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王菲, 赖桂花, 曹建雄.基于TLR4/NF-κB信号通路探讨消肿止痛外敷散对乳腺癌骨转移大鼠的镇痛机制[J].湖南中医药大学学报,2025,45(12):2244-2253[点击复制] |
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| 基于TLR4/NF-κB信号通路探讨消肿止痛外敷散对乳腺癌骨转移大鼠的镇痛机制 |
| 王菲,赖桂花,曹建雄 |
| (湖南中医药大学第一中医临床学院, 湖南 长沙 410007;南华大学附属第一医院, 湖南 衡阳 421001;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 探讨消肿止痛外敷散对乳腺癌骨转移癌痛（BMCP）大鼠的镇痛作用及机制。方法 将48只雌性SD大鼠分为假手术组（8只）和造模组（40只）。造模组通过向大鼠左侧胫骨注入MRMT-1乳腺癌细胞悬液（3×104个细胞）建立骨转移癌痛模型。模型建立后，造模组进一步随机分为模型组、消肿止痛外敷散高剂量组（16.80 g/kg）、消肿止痛外敷散中剂量组（8.40 g/kg）、消肿止痛外敷散低剂量组（4.20 g/kg）和扶他林组（0.50 g/kg），每组8只。每日9:00给药，持续10 h，连续21 d。采用电子压痛仪检测机械痛阈值；使用智能热板仪检测热痛阈值；X线检查大鼠胫骨骨质破坏情况；HE染色观察大鼠胫骨组织病理变化；ELISA检测大鼠血清中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β和IL-6含量；qPCR检测大鼠背根神经节中Toll样受体4（TLR4）、核因子-κB（NF-κB） p65 mRNA表达水平；Western blot检测大鼠背根神经节中TLR4、髓样分化因子88（MyD88）、p-NF-κB p65、p-抑制性κB激酶β（IKKβ）、TNF受体相关因子6（TRAF6）与转化生长因子激酶1（TAK1）蛋白表达水平。结果 与假手术组相比，模型组、扶他林组及消肿止痛外敷散各剂量组大鼠机械痛阈值与热痛阈值均降低（P<0.05）；X线和HE染色均显示大鼠骨质严重破坏；血清TNF-α、IL-1β和IL-6含量、背根神经节中TLR4和NF-κB p65 mRNA表达水平、背根神经节中TLR4、MyD88、TAK1、TRAF6蛋白表达水平以及p-IKKβ/IKKβ、p-p65/p65蛋白表达水平比值均升高（P<0.05）。与模型组比较，扶他林组和消肿止痛外敷散各剂量组大鼠机械痛阈值和热痛阈值均升高（P<0.05）；X线和HE染色均显示大鼠骨质破坏改善，其中消肿止痛外敷散高、中剂量组改善最明显；血清TNF-α、IL-1β和IL-6含量、背根神经节中TLR4和NF-κB p65 mRNA表达水平、背根神经节中TLR4、MyD88、TAK1、TRAF6蛋白表达水平以及p-IKKβ/IKKβ、p-p65/p65蛋白表达水平比值均降低（P<0.05）。与扶他林组相比，消肿止痛外敷散高、中剂量组大鼠机械痛阈值和热痛阈值均升高（P<0.05），血清TNF-α、IL-1β和IL-6含量、背根神经节中TLR4和NF-κB p65 mRNA表达水平、背根神经节中TLR4、MyD88、TAK1、TRAF6蛋白表达水平以及p-IKKβ/IKKβ、p-p65/p65蛋白表达水平比值均降低（P<0.05）；消肿止痛外敷散低剂量组大鼠机械痛阈值和热痛阈值均降低（P<0.05），血清TNF-α、IL-1β和IL-6含量、背根神经节中TLR4和NF-κB p65 mRNA表达水平、背根神经节中TLR4、MyD88、TAK1、TRAF6蛋白表达水平以及p-IKKβ/IKKβ、p-p65/p65蛋白表达水平比值均升高（P<0.05）。与消肿止痛外敷散低剂量组相比，消肿止痛外敷散高、中剂量组大鼠机械痛阈值和热痛阈值均升高（P<0.05）；血清TNF-α、IL-1β和IL-6含量、背根神经节中TLR4和NF-κB p65 mRNA表达水平、背根神经节中TLR4、MyD88、TAK1、TRAF6蛋白表达水平以及p-IKKβ/IKKβ、p-p65/p65蛋白表达水平比值均降低（P<0.05）。结论 消肿止痛外敷散可缓解骨转移癌痛，可能与抑制TLR4/NF-κB信号通路激活，下调TNF-α、IL-1β和IL-6的表达相关。 |
| 关键词: 骨转移癌痛 消肿止痛外敷散 外治 TLR4/NF-κB 背根神经节 乳腺癌 |
| DOI：10.3969/j.issn.1674-070X.2025.12.002 |
| 投稿时间：2025-07-25 |
| 基金项目:湖南省中医药科研计划项目（201946）。 |
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| Analgesic mechanisms of topical Xiaozhong Zhitong Powder in rats with breast cancer bone metastasis based on the TLR4/NF-κB signaling pathway |
| WANG Fei, LAI Guihua, CAO Jianxiong |
| (The First Clinical School of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;The First Hospital of University of South China, Hengyang, Hunan 421001, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To investigate the analgesic effects of topical Xiaozhong Zhitong Powder (XZZTP) and its underlying mechanisms in a rat model of bone metastatic cancer pain (BMCP) secondary to breast cancer. Methods Forty-eight female Sprague-Dawley (SD) rats were allocated into sham-operated group (n=8) and modeling group (n=40). The BMCP model was established in the modeling group by injecting an MRMT-1 breast cancer cell suspension (3×104 cells) into the left tibia of each rat. After successful modeling, the modeling group were further randomized into model group, high-dose (16.80 g/kg), medium-dose (8.40 g/kg), and low-dose (4.20 g/kg) XZZTP groups, and Voltaren group (0.50 g/kg), with eight rats in each group. Treatments were administered daily starting at 9:00 a.m., sustained for ten hours, and continued for 21 consecutive days. Mechanical pain threshold was assessed using an electronic von Frey analgesiometer, while thermal pain threshold was measured with an intelligent hot plate apparatus. Bone destruction in the left tibia was evaluated via X-ray imaging, and histopathological changes in