-CII antibodies (anti-CII Abs) tended to be higher in early stages of diseases. In our extending study, among 997 patients with RA, 269 (27.0%) were positive for circulatory IgG antibody to CII, those levels were fluctuated over time. It is hard to assess the significant amount of T cell responses to CII and CII (255~274) in RA. By using a sensitive method of antigen specific mixed lymphocyte culture, we can detect the presence of CII-reactive T cells in peripheral blood mononuclear cells of RA patients. Sixty seven (46.9%) of 143 patients showed positive CII reactive T cell responses to CII or CII (255~274). The frequencies of CII reactive T cells were more prominent in inflamed synovial fluid (SF) than in peripheral blood. These T cells could be clonally expanded after consecutive stimulation of CII with feeding of autologous irradiated antigen presenting cells (APC). Moreover, the production of Th1-related cytokine, such as IFN-gamma, was strongly up-regulated by CII reactive T cells. These data suggest that T cells responding to CII, which are probably presenting the IFN-gamma producing cells, may play an important role in the perpetuation of inflammatory process in RA. To evaluate the effector function of CII reactive T cells, we investigated the effect of CII reactive T cells and fibroblasts-like synoviocytes (FLS) interaction on the production of pro-inflammatory cytokines. When the CII reactive T cells were co-cultured with FLS, the production of IL-15 and TNF-alpha from FLS were significantly increased (2 to 3 fold increase) and this increase was clearly presented in accord to the expansion of CII reactive T cells. In addition, the production of IFN-gamma and IL-17, T cell derived cytokines, were also increased by the co-incubation of CII reactive T cells with FLS. We also examined the impact of CII reactive T cells on chemokines production. When FLS were co-cultured with CII stimulated T cells, the production of IL-8, MCP-1, and MIP-1alpha were significantly enhanced. The increased production of these chemokines was strongly correlated with increase the frequency of CII reactive T cells. Conclusively, immune response to CII was frequently found in RA. Activated T cells in response to CII contributed to increase the production of proinflammatory cytokines and chemokines, which were critical for inflammatory responses in RA. The interaction of CII-reactive T cells with FLS further augmented this phenomenon. Taken together, our recent studies have suggested that autoimmunity to CII could play a crucial role not only in the initiation but amplification/perpetuation of inflammatory process in human RA.

ULTRASTRUCTURE OF PERINEURIAL CELLS AND FIBROUS LONG-SPACING BODIES (FLS) IN ENDONEURIUM OF NORMAL HUMAN PERIPHERAL NERVE / 解剖学报

The perineurial cells of the small nerve branch of the normal human abdominal wall are observed with electron microscope. The fibrous long-spacing bodies (FLS) are found within the interstitial substance of the endoneurium. 1. 2-5 layers of the perineurial cells surround the nerve fasciculus. The perineurial cells are squamous in shape and the cytoplasm contains microfilaments and pinocytotic vesicles. Each perineurial cell has an obvious basement membrane on its basal surface but the fibroblasts, whatever situated in the endoneurium or on the outer surface of the perineurium, has no basement membrane. Numerous desmosome and some gap junctions between the close attached perineurial cells are demostrated. The collagen fibrils between perineurial cells can be often shown. FLS bodies and collagen fibrils about 45 nm in diameter in the interstitial substance of endoneurium inside perineurial cells are demonstrated. In the connective tissue surrounding the outside of perineurial cells, the collagen fibrils of 80 nm in diameter can be seen, but no FLS bodies present there. 2. FLS bodies in the endoneurial matrix can be demostrated. Most of them closely associated with the basement membrane of the Schwann cells surrounding the unmyelinated nerves but no FLS bodies are found associate with those of the myelinated nerves. A few FLS bodies do not relate to basement membrane and they are invested only with the collagen fibrils. In the longitudinal sections, most of FLS bodies are in spindle shape of various sizes, their longitudinal axes parallel to that of the adjoining collagen fibrils. Sometimes the FLS bodies continue with the collagen fibrils are found. Occasionally, FLS body like a bridge locate between two Schwann cells and its dark bands continue to the basement membrane of the Schwann cells. Two or three FLS bodies may fuse together but their cross bands do not registered at the same level. In the oblique sections, FLS bodies are nearly rectangular in shape, with the cross bands shown, and their appearance do not show greaty difference from those in longitudinal sections. The periodicity of FLS bodies is about 133nm in length; the dark band in it is about 53 nm, which is made of the dense granular substance; the light band is about 80 nm, which is made of a network with approximated parallel microfilaments. There are no further crossstriated structures within the periods. Some problems, such as the role of the perineurial cells, the relationship of FLS bodies with basement membrane, are briefly discussed.