| Objective: Major pathological changes of rheumatoid arthritis (RA) exist in joint,characterized by hyperplasia of synovium, infiltration of abundant inflammatory cells and progressive destruction of cartilage and bone structures, while vasculitis appears outside of joint. Three pathophysiological changes of synovium hyperplasia, inflammation and autoimmunity are comprehensively personificated in the whole process of RA, which interact, correlate and form a reticular network running through pathogenesis of RA. Fibroblast-like synoviocytes (FLSs) isolated from synovium of RA play a major role in the pathogenesis of rheumatoid arthritis (RA) by uncontrolled proliferation and secreting matrix metalloproteinases (MMPs), which is the leading causes of promoting inflammation and joint destruction. IL-1βwhich played a pivotal role in secreting MMPs, has been considered to be the important inflammatory mediator in degradation of cartilage and bone matrix.More and more evidence indicate that MMPs has been considered to be the major reason leading to cartilage destruction in RA. MMP-2 is known as gelatinase A and degrades several types of collagen,including I, IV, V, X and XI,which are impotant constructions of cartilage. Signal transduction mechanisms involved in the expression and synthesis of MMPs are very complicated, which meanwhile participate in inflammatory mediators production, cell proliferation, and so on. Cyclooxygenase-2(COX-2)-related MMP-2 secreting was found to be dependent on prostaglandin E2 (PGE2) production. As a important inducible enzyme in RA, COX-2 is also the rate-limiting enzyme required for the conversion of arachidonic acid to prostaglandins. PGE2 is a mediator of inflammation. COX-2 may develop the function of inflammation via PGE2-dependent pathway in RA. The increasing expression of COX-2 and large scale generation of PGE2 are found in synovial cell, vascular endothelial cell, chondrocyte and monocyte in RA. Selective inhibitors of COX-2 could relieve pain and inflammation of patients by inhibiting the synthesis of COX-2 and PGE2. Mitogen-activated protein kinases (MAPKs) activation is one of the important characters of RA synovitis.There there subfamilies involving in MAPKs, which are extracellular-signal regulatedprotein kinase (ERK), c-Jun amino-terminal kinase (JNK) and p38 MAPK.IL-1βhas been reported to be one of the important inducing mediators. Overactivtion of MAPKs which induce a large amount of inflammatory factors produced by synovial cell, could promote the expression of MMPs and PGs,and pacipate in cell proliferation.Study indicate that secretion of MMP-1 and MMP-3 in RA FLSs could be decreased by supressing the activity of p38 MAPK. However, it is not clear whether COX-2 has a effect on secretion of MMP-2 by PGE2-dependent pathway, and it is needed to further confirm whether MAPKs has effect on expression of MMP-2.Cholecystokinin-octapeptide (CCK-8) is a kind of endogenous brain-gut peptide. Recent studies suggest the anti-inflammatory and immunomodulatory effect of CCK-8. It has been reported that CCK-8 could protect adult rats from joint inflammation induced by carrageenan. Our successional studies demonstrated that CCK-8 could inhibit proliferation of rat fibroblast-like synoviocyte line RSC-364 cells and collagen-induced arthritis (CIA) FLSs, and could induce a decrease in MMP-1, -2, -3 and -9 secretion in RSC-364 cells induced by TNF-α, whose upstream mechanism was associated with reducing AP-1 activity and MAPKs phosphorylation via activated the passway of cAMP-PKA. Recent study has displayed that CCK-8 has a negative regulating effect on phosphorylation of p38 MAPK in RSC-364 stimulated with IL-1β. However, it is not clear whether CCK acted on expression of MMP-2 in RSC-364 and its molecular mechanisms.The present study was designed to observe the regulatory effect of CCK-8 on MMP-2 secretion of rat fibroblast-like synoviocyte line RSC-364 cells induced by recombinant