| Objective:To study the change of PGE2,TNF alpha,IL-6,IL – 8,MMPs,TIMP-1 mRNA for the human intervertebral disc annulus fibrosus cells disturbance by different level of IL-1 beta or(and)HMGB1;To test toll-like receptors-4,advanced glycosylation end products and research the correlation of IL-1 beta/HMGB1 signaling pathways with mediating inflammation of the intervertebral disc annulus damage;To study the correlation of NF-kappa B signaling pathway and IL-1 beta/HMGB1 signaling pathways mediating inflammation of the intervertebral disc annulus damage,and to apply a foundation for studying the mechanism of intervertebral disc degeneration,and provide the basis for the prevention and treatment of intervertebral disc herniation.Methods:Intervertebral disc annulus fibrosus cells were interfered by 0,0.5,1,2 or 5ng/ml IL-1 beta or(and)0,20,40 or 80ng/ml HMGB1 and intervertebral disc annulus fibrosus cells were interfered by in 2ng/ml IL-1 beta and(or)40ng/ml HMGB1 interference for 0,6,12,24 and 48 hours,the change of PGE2,TNF alpha,IL-6,IL-8 was detected by enzyme-linked immunosorbention,compared the difference between different concentrations and different time;intervertebral disc annulus fibrosus cells were interfered by 2ng/ml IL-1 beta and(or)40ng/ml HMGB1 for 12 hours,and MMP-1,3,9 and metal matrix protein inhibiting factor 1 mRNA and protein expression changes were detected by western blot and RT-qPCR.0,0.5,1,2 or 5 ng/ml IL-1 beta interfered fiber ring cell for 12 hours respectively,RT-PCR TLR-2,4,and 6 or RAGE mRNA level were detected,experiment could be divided into four groups,respectively,blank control group,40ng/mlHMGB1 interference group,2ng/ml IL-1 beta interference group and 40ng/ml HMGB1+2ng/ml IL-1 beta interference group interfered on disc annulus fibrosus cells,TLR mRNA level-4 and RAGE were measured by RT-PCR,and compared the differences between the four groups.we knocked down TLR-4 in the in human intervertebral disc cells,and re-evaluated the IL-1β and HMGB1 promoted release of inflammatory cytokines.2ng/ml IL-1 beta and 40ng/ml HMGB1 interfered in the disc annulus fibrosus cells respectively,using the method of Western Blot and RT-PCR,the NF-kappa B,I kappa B alpha protein and mRNA were detected.All experiment were done independently three times,the results used mean + /-standard error,the significance level was as 0.05.Results:Different concentrations of IL-1 beta and HMGB1 interfered inflammatory factors of intervertebral disc annulus fibrosus cells.PGE2 and TNF alpha content increased after 5ng/ml IL-1 disc interference fiber ring cell,1or 2ng/ml IL-1β interfered disc annulus fibrosus cells after,IL-6 and IL-8 levels significantly increased;0,20,40 and 80ng/ml HMGB1 interfered intervertebral disc cells for 24 hours,and inflammatory factors PGE2,TNF alpha,secretion of IL-6 and IL-8 levels were significantly increased,40 and 80ng/ml,PGE2 and TNF alpha significant rise,80ng/ml HMGB1 interfered intervertebral disc annulus fibrosus cells,IL-6 and IL-8 significant increased.IL-1 beta and HMGB1 effected on inflammatory factors of human intervertebral disc cells as synergy.At the same time,we used 2ng/ml IL-1 beta and 40ng/ml HMGB1 interference disc fiber ring cell,6,12,24 and 48 hours respectively,the 2ng/ml IL-1 beta interfered for 12 to 48 hours and 40 ng/ml HMGB1 interfered for 24 to 48 hours,PGE2 significant increased,while at the same time 2ng/ml IL-1 beta and 40ng/ml interfered HMGB1,levels of PGE2 significantly increased,the difference was statistically significant.Also,at the same time 2ng/ml IL-1 beta and 40ng/mlHMGB1 interfered disc fiber ring cell,TNF alpha,IL-6 and IL-8 significantly increased,it was higher than that of single use IL-1 beta or HMGB1 interfering.The effection of IL-1β and HMGB1 on intervertebral disc cells IL-1 beta(2 ng/ml)interfered intervertebral disc cells,MMP-1 and MMP-3 mRNA significant rose,40ng/ml HMGB1 lead to MMPs