Taurine Reduces The Mechanism Of Epidural Fibrosis After Laminectomy

Laminectomy is one of the most common operations for spine surgeons.Epidural scar formation after laminectomy weakened the effects of decompression.It is important to prevent and reduce epidural scar after laminectomy.Previous evidence has demonstrated that epidural scar formation was associated with excessive proliferation and activation of fibroblasts and collagen deposition.Thus,researchers focus on how to inhibit proliferation and activation of fibroblasts.Current major progresses in preventing scar formation include blocking infiltration and accumulation using topical biomaterials,inhibiting fibroblasts proliferation and inducing apoptosis using Mitomycin C,FK506,rapamycin et al.Our studies have revealed that both MMC-C and FK506 have significant effects in inhibiting fibroblasts proliferation,collagen synthesis and promoting cells apoptosis,thus reducing epidural scar formation.However,toxic effects on peripheral nerve tissue by topical use of MMC-C and FK506 seriously hindered clinical application of these reagents.Given this,we further explored to find an alternative safe and effective therapeutics.Taurine is one of the extremely abundant amino acids in mammals and is also a kind of common ingredients in energy drink Taurine has a role in adjusting osmosis pressure,stabilizing membrane and regulating nerve conduction and protecting cells from peroxidation induced by various stresses.Numerous studies have suggested that taurine can markedly inhibit fibrosis in multiple organs.As we all know,fibrosis of liver,lung and heart was actually the process of activation,proliferation of fibroblasts and collagen deposition,In this study,we found that taurine via both topical and intraperitoneal approach remarkably reduced epidural fibrosis in mice models of laminectomy.In vitro experiments demonstrated that the effects of taurine in inhibiting fibroblasts proliferation,activation and collagen synthesis was achieved via downregulating EG3R1.Part 1:MMC-C Induces the Apoptosis of Epidural Scar Fibroblasts after Spinal Cord Decompression via Endoplasmic Reticulum Stress PathwayObjective:The epidural scar adhesion after operation is related to the success of decompression of spinal cord injury.Studies have confirmed that the scar adhesion can be effectively alleviated by the local application of mitomycin C(MMC-C)after operation.The scar adhesion after operation is associated with the cell apoptosis,which is induced by endoplasmic reticulum stress pathway,but the specific mechanism remains unclear.Methods:Experiments were conducted to investigate the mechanism of MMC-C on the proliferation and apoptosis of human epidural scar fibroblasts.The hruman epidural scar fibroblasts were treated with MMC-C(1,5,10,20 and 40?g/mL)for 12,24 and 48h.Results:MMC-C inhibited the growth of human epidural scar fibroblasts in a dose-and time-dependent manner.MMC-C could significantly increase the expressions of pro-apoptotic proteins in cells,including Fas,DR4(death receptor 4),DR5,cleaved caspase-8/9,Bax,Bim and cleaved-3,and significantly decrease the expressions of anti-apoptotic proteins,including Bcl-2 and Bcl-xL.But MMC-C-induced apoptosis could not be completely inhibited by the caspase-8/9 inhibitors(Z-IETD-FMK and Z-LETD-FMK).At the same time,MMC-C could increase the expressions of GRP78,endoplasmic reticulum stress pathway-related protein(CHOP)and Caspase-4 protein in cells in a dose-dependent manner,thus inducing the endoplasmic reticulum stress.In the experiment,the endoplasmic reticulum stress inhibitor Salubrinal(Sal)could significantly inhibit the cell viability after treatment with MMC-C and inhibit the apoptosis through decreasing the CHOP expression in cells.Conclusion:The results confirmed that MMC-C can,through endoplasmic reticulum stress pathway,partially induce the apoptosis of epidural scar fibroblasts.Part 2:FK506 Activates Endoplasmic Reticulum Stress-induced Fibroblast Apoptosis through CHOP PathwayObjective:Hypertrophic scar(HS)is derived from the decreased apoptosis and increased proliferation of fibroblast,so the fibroblast apoptosis is the key task in developing new therapeutic strategies for HS.It has been shown in pre,vious reports that FK506(tacrolimus capsules 506)can reduce the formation of scar in the body and also induce endoplasmic reticulum stress(ER stress).However,the effect of FK506 on ER-mediated fibroblast apoptosis is still unknown.Methods:In this study,rat skin fibroblasts were used.The cell counting kit Kit-8 was adopted to detect the cell viability and Annexin V/Propidium Iodide Double-staining was used to detect the cell apoptosis.And then small interfering RNAs(siRNAs)or lentivirus infection was used to perform the gene silencing.Finally,Western blotting was performed to detect the expression of apoptosis-related proteins,and co-immunoprecipitation was used to investigate the interaction between proteins.Results:The cell viability could be significantly reduced and fibroblast apoptosis could be induced by FK506.Interestingly,FK506 treatment could also activate the ER stress.Our study further confirmed that ER stress triggered by activation of CHOP(C homologous protein 10)could modulate the FK506-induced apoptosis,which had been proved by the decreased cell apoptosis after inhibiting ER stress via TUDCA(tauro ursodesoxy cholic acid)or CHOP expression silencing.In addition,the results of co-immunoprecipitation showed that FK506 treatment induced FKBP12.6 to be separated from RyR2 and migrated from ER membrane to cytosol,thus accelerating the ER stress-mediated apoptosis.Conclusion:FK506-induced fibroblast apoptosis can be,through CHOP signaling pathway,regulated by ER stress.Part 3:Taurine Reduces the Epidural Fibrosis after Laminectomy via Down-regulating EGR1Objective:As a kind of common complication after laminectomy,Epidural fibrosis has been proved to be closely related to poor surgical results.It has been shown in previous studies that taurine has a significant inhibitory effect on the fibrosis in lung and liver.This study aimed to examine the effect of taurine on epidural fibrosis of model rats after laminectomy and investigate its possible molecular mechanisms.Methods:Each rat received laminectomy to establish the epidural fibrosis model.After taurine treatment,the epidural fibrosis was detected via Masson's tri chrome staining and immunohistochemical staining.The cell counting kit was used to detect the cell survival rate.And the apoptosis of fibroblasts was detected via Annexin V/Propidium Iodide Double-staining.The significant changes in mRNA were detected via Microarray.qRT-PCR(quantitative real-time polymerase chain reaction)was used to detect the mRNA expression level.The cell lines with stable knockout and overexpression of related proteins were established via lentivirus infection.And Western blotting was performed to detect the expression of fibrosis-related proteins.Results:Taurine significantly reduced the level of epidural fibrosis in model rats induced by laminectomy.But the expression levels of TGF-? and its receptor did not change,so it was confirmed that this effect of taurine was unrelated to the TGF-?(transforming growth factor-P)/Smad pathway.In addition,taurine had little effect on cell apoptosis.It is interesting that the expression level of EGR1(early growth response protein 1,a kind of fibrotic promoting factor)was significantly reduced by taurine in both in-vivo and in-vitro experiments.Moreover,the activation of fibroblasts was enhanced by the overexpression of EGR1,but the knockout of EGR1 exhibited an opposite effect,suggesting that EGR1 plays an important role in the inhibition of taurine on TGF-?-induced fibrosis.Conclusion:Taurine decreases the epidural fibrosis in in-vivo experiments and reduces the activation of fibroblasts in in-vitro experiments,which are mediated by EGR1.Taurine is expected to be a potential prophylactic agent for epidural fibrosis after laminectomy.