Objectives Previously, we reported the improved transfection efficiency of a plasmid DNA-chitosan (pDNA-CS) complex using a phosphorylatable nuclear localization signal-linked nucleic kinase substrate short peptide (pNNS) conjugated to chitosan (pNNS-CS). the pBudCE4.1-miR-140 pBudCE4.1 group (p = 0.648), pBudCE4.1-miR-140 control group (p = 0.688), or pBudCE4.1 control group (p = 0.955) (Fig. 2b). Chondrocyte proliferation significantly increased in the pBudCE4.1-IL-1Ra+miR-140 group, followed by the pBudCE4.1-miR-140 group and the pBudCE4.1-IL-1Ra group, compared with the control group (p 0.01). There was a significant difference in chondrocyte proliferation between the pBudCE4.1-miR-140 group and the pBudCE4.1-IL-1Ra group (p = 0.048), and there was no significant difference between the pBudCE4.1 group and control groupings (p = 0.759) (Fig. 2c). Open in a separate windows Fig. 2 Graphs showing the effect on rabbit chondrocyte proliferation of a) interleukin-1 receptor antagonist protein (IL-1Ra) and b) miR-140 expression. The relative miR-140 expression level in different groups was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). miR-140 gene expression in each group was normalized to the U6 expression level. The IL-1Ra concentration in the cell supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA). HILDA c) Graph showing rabbit chondrocyte cell proliferation by the MTT method. The data are shown as mean values and standard deviations. *p 0.05; ?p 0.01; NS, not significant. control group; ?p 0.05 pBudCE4.1; ?p 0.05 pBudCE4.1-IL-1Ra; p 0.05 pBudCE4.1-miR-140. control group; ?p 0.05 pBudCE4.1; ?p 0.05 pBudCE4.1-IL-1Ra; p 0.05 pBudCE4.1-miR-140; NS, not significant. As shown in Figures 5b and ?and5c,5c, compared with the sham group, all surgical groups presented with increased GAG and NO levels. The GAG and NO contents significantly decreased in the pBudCE4.1-IL-1Ra+miR-140 group, followed by the pBudCE4.1-IL-1Ra and pBudCE4.1-miR-140 groups, compared with the pBudCE4.1 group (p 0.05). experiments have confirmed that pNNS-CS can transport pDNA into the nucleus and intensively augment exogenous gene expression, which is mainly related to the fact that pNNS can assist the nuclear localization and intra-nuclear disassociation of exogenes. 13 The results encourage us to use pNNS-CS to mediate gene transfection in chondrocytes. Here, we investigated the use of pNNS-CS-mediated gene transfection in chondrocytes to promote efficient expression both and and and and GAG findings, the safranin O/fast green staining results indicated that IL-1Ra and miR-140 increased proteoglycan synthesis. All the results strongly suggest that the synergistic effects of IL-1Ra and miR-140 were clearly superior to the effects of IL-1Ra or miR-140 alone, not only for promoting cartilage proliferation, but also for inhibiting the inflammatory response and cartilage degradation. In conclusion, our NVP-BKM120 kinase inhibitor findings show that pNNS-CS complexes can efficiently carry exogenous genes into rabbit chondrocytes and promote expression both and em in vivo /em NVP-BKM120 kinase inhibitor . Our study also provided direct experimental evidence for the synergistic effect of IL-1Ra and miR-140 on repairing cartilage defects by enhancing chondrocyte proliferation, proteoglycan synthesis, and COL2A1 and ACAN expression. Meanwhile, the combination of these two exogenous genes has better biological effects than either IL-1Ra or miR-140 alone. Further work is necessary to study pNNS-CS transfection efficiency in cells from other species, including human chondrocytes, and the synergistic mechanisms of selected genes NVP-BKM120 kinase inhibitor in cartilage defects. Footnotes Author contributions: R. Zhao: Designed the NVP-BKM120 kinase inhibitor study, Performed the experiments, Analyzed the results, Wrote the manuscript. S. Wang: Performed the experiments.L. Jia: Performed the experiments. Q. Li: Performed the experiments. J. Qiao: Performed the experiments. X. Peng: Designed the study, Performed the experiments, Analyzed the results, Wrote the manuscript. Ethical review statement: The protocols in this research that involved pets had been accepted by the Institutional Pet Care and Make use of Committee of Weifang Medical College or university. Stick to us @BoneJointRes Financing statement This function was supported with the Country wide Natural Science Base of China (No. 81770915, 81301737); the Shandong Provincial Normal Science Base, China (No. ZR2012HQ034, ZR2015HL025); as well as the PhD plan of Weifang Medical College or university, China (Zero. 2017BSQD05). No benefits in virtually any form have already been received or will end up being received from a industrial party related straight or indirectly to the main topic of this article..