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Objective To investigate the effects of chitosan(CS)/pcDNA3.1(+) CrmA on the expression of interleukin-13 (IL-1β3) converting enzyme (ICE) and IL-1β in chondrocytes.Methods Rabbit chondrocytes were isolated and cultured.Chondrocytes were treated with PBS,10 μg/ml CS/pcDNA3.1(+) and CS/pcDNA3.1 (+) CrmA respectively for 6 hours.Then 10 ng/ml IL-1β was added into the culture medium.After 48 hours,the messenger RNA and protein expression of ICE and IL-1β in chondrocytes were detected by using real time polymerase chain reaction and western blotting.Results In CS/pcDNA3.1 (+) CrmA treated group (0.52 ±0.09),the mRNA expression of ICE inchondrocytes was significantly inhibited compared withcorresponding samples of CS/pcDNA3.1 (+) group (0.84±0.11,t=4.42,P＜0.01) and PBS group (1.00±0.10,t=6.58,P＜0.01).ICE protein expression in CS/pcDNA3.1 (+) CrmA treated group (0.20±0.03) was markedly lower than CS/pcDNA3.1 (+) (0.37±0.05,t=4.85,P＜0.01) and PBS treated group (0.44±0.07,t=6.68,P＜0.01).There was no significant difference of ICE mRNA and protein expressions in chondrocytes between CS/pcDNA3.1(+) group and PBS group.Significant difference of IL-1β mRNA expression was found in the three groups.IL-1 β mRNA expression level was significantly lower in CS/pcDNA3.1(+) CrmA treated group (0.55± 0.08) than CS/pcDNA3.1(+) (0.69±0.06,t=3.50,P＜0.01) and PBS group (0.99±0.04,t=11.12,P＜0.01) of chondrocytes.IL-1β protein expression level of chondrocytesin CS/pcDNA3.1(+) CrmA group (0.230±0.020) was significantly lower than CS/pcDNA3.1 (+) (0.450±0.060,t=5.07,P＜0.01)and PBS groups (0.610±0.090,t=8.70,P＜0.01) of cells.Significant difference of IL-1β mRNA and protein expression between CS/pcDNA3.1 (+) and PBS group was also observed (t=7.61,P＜0.01;t=3.63,P＜0.01).Conclusion CrmA mediated by chitosan can significantly suppress the mRNA and protein expression of ICE,thus down regulat the expression of IL-1β,which may be one of the mechanisms of CS/pcDNA3.1 (+) CrmA in the treatment of osteoarthritic cartilage degeneration.
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Objective To investigate the effects of chitosan/pCDNA3.1 (+) CrmA on matrix metalloproteinase (MMP)-1 and tissue inhibitor of metalloproteinase (TIMP)-1 expression of chondrocytes induced by interleukin-1β (IL-1β) in mRNA and protein levels.Methods Rabbit chondrocytes were isolated and cultured.Chondrocytes were treated with PBS,10 μg/ml chitosan (CS) and chitosan/ pCDNA3.1 (+)CrmA respectively for 6 hours.Then 10 ng/ml IL-1β was added into the culture medium.After 48 hours,real time polymerase chain reaction (real time PCR) and Western blot assay were used to examine the changes of MMP1 and TIMP-1 in mRNA and protein levels.Results In CS/pCDNA3.1 (+)CrmA treated group (0.44±0.04),the messenger RNA expression of MMP-1 in chondrocytes was significantly suppressed compared with corresponding samples of PBS treated group (1.00±0.05) and CS treated group (0.76±0.07),There was significant difference between three groups (F=106.93,P＜0.01).The MMP-1 protein expression of chondrocytes in CS/pCDNA3.1 (+)CrmA treated group (0.28±0.03) was lower than that of PBS treated group (0.73±0.06) and CS treated group (0.46±0.05)(F=59.66,P＜0.01).No significant difference of TIMP-1 expression among the three groups was observed in mRNA and protein levels.Conclusion CS/pCDNA3.1(+) CrmA can significantly inhibit the mRNA and protein expressions of MMP-1 of chondrocytes induced by IL-1β,which leads to up-regulation the ratio of TIMPs to MMPs of IL-1β induced chondrocytes.It may be a part of the mechanisms of the therapeutic effects of CS/pCDNA3.1 (+)CrmA on osteoarthritis.
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Objective To study the effect of chitosan-pCrmA nanoparticles on the apoptosis of chondrocytes induced by interleukin-1 beta (IL-1β).Methods Chitosan-pDNA nanoparticles were prepared and characterized.The transfection efficiency of chitosan-mediated pIRES2-EGFP was evaluated using fluorescence microscope.The cytotoxicity of chitosan-pIRES2-EGFP nanoparticles in primary rabbit chondrocytes was analyzed by MTT assay.The expression of chitosan-mediated pCrmA in primary rabbit chondrocytes was verified by Western blotting.The effect of chitosan-mediated CrmA on chondrocytes apoptosis induced by IL-1β were analyzed by TUNEL assay.One-way ANOVA was used to analysis.Results The size of chitosan-pDNA nanoparticles was 50 nm.The pDNA release of chitosan-pDNA nanoparticles appeared as biphasic release at pH 2.0 and pH 7.4 buffer.The expression of CrmA in rabbit primary chondrocytes mediated by chitosan could be detected.The chitosan-pIRES2-EGFP nanoparticles had no cytotoxicity.The apoptosis rate of chondrocytes in the chitosan-pCrmA nanoparticles treated group was significantly lower than that of the chitosan treated group (P＜0.05) and PBS group (P＜0.01).Conclsion Chitosan is an effective non-viral gene transfer vector.The CrmA mediated by chitosan can significantly inhibit chondrocytes apoptosis induced by IL-1β,suggesting that chitosan-pCrmA nanoparticles may be the treatment of osteoarthrifis.