ABSTRACT
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.
ABSTRACT
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.