expression significant rising,2ng/ml IL-1 beta or 40ng/ml HMGB1 interfered intervertebral disc cells alone,TIMP-1 mRNA level rose unconspicuous,at the same time combining IL-1 beta and HMGB1 metal matrix proteins promoted the MMP of intervertebral disc cells.Study found 2 and 5ng/ml IL-1 beta interfered in intervertebral disc after annulus fibrosus cells,TLR-2 and TLR-4 mRNA significant rose,and TLR-6 mRNA level was not changed,in addition,we also study the 40ng/ml HMGB1 interfered in intervertebral disc cells,we found that the levels of both in 40ng/ml HMGB1 interfered in the cell,TLR-2,4 significantly increased,and 40ng/mlHMGB1 + 2ng/ml IL-1 beta interference group had elevated levels of TLR4/RAGE mRNA degree was higher than single drug group(the other three groups),and TLR4/RAGE mRNA level also increased at the same time.The expression of TLR-4 in both mRNA and protein levels was markedly reduced in the siRNA-TLR-4 groups,the promoted PGE2 release by both IL-1β(5ng/ml)and HMGB1(80ng/ml)was significantly inhibited by the transfection with 60 nM si RNA-TLR-4.In addition,the IL-1β and HMGB1 promoted release of TNF-α and IL-6 was also reduced in the siRNA-TLR-4 groups.However,such inhibition was not significant for IL-8.2ng/ml IL-1 beta and 40ng/ml HMGB1 interfered in the disc annulus fibrosus cells,the NF-kappa B/p65 mRNA level significantly increased,and 40ng/ml HMGB1 treatment group,the I kappa B alpha predominate mRNA level significantly decreased,when using 2ng/ml IL-1 beta joint 40ng/ml HMGB1 interfered in the disc annulus fibrosus cells,the NF-kappa B/p65 rose in mRNA and I kappa B alpha predominate mRNA decreased at the same time,we also using western blotting technique to detect the NF-kappa B,I kappa B alpha protein expression of predominate,2ng/ml IL–1 beta and 40ng/ml HMGB1 interfered in the disc annulus fibrosus cells,the NF-kappa B/p65 protein expression significantly raised,with 2ng/ml IL-1 beta joint 40 ng/ml HMGB1 interfered in the disc annulus fibrosus cells,the NF-kappa B/p65 protein expression increased more significantly,by comparison,with only 2ng/ml pure IL-1 beta compared 40ng/ml HMGB1 group,difference was statistical significance.2ng/ml IL-1 beta and 40ng/ml HMGB1 interfered in the disc annulus fibrosus cells,I kappa B alpha predominate protein expression significantly decreased,when using 2ng/ml IL-1 beta joint 40 ng/ml HMGB1 interfered in the disc annulus fibrosus cells,more significant the I kappa B alpha protein predominate,comparing with only pure 2ng/ml IL-1 beta,40ng/ml HMGB1 group,difference has statistical significance.Conclusion:1.With the increase of concentration of IL-1 beta and HMGB1,PGE2,TNF alpha,IL-6 and IL-8 secretion of intervertebral disc annulus fibrosus cells increased,and combing IL-1 beta and HMGB1 in cells,there increased significantly than single.2.With IL-1 beta and HMGB1 increasing,metal matrix proteins(MMP-1,MMP-3)and the secretion of MMP-9 increased,but not for the influence of the TIMP-1.3.In the intervertebral disc annulus fibrosus cells,with increasing of IL-1 beta,TLR-2,TLR-4,RAGE mRNA significant increased besides TLR-6.4.HMGB1 and IL-1 beta interfered joint disc annulus fibrosus cells,TLR4/RAGE mRNA rose with superposition effect.5.IL-1 beta and HMGB1 interfered in intervertebral disc annulus fibrosus cells,the NF-kappa B protein and mRNA expression rose significantly,but the I kappa B alpha protein and mRNA expression predominate dropped significantly.6.HMGB1 and IL-1 beta interfered in joint disc fiber ring cells on the NF-kappa B,rise in I kappa B alpha predominate and inhibited with superposition effect.This study found that in many factors of anulus fibrosus cells injury,inflammatory injury IL-1 beta and HMGB1 synergistic dose dependently induced by TLR4/RAGE signaling pathway,is one of the leading causes of intervertebral disc inflammation injury. |
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