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Aim To observe the influence of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced rat fibroblast-like synovial cell line RSC-364.Methods The secretion levels of MMP-1, MMP-3, MMP-9 and TIMP-1 were determined using ELISA;MMP-3 and MMP-9 mRNA expressions were detected by RT-PCR.Results MMP-3 and MMP-9 could not be examined in RSC-364 incubated with CCK-8 and unstimulated RSC-364, which was able to product a little MMP-1, TIMP-1 and express even less MMP-3,-9 mRNA.CCK-8 inhibited the increase in MMP-1, MMP-3, MMP-9 secretion and MMP-3,-9 mRNA expression in TNF-α-induced RSC-364.TIMP-1 production was also increased in TNF-α-induced RSC-364.CCK-8 had no effect on TIMP-1 production in TNF-α-induced RSC-364, but was able to reduce the ratios of MMP-1, MMP-3, MMP-9 to TIMP-1.Conclusion The inhibitory effect of CCK-8 on MMPs activity may be related to the decrease of MMPs mRNA expression, MMPs secretion and the ratios of MMPs to TIMP-1 in TNF-α-induced RSC-364, which indicates that CCK-8 might be a possible regulator in the pathogenesis of rheumatoid arthritis.
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Seventy-three patients with type 2 diabetes mellitus were divided into atheroselerosis(AS) group and non-AS group according to the intima-media thickness(IMT)of the carotid artery.The plasma visfatin level in AS group was higher than that in non-As group[(44.95±10.14 vs 34.52±9.08)μg/L,P<0.05],and both of them were higher than that of the control [(24.46±7.18)μg/L,both P<0.05 ].The visfatin level Was positively correlated with IMT,waist-to-hip ratio,visceral fat thickness,fasting insulin,and HOMA insulin resistance index.Age,duration of diabetes,HbA_(1C),and visfatin level were the major risk factors for IMT of the carotid artery.
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AIM: To investigate the effect of sulfated cholecystokinin octapeptide (CCK -8 ) on TNF -α induced IL - 6 mRNA expression, NF - κB activation in the rat fibroblast - like synovial cell strain RSC - 364 and its possible receptor mechanisms. METHODS: RSC -364 cells were stimulated with TNF - α( 10 μg/L) in the presence or absence of sCCK- 8( 10-8 - 10-6 mol/L) or/and CCK receptor antagonist proglumide(2 mg/L). IL -6 and CCK receptor A/B (CCK- AR/CCK/BR) mRNA expression were assayed by reverse transcription polymerase chain reaction (RT- PCR) at 3 h after stimulation, and nuclear factor - κB (NF - κB) binding activity was analyzed by electrophoretic mobility shift assay (EMSA) at lh after stimulation. At 30 min of stimulation the IκB protein level in cytoplasma was measured by Western blotting. RESULTS: Both CCK - AR and CCK - BR were constitutively expressed on RSC - 364. sCCK - 8, at concentrations from 10-8 mol/L to 10 -6 mol/L, significantly increased IL - 6 mRNA expression, CCK - AR and CCK - BR mRNA expression, NF - κB binding activity and IκB protein degradation. The effects of sCCK - 8 on NF - κB activity and IκB degradation level were attenuated by CCK receptor antagonist proglumide. CONCLUSION: sCCK - 8 upregulats TNF - α- induced IL - 6 mRNA expression by NF - κB pathway through its receptor on rat synoviocytes, suggesting its possible regulatory role in the pathogenesis of rheumatoid arthritis.
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<p><b>OBJECTIVE</b>To investigate the role of nuclear factor kappa B (NF-kappaB) pathway inhibition in lipopolysaccharide (LPS)-stimulated apoptosis of polymorphonuclear neutrophils (PMNs).</p><p><b>METHODS</b>Rats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate (PDTC) and gliotoxin were used as NF-kappaB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-kappaB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling and DNA fragmentation. IkappaBalpha degradation was analyzed by Western blot. NF-kappaB DNA binding activity was detected by an electrophoretic mobility shift assay.</p><p><b>RESULTS</b>(1) The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS (100 micro g per rat) intratracheal instillation were alleviated by PDTC (50, 100, or 200 mg/kg, i.p.) in a dose-dependent manner. (2) PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment. (3) IkappaBalpha degradation and increased NF-kappaB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment.</p><p><b>CONCLUSION</b>Inhibition of either NF-kappaB itself or the upstream signals in NF-kappaB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role.</p>
Subject(s)
Animals , Humans , Rats , Apoptosis , Cells, Cultured , Lipopolysaccharides , Pharmacology , NF-kappa B , Physiology , Neutrophils , Physiology , Rats, Sprague-Dawley , Signal Transduction , PhysiologyABSTRACT
AIM:To investigate the effect of sulfated cholecystokinin octapeptide(CCK-8) on TNF-? induced IL-6 mRNA expression,NF-?B activation in the rat fibroblast-like synovial cell strain RSC-364 and its possible receptor mechanisms.METHODS:RSC-364 cells were stimulated with TNF-?(10 ?g/L) in the presence or absence of sCCK-8(10-8-10-6 mol/L) or/and CCK receptor antagonist proglumide(2 mg/L).IL-6 and CCK receptor A/B(CCK-AR/CCK/BR) mRNA expression were assayed by reverse transcription polymerase chain reaction(RT-PCR) at 3 h after stimulation,and nuclear factor-?B(NF-?B) binding activity was analyzed by electrophoretic mobility shift assay(EMSA) at 1h after stimulation.At 30 min of stimulation the I?B protein level in cytoplasma was measured by Western blotting.RESULTS:Both CCK-AR and CCK-BR were constitutively expressed on RSC-364.sCCK-8,at concentrations from 10-8 mol/L to 10-6 mol/L,significantly increased IL-6 mRNA expression,CCK-AR and CCK-BR mRNA expression,NF-?B binding activity and I?B protein degradation.The effects of sCCK-8 on NF-?B activity and I?B degradation level were attenuated by CCK receptor antagonist proglumide.CONCLUSION:sCCK-8 upregulats TNF-?-induced IL-6 mRNA expression by NF-?B pathway through its receptor on rat synoviocytes,suggesting its possible regulatory role in the pathogenesis of rheumatoid arthritis.