rat IL-1β(rIL-1β) whether the function was developed by PGE2-dependent pathway, investigate the upstream signal transduction mechanism in vitro, in order to clarify its regulatory effect on synoviocyte secretion and its molecular mechanisms.Methods: 1 Western blot was applied to examine the effects of CCK-8 , MAPKs on COX-2 and MMP-2, and CCK-8 on MAPKs in RSC-364 stimulated with rIL-1β. (1)To examine the effects of rIL-1βon COX-2 and MMP-2 in RSC-364,in order to observe the time-effect relationship. Cells were divided into five groups:①control group;②rIL-1β(30μg/L)1 h group;③rIL-1β(30μg/L)7 h group;④rIL-1β(30μg/L)12 h group;⑤rIL-1β(30μg/L)24 h group. (2)To examine the effects of COX-2 and PGE2 on MMP-2 in RSC-364 stimulated with rIL-1β:To further study wether rIL-1βstimulates MMP-2 expression via a PGE2-dependent-COX-2 mechanism in RSC-364,we set up eight groups:①control group;②rIL-1β(30μg/L) group;③⑤rIL-1β(30μg/L)+NS-398 (2,5,10μmol/L) groups;⑥solo-NS-398 (10μmol/L) group;⑦⑧PGE2(5,10μmol/L) groups. (3)To examine the effects of CCK-8 on COX-2 and MMP-2 in RSC-364 stimulated with rIL-1βat the peak time, cells were divided into six groups:①control group;②rIL-1β(30μg/L) group;③CCK-8 (10-6 mol/L) group;④⑥rIL-1β+CCK-8(10-10,10-8,10-6 mol/L) groups.(4)To examine the effects of MAPKs on COX-2 and MMP-2 in RSC-364 stimulated with rIL-1β, so as to study the upstream signal transduction mechanism. Cells were divided into six groups:①control group;②rIL-1β(30μg/L) group;③⑤rIL-1β+SB203580(1,5,10μmol/L)/PD98059(1,5, 10μmol/L)/SP600125(1,5,20μmol/L) group group;⑥SB203580 (10μmol/L)/PD98059 (10μmol/L)/SP600125(20μmol/L) group.(5)To examine the effects of rIL-1βon MAPKs in RSC-364,in order to obtain the peak time of MAPKs phosphorylation. Cells were divided into six groups:①control group;②rIL-1β(30μg/L) 5 min group;③rIL-1β(30μg/L) 15 min group;④rIL-1β(30μg/L) 30 min group;⑤rIL-1β(30μg/L) 60 min group;⑥rIL-1β(30μg/L) 120 min group. (6)To examine the effects of CCK-8 on MAPKs in RSC-364 stimulated with rIL-1βat the peak time of MAPKs phosphorylation. Cells were divided into six groups:①control group;②rIL-1β(30μg/L) group;③CCK-8 (10-6 mol/L) group;④⑥rIL-1β+CCK-8(10-10,10-8,10-6 mol/L) groups. After the cells were incubated for the indicated periods, whole-cell lysates were prepared and the levels of COX-2,MMP-2 and MAPKs phosphorylation were examined by Western blot. 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.2 Enzyme immunoassay(EIA) was used to examine the effects of CCK-8 and MAPKs on PGE2 in rIL-1β-induced RSC-364 cells. RSC-364 cells in logarithmic growth phase were adjusted to 1×105/ml using DMEM containing 10% FSC and grew to confluence in 24-well plates(1ml/well). Cells were divided into the fellowing six groups:①Control group;②rIL-1β(30μg/L) group;③～⑤rIL-1β+CCK-8(10-10,10-8,10-6 mol/L)group;⑥CCK-8(10-6 mol/L)group.To further study the signal transduction mechanism of PGE2 secretion in rIL-1β-induced RSC-364 cells, we set up groups of SB203580 (10μmol/L)/PD98059(10μmol/L)/SP600125(20μmol/L) added separately 30 min before rIL-1βand solo-SB203580(10μmol/L)/ PD98059(10μmol/L)/SP600125(20μmol/L) groups. Then cells of every group were incubated for 24 h, PGE2 content in the culture supernatants was examined using enzyme immunoassay(EIA) technique.Results: 1 Western blot was applied to examine the effects of CCK-8 , MAPKs on COX-2 and MMP-2 ,and CCK-8 on MAPKs in RSC-364 stimulated with rIL-1β: COX-2 was expressed little in control group,while weak MMP-2 was observed. CCK-8 had no effect on the expression of COX-2 and MMP-2. Compared with control group, there was no change in expression of COX-2 1h after rIL-1βaddition.An increase in COX-2 was detected 7h(P<0.05),most obviously change was observed at 24h(1.70±0.03 vs 0.00±0.00,P<0.05), in a time dependent fashion; Treatment of cells with CCK-8 (10-1010-6mol/L) before rIL-1βabrogated the activation of COX-2 to 13.41%,24.39%,43.90% in RSC-364 cells in a dose dependent