Preventive Effect And Mechanism Of Decorin On Epidural Fibrosis And Epidural Adhesions After Laminectomy

Part Ⅰ The Expression of Decorin in Epidural Scar TissueObjective: To explore the expression level of decorin in the epidural scar tissue after lumbar spine surgery.Methods: The patients who had previously undergone lumbar fenestration or laminectomy were included in the study.At reoperation,epidural scar tissue and adjacent normal tissue were obtained.The total protein was extracted from the scar tissue and adjacent normal tissue,respectively.Then,the expression levels of decorin,transforming growth factor beta 1(TGF-β1)and collagen Ⅰ were detected by Western-blot.In addition,the expression levels of decorin,TGF-β1 and collagen Ⅰ were analyzed by immunohistochemical staining.Results: Western-blot analysis showed that the expression of decorin in the epidural scar tissue was significantly lower than that in the normal tissue.However,the expression of TGF-β1 and collagen Ⅰ in the scar tissue was significantly increased.Immunohistochemical staining showed that the positive expression of decorin was lower than that of normal tissue,while the positive expression of TGF-β1 and collagen Ⅰ was higher than that of normal tissue.Conclusion: The expression of decorin in the epidural scar tissue was significantly decreased,while the expression of TGF-β1 and collagen Ⅰ was significantly increased.The decrease of decorin and the high concentration of TGF-β1 may jointly promote hyperplasia of epidural scar.Part Ⅱ The Effect and Mechanism of Decorin on Proliferation and Transdifferentiation of the Human FibroblastsObjective: To investigate the effects of decorin on proliferation and transdifferentiation of the human fibroblasts in vitro.The molecular mechanisms that might be involved were also explored.Methods: The Cell Counting Kit-8(CCK-8)assay was used to detect different concentrations of decorin(1 μg/mL,2.5 μg/mL,5 μg/mL and 7.5 μg/mL)and TGF-β1(1 ng/mL,2.5 ng/mL,5 ng/mL and 10 ng /mL)on the proliferation of the human fibroblasts in vitro.After the fibroblasts were stimulated with TGF-β1(5 ng/mL)and decorin(5 μg/mL and 7.5 μg/mL),the expression of α-smooth muscle actin(α-SMA)was detected by Western-blot,Polymerase chain reaction(PCR)and immunofluorescence staining to observe the effect of decorin on the transdifferentiation of fibroblasts into myofibroblasts.Meanwhile,the effects of decorin on the synthesis of collagen I,collagen Ⅲ and fibronectin in the fibroblasts induced by TGF-β1 were detected by Western-blot and PCR.Then,immunofluorescence staining was used to further verify the expression of collagen Ⅰ and fibronectin in the fibroblasts stimulated by TGF-β1.In order to explore the effects of decorin on Smad2/3 signaling pathway,the changes of Smad2/3 signaling pathway-related protein molecules including Smad2,p-Smad2,Smad3 and p-Smad3 were detected by Western-blot.The nuclear translocation of Smad2/3 was observed by immunofluorescence staining.Finally,the effects of SIS3(a novel specific inhibitor of Smad3)and decorin(7.5 μg/mL)on the expression of Smad3,p-Smad3,α-SMA and collagen Ⅰ in the fibroblasts stimulated by TGF-β1 were detected by Western-blot.Results: The CCK-8 assay showed that the decorin had no significant effect on the proliferation of fibroblasts,while TGF-β1 promoted the proliferation of fibroblasts in a dose-dependent manner.This promotion effect peaked when the concentration of TGF-β1 was 5 ng/ml.However,the proliferation of fibroblasts induced by TGF-β1(5 ng/mL)was significantly inhibited by the decorin(5 μg/mL and 7.5 μg/mL).The results of Western-blot and PCR showed that decorin could significantly inhibit the expression of α-SMA in fibroblasts induced by TGF-β1.Immunofluorescence staining showed that the expression of α-SMA in the fibroblasts was notably increased after TGF-β1 stimulation,but significantly decreased after decorin intervention.In addition,the Western-blot and PCR showed that decorin significantly inhibited the synthesis of extracellular matrix components(collagen I,collagen Ⅲ and fibronectin)of the fibroblasts after TGF-β1 stimulation.Immunofluorescence staining showed the similarly trend.The activation of the Smad2/3 signaling pathway induced by TGF-β1 was time dependent,and the activation of the Smad2/3 signaling pathway was the strongest after stimulated with TGF-β1 for 1 h.However,the Western-blot analysis showed that the phosphorylation levels of Smad2 and Smad3 were significantly reduced after treatment with decorin(5 μg/mL and 7.5 μg/mL)in the TGF-β1-treated fibroblasts.Moreover,immunofluorescence staining showed that the decorin inhibited the nuclear translocation of Smad2/3.Furthermore,the Western-blot analysis showed that both SIS3(10 μM)and decorin(7.5 μg/mL)significantly inhibited the expression of p-Smad3,α-SMA and collagen Ⅰ in the fibroblasts stimulated by TGF-β1.Conclusion: Decorin can significantly inhibit TGF-β1-induced fibroblasts proliferation and transdifferentiation into myofibroblasts.In addition,decorin significantly reduced the production of collagen fibers and fibronectin in fibroblasts following TGF-β1 stimulation.The Smad2/3 signaling pathway is involved in the epidural fibrosis process induced by TGF-β1.However,decorin can inhibit TGF-β1-induced activation of the Smad2/3 signaling pathway.Part Ⅲ The Preventive Effect of Decorin on Peridural Fibrosis and Adhesions after Laminectomy in Rats: An in vivo StudyObjective: To explore the effect