独活寄生汤延缓人椎间盘髓核细胞退变机制的研究

Mechanism of Duhuo Jisheng decotion in delaying degeneration of nucleus pulposus cells in human intervertebral disc

探讨独活寄生汤（Duhuo Jisheng decotion,DHJSD）延缓人椎间盘退变的作用及其可能的分子机制。术中摘取人椎间盘标本,根据磁共振Pfirrmann退变程度分为正常组和退变组。Western blot和Real-time PCR法检测椎间盘组织中炎症因子TNF-α,IL-1β和胞外基质分解酶MMP-3,MMP-13的表达。体外培养人髓核细胞,用CCK-8法测定在基质细胞衍生因子1（stromal cell-derived factor-1,SDF-1,10μg·L-1）和各种不同浓度的DHJSD共同处理下,髓核细胞的活力情况,并筛选出DHJSD合适的作用浓度。随后分为3组：对照组、SDF-1组、DHJSD＋SDF-1组,检测各组细胞TNF-α,IL-1β,Agg,co IⅡ,MMP-3,MMP-13的表达情况。为进一步探讨其分子机制,采用NF-κB抑制剂（BAY11-7082）或CXCR4-siRNA转染髓核细胞,检测CXCR4,NF-κB主要基团P65磷酸化水平（p-P65）以及P65核转移情况,细胞炎症因子和细胞外基质的表达情况。通过检测2组椎间盘组织发现退变髓核组织中TNF-α,IL-1β,MMP-3,MMP-13的表达明显升高（P〈0.05）。而体外细胞实验发现,CCK-8法检测显示当DHJSD质量浓度为300 mg·L-1时髓核细胞的活力明显增加。将实验分为3组后,检测发现与单独SDF-1组相比,用DHJSD处理后TNF-α,IL-1β,MMP-3,MMP-13含量明显降低,而Agg和co IⅡ表达明显上升。采用CXCR4-siRNA转染髓核细胞后发现,SDF-1可以明显提高CXCR4和p-P65的表达,促进P65向核内转移,而给予DHJSD或CXCR4-siRNA处理后其作用明显被抑制。进一步采用BAY11-7082处理髓核细胞后发现,能明显降低髓核细胞炎症因子TNF-α和IL-1β,以及胞外基质分解酶MMP-3和MMP-13的表达。独活寄生汤可抑制炎症因子表达,促进胞外基质合成,其潜在机制与SDF-1/CXCR4/NF-κB信号通路有关。

This paper aimed to investigate the role of Duhuo Jisheng decotion（DHJSD） in delaying human disc degeneration and its possible molecular mechanism. The intervertebral disc specimens were divided into normal and degenerated groups according to Pfirrmann classfication. The expressions of TNF-α,IL-1β,MMP-3 and MMP-13 in intervertebral disc tissue were detected by Western blot and PCR. Then degenerated human primary NPCs were cultured in vitro,the viability of NPCs treated with stromal cell-derived factor-1（SDF-1,10 μg·L-1） and various concentrations of DHJSD was assessed by the CCK-8 assay,and the appropriate concentration was screened. The experiment was divided into three groups,control group,SDF-1 group and DHJSD plus SDF-1 group. The levels of TNF-α,IL-1β,Agg,co I Ⅱ,MMP-3 and MMP-13 were detected. The levels of CXCR4,NF-κB major groups P65 phosphorylation（p-P65） and nuclear translocation,after treated with CXCR4 siRNA and NF-κB inhibitor（BAY11-7082） were measured by Western blot and immunofluorescence. At the same time,the expression of cell inflammatory factors and extracellular matrix were also measured. The expressions of TNF-α,IL-1β,MMP-3 and MMP-13 in the degenerated intervertebral disc tissue were significantly increased. In vitro study,the results of CCK-8 indicated that the viability of NPCs was significantly increased when DHJSD concentration was 300 mg·L-1. After the experiment was divided into three groups,compared with SDF-1 group,the expressions of TNF-α,IL-1β,MMP-3 and MMP-13 in DHJSD group were significantly decreased,but the expressions of Agg,co I Ⅱ were significantly increased.When CXCR4-siRNA was transfected into NPCs,SDF-1 increased expressions of CXCR4 and p-P65 and inhibited nuclear translocation of P65,whose effect was suppressed by CXCR4-siRNA and DHJSD. In addition,when BAY11-7082 was used to treat NPCs,the expression of TNF-α,IL-1β,MMP-3 and MMP-13 were significantly decreased. DHJSD could inhibit the production of inflammatory factors and promote the synthesis of extracellular matrix. The potential mechanism may be related to the SDF-1/CXCR4/NF-κB signaling pathway.