The Role And Mechanism Of Caveolin-1 In Promoting The Repair Of Annulus Fibrosus By Moderate Mechanical Stimulation

Objective:As an important component of the intervertebral disc(IVD),the structure stability of annulus fibrosus(AF)is the key to maintain the mechanical strength of IVD.It’s known that mechanical loading can regulate the structure and function of AF in either positive or negative ways,depending on the loading mode and level.In general,excessive biomechanical loading leads to wear or tear,causing disc degeneration and inducing the inflammatory responses of AF cells(AFCs).On the other hand,mechanical loading at physiologic level can antagonize inflammatory responses and shows beneficial effects on AF.Recent studies have shown that caveolin-1(Cav1),the core protein caveolae,plays an important role in cellular mechanotransduction.In this study,we aimed to investigate the effects of mechanical loading on AFCs and cheek whether the expression and distribution of Cav1 is related to the effect of external mechanical stimulation on the inflammatory responses of AFCs.Specific objectives:(1)To investigate the effects of cyclic tensile strain(CTS)of different magnitudes on the morphology,proliferation,migration and matrix anabolism of AFCs,and then screen moderate CTS which benefit for cell growth.(2)To detect the effects of CTS of different magnitudes on the inflammatory responses of AFCs,and then screen moderate CTS which has the anti-inflammation function.(3)To confirme the role and mechanism of Cav1 in CTS regulated inflammatory response by overexpressing or inhibiting Cav1 expression.(4)To assess the effects of moderate mechanical traction on the degenerative discs at early stage.Methods:(1)AFCs were subjected to CTS(0%,2%,5%or 12%at 0.5 Hz)for 24 h.Cell morphology,proliferation and migration were observed by F-actin staining,flow cytometric analysis and would-healing assay,respectively.The matrix anabolism of AFCs was detected by immunocytochemistry.(2)The expression of pro-inflammatory genes(Cox2、Tnfa、Il1b及Il6)in AFCs subjected to CTS(0%,5%or 12%at 0.5 Hz)were analyzed by RT-qPCR.To induce inflammation,IL-1β was supplemented in the culture medium during the entire course of 5%CTS.RT-qPCR was performed to detect the expression of proinflammatory genes(Cox2、Tnfa、Il1b及Il6)in AFCs treated with 5%CTS in the presence or absence of IL-1β.(3)The expression of Cavl and integrin β1 in AFCs subjected to CTS(0%,5%or 12%at 0.5 Hz)were analyzed by Western blot.The cellular location of Cav1,integrin β1 and NF-κB in AFCs subjected to CTS(0%,5%or 12%at 0.5 Hz)were detected by immunofluorescence.AFCs were transfected with Cav1 plasmids or Cav1 siRNA to further explore the signaling pathways involved in the inflammatory responses of AFCs under different mechanical loading conditions.The regulatory relationships of Cav1,integrin β1 and NF-κB were assessed by Western blot,immunofluorescence and Coimmunoprecipitation.(4)In vivo studies of magnetic resonance imaging(MRI)and histological staining were performed to evaluate the effects of moderate mechanical loading on degenerative discs.Results:(1)AFCs under static condition showed flatted cell shapes and exhibited random direction.However,the morphology of stretch-treated cells became spindle-like when treated with 2%,5%or 12%CTS.Cells in the 5%CTS group tended to spread along the loading direction,whereas cells treated with 12%CTS grew along the perpendicular direction of mechanical loading.The percentage of cells in S phase significantly increased after being treated with 5%CTS,indicating that 5%CTS yield a higher level of cell proliferation than other groups.In addition 5%CTS promoted AFC migration and the synthesis of extracellular matrix(ECM)significantly.(2)Pro-inflammatory genes,including Cox2,Tnfa,Il1b and Il6,significantly increased in AFCs treated with 12%CTS.Nevertheless,5%CTS had no obvious influence on the expression of pro-inflammatory genes in AFCs compared to those under static condition.Inflammatory response,catabolic process,and apoptotic pathway were activated,while cell proliferation was negatively regulated in the 12%CTS group.On the contrary,small molecule biosynthetic process,ECM organization and cell proliferation were up-regulated in the 5%CTS group compared with the Ctrl group.(3)The expression of Cavl and integrin β1 was down-regulated in the 5%CTS group,which might result in the decrease of cellular sensitivity to physical signals.In contrast,12%CTS significantly increased the expression of these two proteins.Cavl and integrin were presented at the plasma and membrane of AFCs,and no further increase of nuclear Cav1 was measured upon CTS of 5%or 12%.NF-κB was located in the cytoplasm under static and 5%CTS conditions,but was rapidly translocated into the nucleus upon 12%CTS.Knockdown of Cav1 in AFCs treated with 12%CTS led to a reduction of proinflammatory gene level and overexpression of Cav1 abrogated the anti-inflammatory effects induces by 5%CTS and resulted in up-regulation of pro-inflammatory genes.Cav1 over-expression in AFCs increased the protein level of the phosphorylation of p65(p-p65),which is an indicator of p65 activation and acts as the major transcription factor of the NFκB signaling pathway.Immunofluorescence results further confirmed that p65 rapidly accumulated in the nucleus in response to Cavl overexpression.Integrin β1 was precipitated with an anti-Cavl antibody,while Cav1 could also be precipitated with an anti-integrin β1 antibody.However,p65 could not be precipitated with either Cavl or integrin β1 antibody.Conclusion:This study reveals that excessive mechanical stimulation induced inflammatory responses,depending on Cav1 mediated integrin β1 and NF-κB signaling pathway.While moderate mechanical stimulation inhibited the Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs.In vivo results indicated that moderate mechanical stimulation might have therapeutic effects for the repair of degenerative discs.In general,our study expounds the precise molecular mechanisms underlining moderate mechanical stimulation and may provide an opportunity to develop a new physical therapeutic strategy for the treatment of IVD degeneration.Moreover,targeting Cav1 and integrin β1-mediated inactivation of NF-κB signaling pathway is a promising biological therapy for IVD degeneration.