| Part I: The expression of focal adhesion protein Kindlin-2 in degenerative intervertebral disc tissue Objective: To investigate the correlation between the expression change of focal adhesion protein Kindlin-2 and intervertebral disc(IVD)degeneration(IVDD)by analyzing the Kindlin-2 expression in human nucleus pulposus(NP)samples with different degeneration degrees and IVD tissues of mice with different ages.Methods: 1.The degenerative NP specimens of patients with lumbar disc herniation were collected,and Pfirrmann grading system was used based on the patients’ preoperative magnetic resonance imaging(MRI).Grade II/III was classified as mild degeneration group,and grade IV/V was classified as severe degeneration group.The degeneration degree of the specimen was further determined by alcian blue and H/E staining,and the expressions of focal adhesion proteins(Kindlin-1,2,3 and Talin,Vinculin)were detected by immunofluorescence staining.2.The lumbar IVD tissues of young mice(3 months of age)and old mice(20 months of age)were stained with SO&FG and histological scoring was adopted to determine the degeneration degree.The expressions of above focal adhesion proteins were detected by immunofluorescence staining.Results: 1.In human degenerative NP samples,H/E and alcian blue staining showed that there were fewer NP cells and less extracellular matrix(ECM)in severe degeneration group than that in mild degeneration group.Immunofluorescence staining demonstrated that there were almost no Kindlin-1 or Kindlin-3 positive cells in human NP tissues,and Talin or Vinculin positive cells were few,but there were many Kindlin-2 positive cells and the cell number was decreased with the increase IVDD severity.2.In mice IVD samples,SO&FG staining and histological scores revealed that the IVD degeneration of the aged mice was more severe than that of the young mice.Immunofluorescence staining showed that Kindlin-1 or 3 was almost not expressed in mice IVD,and Talin and Vinculin were expressed at low levels,but Kindlin-2 was highly expressed in NP and down-regulated significantly with the increase of age in mice.Conclusions: These results indicated that Kindlin-2 was mainly expressed in the NP tissue,and its expression level was decreased with the increase of degeneration degree and age,which suggested that the decreased Kindlin-2 expression level was related to IVDD.Part II: The effect of conditional knockout of Kindlin-2 on the intervertebral disc degeneration of miceObjective: To explore the role of Kindlin-2 in IVDD by constructing IVD specific Kindlin-2 knockout mice.Methods: 1.Kindlin-2fl/fl;AggrecanCre ERT2 mice were obtained by mating and injected with corn oil(control group)or tamoxifen(c KO group)at 2 months of age.Lumbar and coccygeal vertebra was scanned by computerized tomography(CT)at age of 2,3,5 and 8 months.The mice were sampled at age of 3,5 and 8 months,and immunofluorescence staining,TUNEL staining,SO&FG staining,histological scoring as well as Western Blot detection were performed.2.In order to further explore the involvement of abnormal mechanical stress,coccygeal IVDs needle stab was performed to cause coccygeal vertebrae instability in Kindlin-2fl/fl;AggrecanCre ERT2 mice at 4 weeks after injection of corn oil or tamoxifen.CT scan of the caudal vertebra was performed before operation(12 weeks of age)and 6 weeks after operation(age of 18 weeks),and samples were collected at age of 18 weeks.Immunofluorescence staining,TUNEL staining,SO&FG staining and histological scoring were performed.Results: 1.In lumbar IVD of mice,immunofluorescence staining demonstrated that the Kindlin-2 expression in c KO group was markedly down-regulated at age of 3,5 and 8 months,compared with the control mice,indicating that the knockout effect was good;SO&FG staining and histological scoring demonstrated that the IVD histological score of the c KO mic was markedly higher than the control mice at age of 5 months,and more significantly at age of 8 months;Immunofluorescence staining and WB analysis revealed that the ECM synthesis and catabolism balance in c KO mice was disturbed,which was manifested as decreased expression of Col2a1 and Acan,and increased expression of Mmp13 and Adamts5;Immunofluorescence staining,TUNEL staining and WB indicated that the cell apoptosis was increased in c KO group,which was manifested as up-regulated expression level of active Caspase3 and Bax,decreased Bcl2 expression,and increased TUNEL-positive cells;CT images showed no significant change in IVD height.2.In coccygeal IVD of mice,immunofluorescence staining also suggested that Kindlin-2 knockout effect was good,but SO&FG staining,histological scoring and CT images demonstrated that there was no markedly difference in coccygeal IVD between c KO mice and the control mice.3.Other results showed that the abnormal mechanical stress caused by needle stab could lead to an increase in histological scores of mice coccygeal IVD,a decrease of IVD height,an imbalance of ECM synthesis and catabolism,and an increase in cell apoptosis in control mice,but the changes were more significant in c KO mice with coccygeal IVDs needle stab.Conclusions: The data suggested that Kindlin-2 knockout could result in spontaneous lumbar IVD degeneration of mice,but there was no obvious