基于Nrf2/HO-1信号通路探讨异甘草素改善KOA痛敏的机制研究

Research on the Mechanism of Isoliquiritigenin Improving Pain Sensitivity in KOA Based on the Nrf2/HO-1 Signaling Pathway

    摘要
  • 摘要:
    目的 探讨异甘草素调控核因子E2相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路改善膝骨关节炎（Knee osteoarthritis，KOA）痛敏的作用机制。
    方法 将50只C57BL/6J小鼠随机分成空白组、KOA组、异甘草素低剂量组（10 mg·kg-1）、异甘草素高剂量组（40 mg·kg-1）、塞来昔布组（43.3 mg·kg-1），每组10只；采用半月板失稳术（Destabilization of the medial meniscus，DMM）构建KOA模型，评估小鼠痛敏状态；采用ELISA法检测小鼠血清中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、白细胞介素6（IL-6）表达水平；免疫荧光观察各组小鼠背根神经节（Dorsal root ganglia，DRG）组织中瞬时受体电位（Transient receptor potential channels，TRPs）离子通道荧光强度；Western blot法检测各组DRG中Nrf2、HO-1、PGP9.5、NGF和Substance P蛋白表达水平。体外实验中使用ML385抑制Nrf2/HO-1信号通路，探讨Nrf2/HO-1信号通路被抑制后异甘草素处理对神经元的影响。
    结果 与空白组相比，KOA组小鼠冷刺激阈值以及机械刺激阈值显著下降（P<0.01）；TRPA1、TRPV1、TRPM8荧光强度显著升高（P<0.01）；血清TNF-α、IL-1β、IL-6水平显著升高（P<0.01）；Nrf2和HO-1表达水平显著降低（P<0.01），PGP9.5、NGF和Substance P的表达水平显著升高（P<0.01）。与KOA组相比，异甘草素不同剂量组及塞来昔布组小鼠冷刺激阈值以及机械刺激阈值显著升高（P<0.01）；TRPA1、TRPV1、TRPM8荧光强度显著降低（P<0.01）；血清TNF-α、IL-1β、IL-6水平显著降低（P<0.05，P<0.01）；Nrf2和HO-1的表达水平显著升高（P<0.01），PGP9.5、NGF和Substance P的表达水平显著降低（P<0.05，P<0.01）。体外实验结果显示，与空白组相比，LPS组中Nrf2和HO-1的水平降低（P<0.01），PGP9.5、NGF和Substance P的蛋白表达水平升高（P<0.01）；ROS含量显著升高（P<0.01）。与LPS组相比，异甘草素组Nrf2和HO-1的蛋白表达水平显著升高（P<0.01），PGP9.5、NGF和Substance P的蛋白表达水平显著降低（P<0.01）；ROS含量显著降低（P<0.01）。与异甘草素组相比，异甘草素+ML385组中Nrf2和HO-1的蛋白表达水平显著降低（P<0.01），PGP9.5、NGF和Substance P的蛋白表达水平显著升高（P<0.01）；ROS含量显著升高（P<0.01）。
    结论 异甘草素可能通过Nrf2/HO-1信号通路改善KOA痛敏。

     

    Abstract:
    OBJECTIVE To investigate the mechanism by which isoliquiritigenin (ISL) improves pain hypersensitivity in knee osteoarthritis (KOA) by regulating the Nrf2/HO-1 signaling pathway.
    METHODS Fifty C57BL/6J mice were randomly divided into five groups (n=10): control group, KOA group, low-dose ISL group (10 mg·kg-1), high-dose ISL group (40 mg·kg-1), and celecoxib group (43.3 mg·kg-1). A KOA model was established using the destabilization of the medial meniscus (DMM) surgery, and pain hypersensitivity was assessed. In vivo experiments included ELISA to measure serum levels of TNF-α, IL-1β, and IL-6; immunofluorescence to observe TRPA1, TRPV1, and TRPM8 ion channel fluorescence intensity in dorsal root ganglia (DRG); and Western blot to quantify Nrf2, HO-1, PGP9.5, NGF, and Substance P protein expression in DRG. For in vitro experiments, ML385 was used to inhibit the Nrf2/HO-1 pathway, and the effects of ISL on neurons were evaluated after pathway inhibition.
    RESULTS Compared with the control group, the KOA group exhibited significantly reduced cold and mechanical stimulation thresholds (P<0.01), increased TRPA1, TRPV1, and TRPM8 fluorescence intensity (P<0.01), elevated serum TNF-α, IL-1β, and IL-6 levels (P<0.01), decreased Nrf2 and HO-1 expression, and increased PGP9.5, NGF, and Substance P expression (P<0.01). Compared with the KOA group, both ISL dose groups and the celecoxib group showed significantly improved cold and mechanical stimulation thresholds (P<0.01), reduced TRP channel fluorescence intensity (P<0.01), decreased inflammatory cytokine levels (P<0.05,P<0.01), upregulated Nrf2 and HO-1 expression, and downregulated PGP9.5, NGF, and Substance P expression (P<0.05, P<0.01). In vitro experiments revealed that LPS treatment reduced Nrf2/HO-1 levels (P<0.01), increased PGP9.5, NGF, and Substance P expression (P<0.01), and elevated reactive oxygen species (ROS) (P<0.01). ISL reversed these effects (P<0.01), while ML385 co-treatment abolished ISL’s benefits, reactivating ROS and pain-related proteins (P<0.01).
    CONCLUSION Isoliquiritigenin alleviates KOA pain hypersensitivity by activating the Nrf2/HO-1 signaling pathway.

     

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