Pathomechanism Of ROS（H₂O₂） In The Process Of The Lumbar Ligamentum Flavum Hypertrophy

Objective Hypertrophy of the ligamentum flavum（HLF）is a significant causative factor in lumbar spinal canal stenosis,being one of the most common spinal disorders in the elderly patients,which can cause the symptoms of radiculopathy and neurogenic claudication secondary to compression of the cauda equina or nerve roots.Studies related to HLF mainly focused on the morphology and histology.The main treatment is surgerical decompression to relieve the oppression.The exact pathogenesis for the progressive hypertrophy of the lumbar LF remain poorly understood.The characteristic histologic changes are disappearance and degeneration of elastic fibers as well as changes in fibers such as proliferation of collagen fibers and cleft in the tissue.While several factors have been reported to be associated with hypertrophy of the LF formation and progression,such as TGF-β1,b FGF,VEGF,MMPs/TIMPs,etc,its exact pathogenesis has not been elucidated yet.Mechanical stress and various cytokines may play a crucial role in this disorder,and further studies are needed to expound the mechanism.In this paper,we make attempts to review the histopathology,major pathogenesis,other factors of hypertrophy of the LF.As the growth of the age,the increased reactive oxygen species（ROS）can lead to cellular mitochondria damage.ROS accumulation is associated with tissue degeneration.In a variety of tissues and organs,of TGF-β1 and FGF-2 could promote collagen synthesis through ERK1/2-NF-κB signaling pathway,and subsequently accelerate the degeneration.The purpose of this study is to clarify the influence of ROS on the HLF and to explore if ROS induces TGF-β1 and FGF-2 expression to acceleratedegeneration and HLF via ERK-NF-κB pathways.Method 1.Isolation,culture of human LFC The LF specimens was washed with sterile saline solution and the tissue of impurities was removed.LFCs were isolated and cultured with tissue piece inoculation methods in vitro.Cells were subcultured when cell density was 70% 80% fusion.The fourth to sixth generation cells were used to experiment.2.The expression of TGF-β1,FGF-2,CollagenⅠ,CollagenⅢ,NF-κB m RNA and protein.2.1 Grouping:LFCs are divided into control group,50μmol/L group,100μmol/L group,150μmol/L group,200μmol/L group.2.2 q RT-PCR:Total RNA was extracted,when LFCs were stimulated by H₂O₂ for 72 h according to control group and experimental group.The expressions of TGF-β1,FGF-2,CollagenⅠ,CollagenⅢ,NF-κB m RNA in both groups were measured by q RT-PCR.2.3Western blot:Total protein was extracted,when LFCs were stimulated by H₂O₂ for 72 h according to control group and experimental group.The expressions of TGF-β1,FGF-2,CollagenⅠ,CollagenⅢ,NF-κB protein in groups were measured by Western blot.3.The expression of ERK1/2-NF-κB signal pathway.3.1LFCs were treated by 200μM H₂O₂ for 5min,10 min,15min,20 min,30min and 60 min,resprectively.The expression of p-ERK1/2 were measured by Western blot.3.2 LFCs were treated by 200μM H₂O₂ for 10 min,20min,30 min,60min,90 min and 120 min,resprectively.The expression of p-p65 were measured by Western blot.3.3LFCs are divided into 200μM H₂O₂ group,200μM H₂O₂+HY-15947 group,200μM H₂O₂+ Bay11-7082 group,200μM H₂O₂+S7959 group,200μM H₂O₂+NSC37204 group.All groups were treated for 72 h.The expressions of CollagenⅠand CollagenⅢ protein in groups were measured by Western blot.LFCs are divided into 200μM H₂O₂+S7959 group,200μM H₂O₂+ NSC37204 group.Both groups were treated for15 min,20min or 60 min,90min,resprectively.The expressions of p-ERK1/2 and p-p65 were measured by Western blot.Results 1.LFCs were observed in medium at 14 days.The cultured cells were in a fibrocyte-like form with long fusiform,irregular polygon or disciform,and with moderate volume,shining brilliant cytoplasm,the big and elliptic nucleus.2.Compared with control group,the expressions of CollagenⅢ m RMA in all groups,CollagenⅠm RMA in 200μM H₂O₂ group,TGF-β1,FGF-2 and NF-κB m RNA in 150μM H₂O₂ group and 200μM H₂O₂ group are significantly higher.（p<0.05）The expressions of TGF-β1,FGF-2,CollagenⅠ,CollagenⅢ,NF-κB protein in experimental groups are significantly higher than control group.3.The expressions of p-ERK1/2 and p-p65 are significantly higher when treated by 200μM H₂O₂.When the TGF-β1 inhibitor or FGF-2 inhibitor is added,the expressions of p-ERK1/2 and p-p65 are significantly reduced when treated by 200μM H₂O₂.The expressions of CollagenⅠand CollagenⅢ protein in LFCs which are treated by 200μM H₂O₂ are significantly reduced when the p-ERK1/2 inhibitor,p-p65 inhibitor,TGF-β1 inhibitor or FGF-2 inhibitor is added.Conclusion 1.Tissue piece inoculation can successfully isolate human lumbar ligamentum flavum cells,which is the preferable method used in the molecular biology research.2.ROS accumulation can lead to increased expressions of TGF-β1,FGF-2,CollagenⅠ,CollagenⅢ,NF-κB.3.ROS（H₂O₂）induces TGF-β1 and FGF-2 expression to accelerate degeneration and hypertrophy of the ligamentum flavum via ERK-NF-κB pathways...