补肾活血汤对椎间盘退变模型大鼠巨噬细胞极化介导炎症-焦亡机制的影响*

作者：宁子柳1,2,3,4，冯帅华1,2,3，吴官保1,2,3，肖 赛1,2,3，李泽湘1,2，杨 届1,2，谢 芳1,2，文 哲1,2，肖宇旃1,2,3， 苏 忱1,2,3，巴拉提•买提卡斯木1,2,3，许铮荣1,2,3

单位：1.湖南省中西医结合医院，湖南 长沙 410006； 2.湖南省中医药研究院，湖南 长沙 410013； 3.湖南中医药大学，湖南 长沙 410208； 4.广安门医院保定医院，河北 保定 071000

引用：引用：宁子柳，冯帅华，吴官保，肖赛，李泽湘，杨届，谢芳，文哲,肖宇旃，苏忱，巴拉提•买提卡斯木，许铮荣.补肾活血汤对椎间盘退变模型大鼠巨噬细胞极化介导炎症-焦亡机制的影响[J].中医药导报,2025,31(9):1-8,23.

DOI：10.13862/j.cn43-1446/r.2025.09.001

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摘要：

目的：基于巨噬细胞极化反应和核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3（NLRP3）/胱天蛋白酶-1（Caspase-1）/白细胞介素-1β（IL-1β）信号通路之间的互作机制，探讨补肾活血汤（BHD）对大鼠椎间盘退变（IDD）模型的抗炎、抗焦亡作用。方法：从44只SPF级SD雄性大鼠中随机取12只大鼠为空白对照组，其余32只为手术造模组。手术造模组大鼠采用新式纤维环穿刺造模方法建立IDD大鼠模型。将30只造模成功的大鼠按随机数字表分成模型对照组、中药组、抑制剂（MCC950）组，每组10只。各组予以相应药物干预4周。干预周期结束后全部处死，取出其尾椎椎间盘组织。苏木精-伊红（HE）染色观察椎间盘病理形态并进行组织学分级评分；透射电镜（TEM）观察髓核细胞超微结构及焦亡情况；酶联免疫吸附试验（ELISA）检测椎间盘组织中肿瘤坏死因子-α（TNF-α）水平；免疫组化法（IHC）检测椎间盘组织中M0型巨噬细胞标记蛋白CD68、M1型巨噬细胞标记蛋白CD197、NLRP3、Caspase-1、IL-1β蛋白阳性表达水平；蛋白质印迹法（Western blotting）检测椎间盘中组织中NLRP3、Caspase-1、IL-1β蛋白相对表达量。结果：空白对照组大鼠椎间盘组织形态结构正常，纤维环排列整齐致密；模型对照组大鼠椎间盘形态结构混乱，髓核组织变形、排列混乱，纤维环裂隙；中药组及MCC950组大鼠椎间盘组织结构基本完整，可见髓核组织少量肿胀、变形，纤维环稍有裂隙。模型对照组大鼠组织学分级评分高于空白对照组（P<0.01）；中药组及MCC950组大鼠组织学分级评分低于模型对照组（P<0.01）。模型对照组大鼠髓核细胞焦亡小体数量高于空白对照组（P<0.01）；中药组及MCC950组大鼠髓核细胞焦亡小体数量均低于模型对照组（P<0.01）。模型对照组大鼠椎间盘组织中TNF-α水平高于空白对照组（P<0.01）；中药组及MCC950组大鼠椎间盘组织中TNF-α水平低于模型对照组（P<0.01）。模型对照组大鼠椎间盘组织中CD68、CD197、NLRP3、Caspase-1、IL-1β阳性表达水平高于空白对照组（P<0.01）；中药组及MCC950组大鼠椎间盘组织中CD68、CD197、NLRP3、Caspase-1、IL-1β阳性表达水平均低于模型对照组（P<0.01）。模型对照组大鼠椎间盘组织中NLRP3、Caspase-1、IL-1β蛋白相对表达量高于空白对照组（P<0.01）；中药组及MCC950组大鼠椎间盘组织中NLRP3、Caspase-1、IL-1β蛋白相对表达量均低于模型对照组（P<0.01）。结论：补肾活血汤可能通过抑制巨噬细胞极化介导NLRP3/Caspase-1/IL-1β焦亡通路，抑制巨噬细胞浸润、极化，减少促炎因子释放及髓核细胞焦亡，从而减轻IDD模型大鼠椎间盘组织病理损伤。