the tibial tissue were observed using HE staining. Serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were measured by ELISA. The mRNA expression levels of toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) p65 in the dorsal root ganglion (DRG) were quantified by qPCR. The protein expression levels of TLR4, myeloid differentiation primary response 88 (MyD88), phosphorylated NF-κB p65 (p-p65), phosphorylated inhibitor of nuclear factor kappa-B kinase beta (p-IKKβ), TNF receptor-associated factor 6 (TRAF6), and transforming growth factor beta-activated kinase 1 (TAK1) in the DRG were determined by Western blot. Results Compared with the sham-operated group, the model group, the Voltaren group, and all dose groups of XZZTP showed significant decreases in both mechanical and thermal pain thresholds (P<0.05). Both X-ray imaging and HE staining revealed severe bone destruction. Serum levels of TNF-α, IL-1β, and IL-6 were significantly elevated (P<0.05). Furthermore, the mRNA expression levels of TLR4 and NF-κB p65 in the DRG significantly increased (P<0.05). The protein expression levels of TLR4, MyD88, TAK1, and TRAF6 in the DRG, as well as the ratios of p-IKKβ/IKKβ and p-p65/p65, were all significantly higher (P<0.05). Compared with the model group, both the Voltaren group and all dose groups of XZZTP exhibited significant increases in mechanical and thermal pain thresholds (P<0.05). X-ray imaging and HE staining showed improved bone architecture, with the most pronounced improvements observed in the high- and medium-dose XZZTP groups. Serum levels of TNF-α, IL-1β, and IL-6 were significantly reduced (P<0.05). The mRNA expression levels of TLR4 and NF-κB p65 in the DRG were significantly downregulated (P<0.05). Additionally, the protein expression levels of TLR4, MyD88, TAK1, and TRAF6 in the DRG, along with the p-IKKβ/IKKβ and p-p65/p65 ratios, all significantly decreased (P<0.05). In comparison to the Voltaren group, the high- and medium-dose XZZTP groups demonstrated significantly higher mechanical and thermal pain thresholds (P<0.05). Both groups also had significantly lower serum levels of TNF-α, IL-1β, and IL-6 (P<0.05). The mRNA expression levels of TLR4 and NF-κB p65 in the DRG, as well as the protein expression levels of TLR4, MyD88, TAK1, TRAF6 and the p-IKKβ/IKKβ and p-p65/p65 ratios, were all significantly reduced (P<0.05). In contrast, the low-dose XZZTP group exhibited decreased mechanical and thermal pain thresholds (P<0.05), while increased serum levels of TNF-α, IL-1β, and IL-6, mRNA expression levels of TLR4 and NF-κB p65, protein expression levels of TLR4, MyD88, TAK1, and TRAF6, as well as the ratios of p-IKKβ/IKKβ and p-p65/p65 (P<0.05) compared to the Voltaren group. Compared with the low-dose XZZTP group, the high- and medium-dose XZZTP groups exhibited significantly elevated mechanical and thermal pain thresholds (P<0.05). Serum levels of TNF-α, IL-1β, and IL-6 were significantly lower (P<0.05). Additionally, both groups also showed significant downregulation of mRNA expression of TLR4 and NF-κB p65, as well as protein expression levels of TLR4, MyD88, TAK1, TRAF6 and the p-IKKβ/IKKβ and p-p65/p65 ratios in the DRG (P<0.05). Conclusion XZZTP can alleviate BMCP, potentially mediated through the inhibition of the TLR4/NF-κB signaling pathway activation and the subsequent downregulation of TNF-α, IL-1β, and IL-6 expression. |
| Key words: bone metastatic cancer pain topical Xiaozhong Zhitong Powder topical application TLR4/NF-κB dorsal root ganglion breast cancer |
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