Effect Of Functional Self-assembly Polypeptide Nanofiber Hydrogel On The Apoptosis Of Rat Nucleus Pulposus Cells Induced By IL-1β

Objective 1.To investigate the effect of IL-1β on the apoptosis of rat nucleus pulposus cells in vitro;2.To investigate whether the functionalized self-assembled polypeptide nanofiber hydrogel(RADKPS)which is modified with BMP-7 functional fragment could inhibit the apoptosis of NPCs induced by IL-1β in vitro.Methods NPCs were isolated from Sprague-Dawley Rats and cultured to the third passage in vitro.NPCs were cultured in the presence of RADA16-I which was without BMP-7 functional fragmenton or RADKPS which was combined with BMP-7 functional fragmenton.Then IL-1β was added to induce apoptosis of rat NPCs.Experiments were divided into three groups: group A(RADA16-I without IL-1β),group B(RADA16-I with IL-1β)and group C(RADKPS with IL-1β).After 48 hours,cell proliferation ability was observed by CCK-8 and DAPI staining respectively.Cell viability was observed by Calcein-AM / PI double staining.Apoptosis of NPCs was observed by DAPI staining and TUNEL staining respectively.The relative expression of mRNA of the apoptosis-related genes were detected by RT-PCR,namely Fas,FasLg,Bcl-2,Bax and Casepase-3;The relative expression of mRNA of the extracellular matrix-related genes were detected by RT-PCR,namely AGG,COLII and SOX-9;The activity of Caspase-3 and Caspase-9 were detected by Caspase-3 and Caspase-9 activity kits respectively.Results 1.Cell proliferation ability: The cell proliferation ability in group B and group C was significantly lower than that in group A(P<0.05),and the cell proliferation ability in group C was significantly higher than that in group B(P<0.05);2.Cell viability(Calcein-AM / PI double staining): The proportion of dead cells in group B and group C were significantly higher than that in group A(P<0.05),and the proportion of dead cells in group C was significantly lower than that in group B(P<0.05);3.The apoptosis of cells(TUNEL staining): The proportion of apoptotic cells in group B and group C was significantly higher than that in group A(P<0.05),and the proportion of apoptotic cells in group C was significantly lower than that in group B(P<0.05);4.The relative expression of mRNA of apoptosis-related genes: The relative expression of mRNA of Fas,Bax and Caspase-3 in group B and group C were significantly higher than those in group A(P<0.05),and The relative expression of mRNA of Fas,Bax and Caspase-3 in group C was significantly lower than those in group B(P<0.05);The relative expression of mRNA of Bcl-2 in group B and group C were significantly lower than that in group A(P<0.05),and the relative expression of mRNA of Bcl-2 in group C was significantly higher than that in group B(P<0.05);There was no significant difference in the relative expression of mRNA of FasLg between the three groups(P>0.05);5.The relative expression of extracellular matrix-related genes: The relative expression of mRNA of AGG,COLII and SOX-9 in group B and group C were significantly lower than those in group A(P<0.05),and the relative expression of mRNA of AGG,COLII and SOX-9 in group C was significantly higher than those in group B(P<0.05);6.The activity of Caspase-3 and Caspase-9: The activity of Caspase-3 and Caspase-9 in group B and group C were significantly higher than those in group A(P<0.05),and the activity of Caspase-3 and Caspase-9 in group C were significantly lower than those in group A(P <0.05).Conclusions In this study,we investigated that the main mechanism of IL-1β-induced apoptosis of rat NPCs may be type II apoptotic pathway which was mediated by Fas and with the involvement of mitochondrial.The functionalized self-assembled polypeptide nanofiber hydrogel(RADKPS)which is modified with BMP-7 functional fragment could inhibit the IL-1β-induced apoptosis of rat NPCs in vitro.It shows that RADKPS has a certain potential in repairing degenerative discs.