The Roles And Mechanisms Of P16 Knockout In Intervertebral Disc Degeneration

Aim:To clarify whether the expression of p16 gene was increased in the senescent nucleus pulposus（NP）cells.To determine whether the increase of p16 gene expression in the NP cells can accelerate its aging process.To determine whether the decrease of p16 gene expression in the NP cells can slow down its aging process.To clarify whether p16 gene knockout can inhibit the intervertebral disc degeneration（IDD）in mice and whether this phenomenon is related to the inhibition of oxidative stress and cell senescence.To determine whether the effect of p16 gene knockout in the intervertebral disc degeneration is associated with regulating the cell cycle through p16-CDK4/6-p Rb-E2 F pathway.To clarify the effect of p16 gene knockout on the expression of proliferation proteins in mice.Methods:We used si RNA transfection technique to build low p16 gene expression NP cells and used overexpression plasmid to construct high p16 gene expression NP cells.Besides,we constructed NP cells degeneration model by the stimulation of IL-1β.Human NP cells were divided into four groups: control,IL-1β stimulated,IL-1β stimulated with p16 gene high expression,IL-1β stimulated with p16 gene low expression.The phenotype of all NP cells was compared and analyzed by using the immunofluorescence,western-blot,rt-qpcr,beta-galactosidase staining,and flow cytology.12-week-old Wild Type（WT）and p16 knockout（p16-/-）mice were used tail suspension model to construct IDD.The mice without tail suspension were chosen as control.The mice were divided into four groups: Control-T,Control-p16-/-,Tail suspension-WT,Tail suspension-p16-/-.And mice were killed and the intervertebral discs were collected after 4 weeks suspension.We used X-ray,MRI,HE staining,and red O staining to verify the success of IDD model.Besides,we used flow cytology,ELISA,immunohistochemistry,RT-PCR,Western-blot methods to compared the differences in the cell cycle,proliferation,senescence,apoptosis,oxidative stress,inflammation,and p16-CDK4/6-p Rb-E2 F pathway related genes expression.Results: Compared with the control group,IL-1β stimulation increased the aging degree of nucleus pulposus cells with high expression levels of p16 gene.At the same time,the percentage of the cells in the G1 phase,cell proliferation and apoptosis cell percentage increased.This phenomenon was much more serious in the IL-1β stimulated with p16 gene high expression group,but situation turned for the better in the IL-1β stimulated with p16 gene low expression group.There was no significant difference in oxidative stress levels in the 4 groups.Compared with WT groups,disc degeneration was mild in p16-/-mice.In p16-/-mice,the age-related gene expression such as IL-1β,IL-6,TNF-α,MMP,p53 and p19 were significantly down-regulated,while the expression of anti-oxidative stress gene such as SOD、GPX、CAT was up-regulated.The results also showed the cell proliferation related gene such as Ki67 and PCNA up-regulated in p16-/-mice,as well as the cell cycle related protein cdk4/6,p RB,E2F1/E2F2 expression.Conclusion:The results of this study demonstrated that the degree of senescence of nucleus pulposis is positively correlated with p16 gene expression level.The increase of p16 gene expression can promote the senescence of NP cells,and the down-regulation of p16 gene expression can slow down its aging process.p16 gene can regulate the NP cell cycle through p16-CDK4/6-p Rb-E2 F pathway.What’s more,p16 knockout can slowdown IDD by inhibiting the level of oxidative stress,inhibiting inflammation,reducing DNA damage,promoting proliferation,,and inhibiting aging process in the disc.We believe that the research results of this project will provide theoretical and experimental basis for the development of effective and safe treatment of intervertebral disc degeneration diseases.