补肾活血汤对椎间盘退变模型大鼠Wnt/β-catenin信号通路的影响及机制*

作者：肖 赛1,2，宁子柳1,2，冯帅华2，杨 届2，谢 芳2，文 哲2，李泽湘2，吴官保2

单位：1.湖南中医药大学，湖南 长沙 410208； 2.湖南省中西医结合医院，湖南 长沙 410006

引用：引用：肖赛，宁子柳，冯帅华，杨届，谢芳，文哲，李泽湘，吴官保.补肾活血汤对椎间盘退变模型大鼠Wnt/β-catenin 信号通路的影响及机制[J].中医药导报,2025,31(12):20-26.

DOI：10.13862/j.cn43-1446/r.2025.12.004

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摘要：目的：探讨补肾活血汤通过抑制Wnt/β-catenin信号通路调控椎间盘退变模型大鼠椎间盘退变的分子机制，明确其在抑制炎症反应、减少细胞凋亡中的作用。方法：将54只6周龄SPF级雄性SD大鼠随机分为空白组（n=12）和手术造模组（n=42）。手术造模组通过纤维环穿刺法建立椎间盘退变模型。将40只造模成功大鼠随机分为模型组、中药组、FH535组及中药+FH535组，空白组与模型组每日灌胃生理盐水（10 mL/kg，2次/d）；中药组灌胃补肾活血汤（10.44 g/kg，2次/d）；FH535组灌胃生理盐水并腹腔注射FH535（25.00 mg/kg，隔日1次，持续20 d）；中药+FH535组灌胃补肾活血汤（10.44 g/kg，2次/d）并腹腔注射FH535（25.00 mg/kg，隔日1次，持续20 d）。4周后处死大鼠，摘取大鼠Co4/5、Co5/6、Co6/7节段的椎间盘组织，Masson染色观察椎间盘组织形态结构，TUNEL染色分析大鼠髓核细胞凋亡率，免疫组织化学（IHC）检测椎间盘组织中Ⅱ型胶原蛋白（Col2a1）、性别决定区Y框蛋白9（Sox9）、B细胞淋巴瘤-2（Bcl-2）及Bcl-2相关X蛋白（Bax）表达水平，酶联免疫吸附试验（ELISA）法检测椎间盘组织中白细胞介素-6（IL-6）水平，蛋白质印迹法（Western blotting）检测椎间盘组织中β-catenin和Wnt-3α蛋白表达水平。结果：与空白组比较，模型组大鼠椎间盘呈现典型退变特征，纤维环结构紊乱，髓核纤维化明显；与模型组比较，中药组、FH535组及中药+FH535组大鼠椎间盘退变均改善。模型组大鼠椎间盘组织中IL-6水平、髓核细胞凋亡率高于空白组（P<0.05）；中药组、FH535组及中药+FH535组大鼠椎间盘组织中IL-6水平、髓核细胞凋亡率低于模型组（P<0.05）。模型组大鼠椎间盘组织中Col2a1、Sox9、Bcl-2表达水平低于空白组（P<0.05），Bax表达水平高于空白组（P<0.05）；中药组、FH535组及中药+FH535组大鼠椎间盘组织中Col2a1、Sox9、Bcl-2表达水平高于模型组（P<0.05），Bax表达水平低于模型组（P<0.05）。模型组大鼠椎间盘组织中Wnt-3α和β-catenin蛋白相对表达量高于空白组（P<0.05）；中药组、FH535组及中药+FH535组大鼠椎间盘组织中Wnt-3α和β-catenin蛋白相对表达量低于模型组（P<0.05）。结论：补肾活血汤可通过调控Wnt/β-catenin信号通路，抑制炎症和细胞凋亡，从而延缓椎间盘退变模型大鼠的椎间盘退变。

关键词：椎间盘退变；补肾活血汤；Wnt/β-catenin信号通路；细胞凋亡；炎症；大鼠

Abstract：

Objective: To investigate the molecular mechanism by which Bushen Huoxue decoction regulates intervertebral disc degeneration through inhibition of the Wnt/β-catenin signaling pathway and clarify its role in suppressing inflammatory responses and reducing apoptosis. Methods: Totally 54 6-week-old male Sprague-Dawley (SD) rats of specific pathogen-free (SPF) grade were randomly divided into blank control group (n=12) and surgical modeling group (n=42). The intervertebral disc degeneration model was established in the surgical group via annulus fibrosus puncture. After successful modeling, the 40 successfully modeled rats were randomly divided into model group, traditional Chinese medicine (TCM) group, FH535 group, and TCM+FH535 group. The blank control group and the model group received intragastric administration of normal saline (10 mL/kg, twice daily). The TCM group received intragastric administration of Bushen Huoxue decoction (10.44 g/kg, twice daily). The FH535 group received intragastric administration of normal saline and intraperitoneal injection of FH535 (25.00 mg/kg, every other day for 20 days). The TCM+FH535 group received intragastric administration of Bushen Huoxue decoction (10.44 g/kg, twice daily) and intraperitoneal injection of FH535 (25.00 mg/kg, every other day for 20 days). The rats were euthanized in four weeks, and intervertebral disc tissues from the Co4/5, Co5/6, and Co6/7 segments were harvested. The morphological structure of the intervertebral disc tissues was observed using Masson's trichrome staining. Apoptosis rates of nucleus pulposus cells were analyzed via TUNEL staining. Immunohistochemistry (IHC) was performed to detect the expression levels of Collagen Type Ⅱ (Col2a1), SRY-related HMG-box 9 (Sox9), B-cell lymphoma-2 (Bcl-2), and Bcl-2 Associated X Protein (Bax) in the intervertebral disc tissues. The level of interleukin-6 (IL-6) in the intervertebral disc tissues was measured using enzyme-linked immunosorbent assay (ELISA). Western blotting was used to assess the protein expression levels of β-catenin and Wnt-3α in the intervertebral disc tissues. Results: Compared with the blank control group, the model group exhibited typical intervertebral disc degeneration characteristics, including disordered annulus fibrosus structure and obvious nucleus pulposus fibrosis. Compared with the model group, the TCM group, FH535 group, and TCM+FH535 group showed varying degrees of improvement in intervertebral disc degeneration. The model group showed significantly higher levels of IL-6 and apoptosis rate of nucleus pulposus cells in the intervertebral disc tissues than the blank control group (P<0.05). The levels of IL-6 and the apoptosis rate of nucleus pulposus cells in the intervertebral disc tissues of the TCM group, FH535 group, and TCM+ FH535 group were lower than those in the model group (P<0.05). The expression levels of Col2a1, Sox9, and Bcl-2 in the intervertebral disc tissues of the model group were lower than those in the blank control group (P<0.05), while the expression level of Bax was higher than that in the blank control group (P<0.05). The expression levels of Col2a1, Sox9, and Bcl-2 in the intervertebral disc tissues of the TCM group, FH535 group, and TCM+FH535 group were higher than those in the model group (P<0.05), while the expression levels of Bax were lower than those in the model group (P<0.05). The relative protein expression levels of Wnt-3α and β-catenin in the intervertebral disc tissues of the model group were higher than those in the blank control group (P<0.05). The relative protein expression levels of Wnt-3α and β-catenin in the intervertebral disc tissues of the TCM group, FH535 group, and TCM + FH535 group were lower than those in the model group (P<0.05).

Key words：intervertebral disc degeneration; Bushen Huoxue decoction; Wnt/β-catenin signaling pathway; apoptosis; inflammation; rat

发布时间：2025-12-31

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