of decorin on prevention of epidural fibrosis and adhesions in rat laminectomy model.Methods: A total of 128 rats were randomly divided into four groups: sham group,model group,spongostan group and decorin treatment group.Sham group(n = 32): accepted sham operation without laminectomy;model group(n = 32): only a laminectomy was performed;spongostan group(n = 32): a spongostan impregnated with 0.4 mL of saline solution was retained on the dura mater;decorin treatment group(n = 32): a spongostan impregnated with decorin in 0.4 mL of saline solution was left on the dura mater.The hind limb motor function of the rats was scored and recorded according to BBB Locomotor Rating Scale before and after operation.Images of the laminectomy sites were obtained by Bruker 7.0 T magnetic resonance imaging(MRI)at 4 and 8 weeks postoperatively.Epidural scar hyperplasia and fibrosis were scored by the radiologists independently on the MRIs.At 4 and 8 weeks postoperatively,8 rats were randomly selected from each group and dissected at the previous operative incision to observe epidural fibrosis and adhesions.Then,the epidural adhesions were evaluated according to Rydell standard.Another 8 rats were selected from each group.The spine columns,including surrounding muscle tissue and epidural fibrotic tissue,were resected en bloc between the cephalad and caudal vertebral bodies with the laminectomy site as the center.After decalcification,the specimens were sectioned for H&E staining,Masson staining and immunohistochemical staining.H&E staining was used to assess the fibroblast infiltration.Masson staining was used to assess the epidural collagen deposition,dural thickness and epidural fibrosis.In addition,the effects of decorin on collagen Ⅰ and fibronectin synthesis in vivo were detected by immunohistochemical staining.Results: There was no significant difference in motor function scores among the four groups preoperatively and postoperatively immediately.At 4 and 8 weeks,BBB Locomotor Rating scores were lower in the model group and the spongostan group than in the sham group.However,the motor function scores were not significantly decreased in the decorin treatment group,and were significantly better than those in the model group and the spongostan group.Gross observation results showed that a large number of scar tissues were formed and adhered to the dura mater in the model group,which were difficult to dissect and separate.In the spongostan group,the epidural scar tissues sticking with the dura mater were easy to dissect at 4 weeks.By the 8th week,the epidural scar tissues were difficult to dissect.By contrast,the decorin treatment group showed mild epidural adhesions,which can be bluntly separated by minimal manual traction without damaging the dura.The results of epidural adhesions score based on Rydell standard showed that the decorin treatment group had the lowest epidural adhesions scores at both 4 and 8 weeks,indicating that the epidural adhesion was the least severe compared with other groups.The results of MRI scanning showed the rats in the model group exhibited noticeable epidural scar adhesions and dense scar tissues compression on the dural sac.At the 4th week,a narrow gap between dense scar tissues and dura mater could be observed in the spongostan group.The dura mater was not obviously compressed.However,in the 8th week,the dense scar tissues increased significantly,causing compression on the dural sac.In the decorin treatment group,there was only a small amount of dense scar tissues around the dural sac at 4 and 8 weeks,and there was no compression on the dural sac.MRIs-based grading of epidural scar hyperplasia and fibrosis showed that the decorin treatment group received the lowest rating,representing the lowest degree of epidural scar hyperplasia and fibrosis.H&E staining and Masson staining showed that the outside dural canal was filled with abundant scar tissues,and the epidural adhesions were extensive and serious in the model group.In the spongostan group,although there was a fuzzy gap between dura mater and epidural scar tissues,it was filled by many fibrous connections at 8 weeks.By contrast,the gap in histological sections corresponding to the decorin treatment group was more evident at 4 and 8 weeks.In addition,the model and spongostan groups exhibited more abundant fibroblasts than the decorin treatment group.There were significant differences between the decorin and model groups,and decorin and spongostan groups.This indicated that decorin could inhibit the proliferation of fibroblasts in vivo.Furthermore,significantly higher mean thickness values of dura mater were observed in the model and spongostan groups than in the sham group at 4 and 8 weeks.There was no significant difference between the model and the spongostan group.However,there were significant differences in the decorin treatment group compared with the model and the spongostan group at 4 and 8 weeks.Accordingly,the lowest epidural fibrosis score was observed in the decorin treatment group,indicating significantly less epidural fibrosis and epidural adhesions in this group.The immunohistochemical staining showed that decorin inhibited the synthesis of collagen Ⅰ and fibronectin in vivo.Conclusion: Epidural fibrosis and adhesions after laminectomy may impair neurological function.Decorin can prevent epidural fibrosis and adhesions by reducing fibroblast infiltration and inhibiting the synthesis of extracellular matrix in vivo.