manner(P<0.05); PD98059, specific inhibitor of ERK,exerted the similar effect as CCK-8, could dose dependently abrogated the activation of COX-2,the inhibition ratio were respectively 5.95%,19.05% and 35.71%; however, SB203580, specific inhibitor of p38 MAPK, and SP600125, specific inhibitor of JNK had no effect same as ERK. Compared with control group, there was no change in expression of MMP-2 1 h and 7 h after rIL-1βaddition.An increase in MMP-2 was detected 12 h(P<0.05),most obviously change was observed at 24h(1.26±0.09 vs 0.55±0.07,P<0.05),in a time dependent fashion;NS398 could inhibit the expression of MMP-2 in rIL-1β-induced RSC-364 cells.Compared with control group, MMP-2 in NS 2,5,10μmol/L group were reduced to 15.38%, 35.90%, 43.59%,in a dose dependent manner.As compared to contorl group, the level of MMP-2 in PGE2 5,10μmol/L group were respectively 0.39±0.03,0.64±0.06 (P<0.05 vs control group). Treatment of cells with CCK-8 (10-1010-6mol/L) before rIL-1βabrogated the activation of MMP-2,the inhibition ratio were respectively 13.10%,29.76% and 52.38% in RSC-364 cells in a dose dependent manner(P<0.05);PD98059, a specific inhibitor of ERK, exerted the similar effect as CCK-8, could dose dependently abrogated the activation of COX-2,the inhibition ratio were respectively 20.00%,23.08% and 27.69%; however,SB203580,a specific inhibitor of p38 MAPK,and SP600125,a specific inhibitor of JNK had no effect same as ERK.The MAPK phosphorylation and non-phosphorylation were expressed moderately in the non-stimulated cells. The expressions of the non-phosphorylation p38MAPK,ERK,JNK had no apparent difference among all the groups(P>0.05). Compared with control group, an increase in p38 MAPK,ERK,JNK phosphorylation was detected 5 min after rIL-1βaddition, and reached a plateau respectively at 30 min, 15 min and 30 min, the results were respectively 0.37±0.09, 0.34±0.07 and 0.38±0.09 (P<0.05 vs control group),finally returned to the basic level at 2 h (P>0.05). Treatment of cells with CCK-8 (10-6mol/L) before rIL-1βabrogated the activation of p38 MAPK,ERK,JNK to 47.83%,43.24% and 45.71% in RSC-364 cells,but CCK-8 had no effect on the activation of p38MAPK,ERK,JNK in resting cells.2 Enzyme immunoassay (EIA) was used to examine the effect of CCK-8 and MAPKs on PGE2 in rIL-1β-induced RSC-364 cells: Compared with control group, stimulation with rIL-1βinduced an increase in PGE2 secretion in medium (1385.76±238.28pg/ml vs 163.64±33.07 pg/ml , P<0.05). Pretreatment with CCK-8 (10-10,10-8,10-6 mol/L) had a tendency of inhibiting the increased PGE2 secretion of rIL-1β-induced RSC-364 cells in a dose dependent manner. PD98059,a specific inhibitor of ERK, exerted the similar effect as CCK-8, could dose dependently inhibit the secretion of PGE2 in RSC-364 stimulated with rIL-1β. However,SB203580,a specific inhibitor of p38 MAPK,and SP600125,a specific inhibitor of JNK had no effect same as ERK.Conclusion: In the present study, we systematically investigated the modulatory effect of CCK-8 on rIL-1β-activated RSC-364 cells expression of MMP-2 at multi-links from a prostaglandin E2-dependent mechanism and the passway of MAPKs in vitro, the conclusions were as follows:1 rIL-1βstimulated an increase in expression of COX-2 and PGE2 in RSC-364 cells, promoting protein expression related to MMP-2 which played a important role in rIL-1β-activated RSC-364 cells MMP-2 seceration; at the same time, rIL-1βcould induced p38 MAPK,ERK,JNK activation. Among the total ERK activation play a negatively regulated role on expression of MMP-2, indicating that it was probable a important mechanism regulating rIL-1β-induced cell MMP-2 expression.2 This study demonstrated that the inhibitory modulation of CCK-8 on the PGE2-dependent COX-2 mechanism which was mediated by ERK activation,inducing a decrease in rIL-1β- induced MMP-2 secretion by obviously downregulating MMP-2 protein expression.
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