1.Effect of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced RSC-364
Jinrong XU ; Bin CONG ; Shujin LI ; Yuhuai JIN ; Zhansheng ZHAO
Chinese Pharmacological Bulletin 2017;33(4):567-571
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.
2.Effects of cholecystokinin octapeptide on TNF- α- induced IL- 6 expression and its possible molecular mechanismin rat synovial cell strain RSC-364
Zhansheng ZHAO ; Yuhuai JIN ; Bin CONG ; Shujin LI ; Jinrong XU ; Yuxia YAO ; Yiling LING
Chinese Journal of Pathophysiology 2007;23(7):1396-1401
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.
3.Effects of cholecystokinin octapeptide on TNF-?-induced IL-6 expression and its possible molecular mechanism in rat synovial cell strain RSC-364
Zhansheng ZHAO ; Yuhuai JIN ; Bin CONG ; Shujin LI ; Jinrong XU ; Yuxia YAO ; Yiling LING
Chinese Journal of Pathophysiology 2000;0(07):-
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.
4.The immunological effect of Ad/MDC-VP1 combined with DNA vaccine against Coxsackievirus infection
Lijing YAN ; Jian LI ; Chan WEN ; Jia LI ; Jiaming LAN ; Xia CHUAI ; Zhiyun GAO ; Yonghong ZHANG ; Yuhuai JIN ; Yongxiang WANG
Chinese Journal of Microbiology and Immunology 2009;29(6):533-537
Objective To construct recombinant adenovirus Ad/MDC-VP1 and investigate its im-muno-boosting effect of the mice primed with the experimental DNA vaccine against Coxsackievirus infection. Methods The recombinant adenovirus Ad/MDC-VP1 was constructed and packaged. The Western blot analysis was used to verify the target protein. BALB/c mice were divided into four groups: Ad/MDC-VP1 group, pcDNA3/MDC-VP1 group, pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group and PBS group. The mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assay, respectively. The lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with le-thal dose of Coxsackievirus, and the assay of the serum virus titers and the observation of protection efficacy against Coxsackievirus infection were carried out. Results The recombinant adenovirus Ad/MDC-VP1 was successfully constructed and the target protein was expressed. It was observed that the titers of CVB3 VP1 specific antibody, lymphocyte stimulation index, CTL cytotoxicity activities and protection rate of the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group were much higher than those of the rest groups( P < 0.05), and the titer of serum virus was lower after CVB3 challenged ( P < 0.05 ). Conclusion Both the cellular and humoral immune responses in mice could been significantly enhanced by the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost strategy.
5.Comparison of the immune effects of Coxsackievirus B3 VP1 protein, rAd/VP1 and pcDNA3/VP1 in mice
Jiaming LAN ; Zhiyun GAO ; Jia LI ; Yuhuai JIN ; Chan WEN ; Wei LI ; Lijing YAN ; Guixia LIU ; Lixin XIE ; Yongxiang WANG
Chinese Journal of Microbiology and Immunology 2011;31(1):25-29
Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P <0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P <0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P < 0.05 ), while survival rate was significantly higher than these two groups ( P < 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.