Clusterin Negatively Modulates Mechanical Stress-mediated Ligamentum Flavum Hypertrophy

ObjectiveTo explore the mechanism of Clusterin on mechanical stress-mediated ligamentum flavum hypertrophy(LFH),and to provide theoretical and potential therapeutic targets for clinical treatment of ligamentum flavum hypertrophy.Methods1.Clinical collection of LFH tissues and Non-LFH tissues were screened for the differentially expressed proteins Clusterin by iTRAQ technology and proteomic.Profibrotic protein TGF-β1 was predicted through protein-protein interaction in String.Clusterin,TGF-β1 and fibrotic metabolite markers COL1A2 and α-SMA were verified by immunohistochemical staining,RT-PCR and Western blot.2.Mechanical stress-LFH cell model was constructed by Flexcell FX-5000 system in vitro.Clusterin,TGF-β1 and fibrotic metabolite markers COL1A2,α-SMA were verified by Immunohistochemical staining,RT-PCR,Western blot and ELISA..3.Inflammatory-LFH cell model was constructed by TGF-β1 in vitro.Recombinant Clusterin protein was used to observe the effects of TGF-β1-LFH.Phosphorylation of SMAD3 pathway and markers of fibrosis COL1A2 and α-SMA were tested by immunofluorescence staining,RT-PCR and Western blot.4.Overexpression or silenced ALK5,the effects of recombinant Clusterin protein and/or TGF-β1 stimulation on LF fibroblasts were observed.The phosphorylation of SMAD3 pathway and fibrosis markers COL1A2 and α-SMA were verified by Western blot.5.Effects of PRKD3 on Clusterin gene,protein and function were observed after overexpression,silencing of PRKD3 or use of PRKD3 inhibitor CRT0066101.Fibrosis markers COL1A2 and α-SMA were detected by immunofluorescence staining,RT-PCR and Western blot.6.A mechanical stress-ligamentum flavum hypertrophy model of mice in vivo was established.Recombinant Clusterin protein was injected into the tail vein to observe the function of Clusterin on LFH.HE staining was used to measure the area of LF.The ratio of collagen fiber to elastic fiber was verified by EVG and Masson staining.Immunohistochemical staining was used to verify the phosphorylation of SMAD3 pathway and a marker α-SMA for fibrosis.7.Serum samples were collected from patients with and without LFH,and Clusterin,PRKD3 protein levels were measured using ELISA.Results1.The differentially expressed proteins Clusterin in high throughput sequencing increased significantly in LFH(Fold Chang=30.33,P=2.20E-09).Bioassay Clusterin predicts the profibrotic protein TGF-β1 through protein-protein interaction.The level of Clusterin,TGF-β1 and fibrotic metabolite markers COL1A2 and α-SMA was significantly increased in LFH,with statisticslly significance differences.2.The genes and proteins of Clusterin,TGF-β1 and fibrotic markers COL1A2 and α-SMA were significantly increased under mechanical stress,with statisticslly significance differences..3.Recombinant Clusterin protein inhibited TGF-β1 stimulation of LF fibroblast fibrosis,and decreased metabolite markers COL1A2 and α-SMA of SMAD3 pathway phosphorylation and fibrosis.Overexpression of ALK5 enhanced TGF-β1 to stimulate LFH,and silencing of ALK5 inhibited TGF-β1 to stimulate fibroblast fibrosis.4.Overexpression of ALK5 enhanced TGF-β1 to stimulate LFH,and silencing of ALK5 inhibited TGF-β1 to stimulate fibroblast fibrosis.Recombinant Clusterin protein inhibited TGF-β1 to stimulate fibroblast fibrosis.5.Silencing of PRKD3 inhibited lysosome to degrade Clusterin protein,and did not affect Clusterin mRNA.Clusterin protein lost its biological activity after lysosome degradation6.Recombinant Clusterin protein alleviated the fibrosis of LF caused by mechanical stress in vivo.7.Human serum Clusterin and PRKD3 protein concentrations did not differ significantly between patients with and without LFH.Clusterin protein acted locally in LF.ConclusionsClusterin alleviates mechanical stress-induced ligamentum flavum hypertrophy in vivo and in vitro through inhibiting TGF-β1/ALK5/SMAD3 signaling pathway.