Liu Zong-Chao, Jiang Yan, Huang Chen-Yi, Liu Yong, Wei Zhang-Chao, Liu Shi-Gui, Fu Zhi-Jiang, Ma Chuan
Department of Orthopedic Surgery, Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, Luzhou 646000, China.
The First Affiliated Hospital of Southwest Medical University, Luzhou 646000, China.
Zhongguo Zhong Yao Za Zhi. 2018 Jul;43(13):2764-2769. doi: 10.19540/j.cnki.cjcmm.20180503.001.
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, coIⅡ, 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, coIⅡ 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.
本文旨在探讨独活寄生汤（DHJSD）在延缓人类椎间盘退变中的作用及其可能的分子机制。根据Pfirrmann分级将椎间盘标本分为正常组和退变组。采用蛋白质印迹法和聚合酶链反应检测椎间盘组织中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、基质金属蛋白酶-3（MMP-3）和基质金属蛋白酶-13（MMP-13）的表达。然后体外培养退变的人原代髓核细胞（NPCs），通过细胞计数试剂盒-8（CCK-8）法评估经基质细胞衍生因子-1（SDF-1，10 μg·L⁻¹）和不同浓度独活寄生汤处理的NPCs的活力，并筛选出合适的浓度。实验分为三组，对照组、SDF-1组和独活寄生汤加SDF-1组。检测TNF-α、IL-1β、聚集蛋白聚糖（Agg）、Ⅱ型胶原蛋白（coIⅡ）、MMP-3和MMP-13的水平。采用蛋白质印迹法和免疫荧光法检测用CXCR4小干扰RNA（siRNA）和核因子-κB（NF-κB）抑制剂（BAY11-7082）处理后CXCR4、NF-κB主要亚基P65磷酸化（p-P65）水平及核转位情况。同时，检测细胞炎性因子和细胞外基质的表达。退变椎间盘组织中TNF-α、IL-1β、MMP-3和MMP-13的表达显著增加。体外研究中，CCK-8结果表明当独活寄生汤浓度为300 mg·L⁻¹时NPCs活力显著增加。实验分为三组后，与SDF-1组相比，独活寄生汤组TNF-α、IL-1β、MMP-3和MMP-13的表达显著降低，但Agg、coIⅡ的表达显著增加。当将CXCR4-siRNA转染到NPCs中时，SDF-1增加了CXCR4和p-P65的表达并抑制了P65的核转位，CXCR4-siRNA和独活寄生汤可抑制其作用。此外，当用BAY11-7082处理NPCs时，TNF-α、IL-1β、MMP-3和MMP-13的表达显著降低。独活寄生汤可抑制炎性因子的产生并促进细胞外基质的合成。其潜在机制可能与SDF-1/CXCR4/NF-κB信号通路有关。