关键词：椎间盘退变；补肾活血汤；巨噬细胞；炎症；NLRP3；Caspase-1；IL-1β；细胞焦亡；大鼠

Abstract：

Objective: To explore the anti-inflammatory and anti-pyroptosis effects of Bushen Huoxue Decoction (BHD) on a rat model of intervertebral disc degeneration (IDD), based on the interaction mechanism between macrophage polarization response and nucleotide oligomerization domain-like receptor thermal protein domain 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1) / interleukin-1β (IL-1β) signaling pathway. Method: Among 44 SPF-grade male SD rats, 12 rats were randomly selected as blank control group, and the remaining 32 were the surgical model group. The surgical mode group rats were modeled using a new annulus fibrosus puncture modeling method to establish the IDD rat model. Totally 30 successfully modeled rats were randomly divided into model control group, traditional Chinese medicine group and inhibitor (MCC950) group according to the random number table, with 10 rats in each group. Each group was given corresponding drug intervention for 4 weeks. After the intervention cycle ended, all rats were sacrificed and the caudal intervertebral disc tissues were removed. Hematoxylin-eosin (HE) staining was used to observe the pathological morphology of intervertebral discs and conduct histological grading and scoring. The ultrastructure and pyroptosis of nucleus pulposus cells were observed by transmission electron microscopy (TEM). The level of tumor necrosis factor-α (TNF-α) in intervertebral disc tissue was detected by enzyme-linked immunosorbent assay (ELISA). The positive expression levels of M0-type macrophage marker protein (CD68) and M1-type macrophage marker protein (CD197, NLRP3, Caspase-1 and IL-1β) in intervertebral disc tissues were detected by immunohistochemistry (IHC). The relative expression levels of NLRP3, Caspase-1 and IL-1β proteins in the tissues of intervertebral discs were detected by Western blotting. Result: The morphological structure of the intervertebral disc tissue in the blank control group rats was normal, and the annulus fibrosus was neatly and densely arranged. In the model control group, the intervertebral disc morphology and structure of rats were disordered, and the nucleus pulposus tissue was deformed and disordered in arrangement. There were fissures in the annulus fibrosus in model control group. The intervertebral disc tissue structure was basically intact in traditional Chinese medicine group and MCC950 group. A small amount of swelling and deformation of the nucleus pulposus tissue could be observed, and there was a slight fissure in the annulus fibrosus in  traditional Chinese medicine group and MCC950 group. The model control group showed higher histological score than  blank control group (P<0.01). The traditional Chinese medicine group and MCC950 group showed lower histological scores than model control group (P<0.01). The model control group showed higher number of pyroptosis bodies in the nucleus pulposus cells, than blank control group (P<0.01). The traditional Chinese medicine group and MCC950 group showed lower number of pyroptosis bodies in the nucleus pulposus cells, than model control group (P<0.01). The model control group showed higher level of TNF-α in the intervertebral disc tissue, than blank control group (P<0.01). The traditional Chinese medicine group and MCC950 group showed lower level of TNF-α in the intervertebral disc tissues, than model control group (P<0.01). The model control group showed higher positive expression levels of CD68, CD197, NLRP3, Caspase-1 and IL-1β proteins in the intervertebral disc tissues, than blank control group (P<0.01). The traditional Chinese medicine group and MCC950 group showed lower positive expression levels of CD68, CD197, NLRP3, Caspase-1 and IL-1β proteins in the intervertebral disc tissues, than model control group (P<0.01). The model control group showed higher relative expression levels of NLRP3, Caspase-1 and IL-1β proteins in the intervertebral disc tissues, than blank control group (P<0.01). The traditional Chinese medicine group and MCC950 group showed lower relative expression levels of NLRP3, Caspase-1 and IL-1β proteins in the intervertebral disc tissues, than model control group (P<0.01). Conclusion: Bushen Huoxia Decoction may inhibit the NLRP3/Caspase-1/IL-1β pyroptosis pathway mediated by macrophage polarization, thereby inhibiting macrophage infiltration and polarization, reducing the release of pro-inflammatory factors and pyroptosis of nucleus pulposus cells, and alleviating the pathological damage of intervertebral disc tissue in IDD.

Key words：intervertebral disc degeneration; Bushen Huoxia Decoction; macrophage; inflammation; NLRP3; Caspase-1; IL-1β; pyroptosis of cells; rat

发布时间：2026-01-08

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