change in coccygeal IVD.Additionally,Kindlin-2 knockout could accelerate coccygeal IVD degeneration of mice suffered from abnormal mechanical stress.The results suggested that Kindlin-2 loss played an important role in IVDD initiation and progression,especially in the presence of abnormal mechanical stress.Part III: The mechanisms of intervertebral disc degeneration caused by Kindlin-2 lossObjective: To investigate whether Nlrp3 inflammasome signaling pathway is involved in cell and tissue degeneration of IVD caused by Kindlin-2 loss.Methods: 1.Immunol stainings were performed to evaluate Nlrp3 inflammasome-related protein level in NP tissues of human with different degeneration degrees and in NP tissues of Kindlin-2 c KO and control mice.2.In rat NP cell line,a pressurizing device was applied to simulate the abnormal compression loading in IVD,and Kindlin-2 si RNA or Kindlin-2 plasmid was used to knock down or overexpress Kindlin-2,respectively.The expressions of proteins related to Nlrp3 inflammasome and ECM synthesis and catabolism were detected,and cell apoptosis was evaluated.3.The expressions of Nlrp3 inflammasome-related proteins and Kindlin-2 were analyzed in IL-1β-treated NP cells with or without abnormal compression loading treatment.4.The Nlrp3 inflammasome pathway activation inhibitor MCC950 was used to inhibit Nlrp3 inflammasome activation under in vivo and in vitro conditions,respectively.The repair effect of MCC950 on the Kindlin-2 loss-induced degeneration of NP cells and IVD tissues with or without abnormal mechanical stress was detected.Results: 1.In human degenerative NP tissues,compared with mild degeneration group,the Nlrp3 inflammasome-related protein levels were markedly up-regulated in severe degeneration group.Similarly,compared with the control mice,Nlrp3 inflammasome-related protein expression was obviously up-regulated in the Kindlin-2 knockout group of mice.2.Abnormal compression loading resulted in decreased Kindlin-2 and Col2a1 expression,up-regulated Mmp13 expression,increased cell apoptosis,and activation of Nlrp3 inflammasome pathway.Kindlin-2 knockdown aggravated these changes,while Kindlin-2 overexpression ameliorated these changes.3.IL-1β down-regulated Kindlin-2 and up-regulated Nlrp3 inflammasome-related protein level in time-and concentration-dependent manner,and abnormal compression could aggravate these changes.4.In vitro,MCC950 inhibited the activation of Nlrp3 inflammasome pathway,decreased Col2a1 expression,increased Mmp13 expression,and increased cell apoptosis induced by Kindlin-2 knockdown with or without abnormal compression loading treatment.5.In vivo,MCC950 inhibited the activation of Nlrp3 inflammasome pathway,increased histological scores,decreased IVD height,down-regulated Col2a1 expression,up-regulated Mmp13 expression,and increased cell apoptosis induced by Kindlin-2 knockdown with or without abnormal mechanical stress.Conclusions: Results suggested that Kindlin-2 loss could lead to the degeneration of IVD cells and tissues via Nlrp3 inflammasome activation.Additionally,activated Nlrp3 inflammasome released IL-1β,which resulted in decreased Kindlin-2 expression and further activation of Nlrp3 inflammasome pathway,forming a Kindlin-2/Nlrp3 inflammasome /IL-1β vicious cycle,and causing IVD degeneration.Part IV: The repair effect of Kindlin-2 overexpression on the degeneration of intervertebral discObjective: To investigate protective effect of adeno-associated virus(AAV)-mediated Kindlin-2 overexpression on compression loading-induced human primary NP cell degeneration and coccygeal IVDs compression model-induced rat IVD degeneration,and to preliminarily explore the clinical transformation significance of Kindlin-2 in IVDD treatment.Methods: 1.Control AAV or Kindlin-2 AAV was used to infect human primary NP cells,then cells were treated with or without compression loading.The expressions of Kindlin-2,Nlrp3,Col2a1,Mmp13 and IL-1β were evaluated,and cell apoptosis was detected.2.SD rats(3 months old)were divided into sham group,coccygeal IVDs compression + control AAV group,and coccygeal IVDs compression + Kindlin-2 AAV group.The operation was performed 3 weeks after AAV infection,and MRI evaluation and sampling were performed 2 weeks after the operation.SO&FG staining,histological scoring,immunofluorescence staining and TUNEL staining were performed.Results: 1.In human primary NP cells,AAV-mediated Kindlin-2 overexpression partially inhibited abnormal compression loading-induced decreased Kindlin-2 expression,activation of Nlrp3 inflammasome pathway,down-regulated Col2a1 expression,up-regulated Mmp13 expression,and increased cell apoptosis.2.In rat coccygeal IVDs compression model,AAV-mediated Kindlin-2 overexpression partially reversed the abnormal compression loading-induced decreased T2 signal in MRI imaging of rat IVD,increased histological scores,decreased Kindlin-2 expression,activation of Nlrp3 inflammasome pathway,down-regulated Col2a1 expression,up-regulated Mmp13 expression,and increased cell apoptosis.Conclusions: The results demonstrated that AAV-mediated Kindlin-2 overexpression could partially repair IVD degeneration,suggesting Kindlin-2 AAV had certain clinical translational value in IVDD treatment. |
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