IGF-1 Promotes Hypertrophy Of The Ligamentum Flavum Via The IGF-1R/AKT/mTORC1 Signaling Pathway

Background:Hypertrophy of the ligamentum flavum is one of the main causes of lumbar spinal stenosis.Its pathological expression is decrease of elastic fiber and proliferation and accumulation of the collagen fibers.Although there are many explanations for the occurrence of Hypertrophy of the ligamentum flavum,its pathologic mechanism is still not fully elucidated and needs further study.Objective:To investigate the role of IGF-1 in hypertrophy of the ligamentum flavum and its potential molecular mechanism of action.Meanwhile,to increase the understanding of pathological mechanism of hypertrophy of the ligamentum flavum.Finally,to provide effective ideas and methods to prevent hypertrophy of the ligamentum flavum and lumbar spinal stenosis.Methods:Preoperative MRI was used to measure the thickness of the ligamentum flavum.Normal and hypertrophic ligamentum flavum tissue specimens were collected during the operation.H&E staining and Masson staining observed the differences in the structure,morphology and fibrosis of the two groups.Cell metabolism status of the two groups were compared under transmission electron microscope.In addition,through immunohistochemistry and Western Blot to detect IGF-1 and IGF-1R/AKT/mTORC1 signaling pathways related proteins of the two groups.At the same time,detected expression of collagen ? and collagen ? of the two groups.The normal ligamentum flavum tissue was taken to culture ligamentum flavum cells.The cell types were identified by immunofluorescence staining of collagen I and vimentin.Effect of IGF-I in the proliferation of ligamentum flavum cells was observed by adding exogenous IGF-1 in different concentrations to stimulate the ligamentum flavum cells 24 h.At the same time,observe the ligamentum flavum cells in different concentrations of exogenous IGF-1,activity change of IGF-1R/AKT/mTORC1 signaling pathways and expression of collagen ? and collagen ?.In addition,NVP-AEW541(inhibitor of IGF-1R)and rapamycin(inhibitor of mTORC1)were added to block the IGF-IR/AKT/mTORC1 signaling pathway and to observe its effect on the biological effects of IGF-1.In addition,mechanical stress was applied on the ligamentum flavum cells to observe change in secretion of endogenous IGF-1 and activity change of IGF-1R/AKT/mTORC1 signaling pathways.Meanwhile,observed change of collagen I and collagen ? synthesis.Results:1.The content of elastic fiber decreased and collagen fiber increased in the hypertrophic ligamentum flavum tissues;high level of metabolism in ligamentum flavum cellsThe normal ligamentum flavum tissues mainly consist by elastic fibers and a small amount of collagen fibers.In hypertrophic ligamentum flavum tissues,the elastic fibers were degraded,broken and arranged in disorder.Meanwhile,collagen fibers proliferated and accumulated.There were few cytoplasm in the normal group and the number of mitochondria,ribosomes and rough endoplasmic reticulum were relatively small in the normal group.These phenomenons indicated that the metabolism of ligamentum flavum cells was weak.The cytoplasm content of ligamentum flavum cells increased significantly in the hypertrophic group and the number of mitochondria,ribosomes and rough endoplasmic reticulum were significantly higher than that in the normal group.2.IGF-1,collagen ? and collagen ? had high expression in the hypertrophic ligamentum flavum tissues;activity of IGF-1R/AKT/mTORCl signaling pathway was higher in hypertrophic ligamentum flavum tissuesImmunohistochemistry and Western Blot results showed that the expression of IGF-1,collagen ? and collagen ? were significantly enhanced in hypertrophic ligamentum flavum tissues,compared with normal ligamentum flavum tissues.At the same time,IGF-1R/AKT/mTORCl signaling pathway related proteins,such as phosphorylated IGF-1 receptor(pIGF-1R),phosphorylated AKT(pAKT),phosphorylated S6(pS6)were also significantly higher than that of normal group.3.High purity of ligamentum flavum cells;IGF-1 did not promote ligamentum flavum cells proliferation.Collagen I and vimentin immunofluorescence staining of primary cultured ligamentum flavum cells were showing strong positive.That indicated that ligamentum flavum cells had high purity.After 24 h exposure of exogenous IGF-1,the absorbance optical density value of the ligamentum flavum cells in each group did not change significantly.4.IGF-1 can activate IGF-1R/AKT/mTORCl signaling pathway of ligamentum flavum cells and promote the synthesis of collagen ? and collagen ?After 24 h exposure of different concentration of exogenous IGF-1,ligamentum flavum cells pIGF-1R,pAKT,pS6 expression were obviously increased.These indicated that IGF-1 activated IGF-1 R/AKT/mTORC1 signaling pathway and existed concentration-response relationship.With increasing of concentration of IGF-1,pIGF-1R,pAKT,pS6 expression gradually enhanced.At the same time,the expression of collagen ? and collagen ? were stimulated by IGF-1.Collagen ? and collagen ? also significantly enhanced and there was a concentration-response relationship with IGF-5.NVP-AEW541 and rapamycin can attenuate collagen ? and collagen ?synthesisNVP-AEW541(inhibitor of IGF-1R)and rapamycin(specific inhibitor of mTORC1),IGF-1R/AKT/mTORC1 signaling pathways related protein expression were decreased significantly than the sole IGF-1 group.The expression of IGF-1,collagen ? and collagen ? were also significantly reduced.These phenomenon indicated that NVP-AEW541 and rapamycin inhibited collagen ? and collagen ?synthesis by blocking IGF-1R/AKT/mTORCl signaling pathway.6.Mechanical stress promoted ligamentum flavum cells to secrete endogenous IGF-land synthesis of collagen ? and collagen ?Under the stimulation of mechanical stress,ligamentum flavum cells increased IGF-1 secretion and activity of IGF-1R/AKT/mTORC1 signaling pathway.Meanwhile,mechanical stress increased synthesis of collagen ? and ?.NVP-AEW541 and rapamycin inhibited activity of IGF-1R/AKT/mTORC1 signaling pathway,then reduced synthesis of collagen ? and ?,but did not affect ligamentum flavum cells to secrete IGF-1.Conclusion:To sum up,IGF-1 can promote synthesis of collagen ? and collagen ? via IGF-1 R/AKT/mTORC1 signaling pathway.Collagen ? and collagen ? proliferated and accumulated lead to hypertrophy of the ligamentum flavum.Consequently,IGF-1 was believed to play a crucial role in hypertrophy of the ligamentum flavum.