| Objective: Rheumatoid arthritis(RA), a chronic inflammatory autoimmune disease characterized by the continuous inflammation, hyperplasia of synovial tissue and pannus, progressively invades cartilage and bone and ultimately results in the joint destruction and disability. The pathogenesis of RA remains poorly understood. Recent studies showed that the fibroblast-like synoviocytes(FLS) played an important role in the initiation of pathogenesis, maintenance of chronic inflammation and the destruction of cartilage and bone. Over-expression of IL-1βin RA is a potent factor to the proliferation and activation of FLS. In the signal pathways of FLS activation stimulated by IL-1β, IL-1βfunctions rely on the phosphorylation of protein tyrosine and activation of the mitogen-activated protein kinase(MAPK) after IL-1βbinds to IL-1 receptor. MAPK family is a group of evolutionary conservative proline-directed serine/threonine kinase. It has been illustrated that p38MAPK(a member of MAPK families) plays an important role in cell proliferation. Cholecystokinin octapeptide(CCK-8), a kind of neuropeptides, has been identified to have the effect of anti-inflammation and immune system regulation. It was reported that CCK-8 could inhibit the proliferation of RSC-364 induced by TNF-αas well as collagen-induced arthritis(CIA). It is unknown whether the CCk-8 has the effects on the proliferation of RSC-364 induced by IL-1β. The purpose of our study is to investigate the effects of CCK-8 on the proliferation of RSC-364 under the stimulation of IL-1βand its possible mechanism. Methods: 1. MTT colorimetric assay was used to examine the effect of CCK-8 on the proliferation of RSC-364 stimulated by IL-1β. Synovial cells in logarithmic growth phase(2×10-4cells/ml) were seeded in 96-well plates. The cells were divided into eight groups(five replicated wells/group): ①control group: DMEM containing 10% FCS as vehicle; ②IL-1βgroups: cells were cultured respectively in the presence of 1, 3, 10, 30 μg/L IL-1β; ③CCK-8 group: cells were treated with 10-6 mol/L CCK-8; ④IL-1β+CCK-8 groups: cells were incubated with CCK-8, at 10-12, 10-10, 10-8, 10-6 mol/L respectively, for 1h, then treated with 30 μg/L IL-1β; ⑤IL-1β+CCK-8+CR1409 group: cells were treated with 10-4 mol/L CR1409 for 30min and with 10-6 mol/L CCK-8 for another 1h, then incubated with 30 μg/L IL-1β; ⑥IL-1β+CCK-8+CR2945 group: the protocol was similar to the one described in group⑤, except for CR1409 replaced by CR2945; ⑦SB groups: SB was used at concentration of 1, 5, 10, 20 μmol/L, respectively; ⑧SB+IL-1βgroup: 5μmol/L SB was applied to the cells, 30min later, these cells were further treated with 30 μg/L IL-1β. After 3 days incubation for every group, the proliferation rate of RSC-364 was detected. 2. Western blottingwas applied to examine the effects of CCK-8 on p38MAPK phosphorylation in RSC-364 stimulated with IL-1β. The cells were divided into six groups: ①control group: serum-free DMEM as vehicle; ②IL-1βgroup: cells were incubated with 30 μg/L IL-1βfor 1, 15, 30, 60, 120min, respectively; ③CCK-8 group: cells were incubated with for 10-10, 10-8, 10-6 mol/L CCK-8 for 10min, respectively; ④IL-1β+CCK-8 groups: cells were incubated respectively with 10-10, 10-8, 10(-60 mol/L CCK-8 for 10min, and with 30 μg/L IL-1βfor additional 30min; ⑤IL-1β+CCK-8+CR1409 group: cells were subjected to 10-4 mol/L CR1409 for 5min and to 10-6 mol/L CCK-8 for 10min, Then, 30 μg/l IL-1βwas applied to these cells for 30min; ⑥IL-1β+CCK-8+CR2945 group: the procedure is similar to the are described in group⑤, except for CR2945 instead of CR1409. And then the total protein of the cells was extracted. Western blotting was used to examine the change of p38MAPK. Data were presented as x ±s and analyzed with ANOVA and least significant difference (LSD) using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: 1. Effects of CCK-8 on the RSC-364 Proliferation of induced by IL-1β. IL-1β, at c...
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