Mechanism by which Tongdu Huoxue Decoction inhibits macrophage inflammation to delay intervertebral disc degeneration in rats

doi:10.12307/2025.943

Abstract

Abstract: BACKGROUND: Intervertebral disc degeneration is the main pathological factor causing low back pain, which is closely related to macrophage-mediated immune inflammation. Tongdu Huoxue Decoction is a proven prescription for the treatment of intervertebral disc degeneration, but it is still unclear whether it can treat intervertebral disc degeneration by regulating the polarization phenotype of macrophages to inhibit inflammation in intervertebral disc tissue.
OBJECTIVE: To investigate the regulatory effects of Tongdu Huoxue Decoction on the expression of macrophage-related inflammatory factors in intervertebral disc tissues of rats, as well as its mechanisms for the treatment of intervertebral disc degeneration.
METHODS: Twenty-four Sprague-Dawley rats were randomly divided into sham operation group, model group, and Tongdu Huoxue Decoction group, with eight rats in each group. An intervertebral disc degeneration model was established using the annulus fibrosus puncture method in the latter two groups. On the 1st postoperative day, 10.8 g/kg Tongdu Huoxue Decoction was given by gavage in the Tongdu Huoxue Decoction group, and the same dose of saline was given by gavage in the sham operation group and the model group, once a day. After 4 weeks of intervention, histopathological changes in the intervertebral disc tissues were assessed using hematoxylin-eosin. Immunohistochemistry and quantitative real-time PCR were performed to detect the relative expression levels of CD68, CD206, interleukin 1β, interleukin 10, type II collagen, and matrix metalloproteinase 13 proteins or mRNA in the intervertebral disc tissues.
RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining results revealed that the model group exhibited a significant decrease in intervertebral disc height, disorganized annulus fibrosus structure with fissures, and unclear demarcation between the nucleus pulposus and the annulus fibrosus. The Tongdu Huoxue Decoction group showed organized arrangement of the annulus fibrosus with pyknosis. (2) Immunohistochemical results demonstrated that, compared with the model group, the Tongdu Huoxue Decoction group had significantly lower expressions of CD68, interleukin 1β, and matrix metalloproteinase 13, and significantly higher expressions of CD206, type II collagen and interleukin 10 (P < 0.05 or P < 0.01). (3) qPCR results showed that there were significant differences in the mRNA expression of CD68, CD206, interleukin 1β, matrix metalloproteinase 13, type II collagen, and interleukin 10 between the three groups (P < 0.001). To conclude, Tongdu Huoxue Decoction can improve intervertebral disc degeneration in the rat model of intervertebral disc degeneration, and its mechanism is associated with the inhibition of macrophage-related inflammatory responses in the intervertebral discs.

Key words: Tongdu Huoxue Decoction, intervertebral disc degeneration, nucleus pulposus, macrophages, inflammation, mechanism, engineered tissue construction

CLC Number:

Yan Laijun, Ge Haiya, Wang Zhengming, Yang Zongrui, Niu Lifeng, Zhan Hongsheng. Mechanism by which Tongdu Huoxue Decoction inhibits macrophage inflammation to delay intervertebral disc degeneration in rats[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(32): 6851-6857.

Figures/Tables 4

2.1 实验动物数量分析此次实验共纳入大鼠24只，每组8只。手术造模后12 h内2只大鼠死亡(模型组与通督活血汤组各1只)，其余大鼠在实验期间均无死亡、下肢瘫痪或切口感染等情况发生。最终每组7只大鼠计入结果分析，4只进行免疫组化检测，3只进行qPCR检测。 2.2 大鼠椎间盘组织病理苏木精-伊红染色结果图1苏木精-伊红染色结果显示，假手术组椎间盘形态完好，纤维环排列整齐，无明显断裂或紊乱迹象，髓核基本呈椭圆形、体积饱满，无明显炎症细胞浸润，上下终板厚度一致，连续性好；模型组椎间盘纤维环结构紊乱，髓核形态破碎、缩小，软骨终板变薄、不连续，髓核与纤维环分界不清晰、可见较大裂隙；通督活血汤组椎间盘组织中纤维环排列相较于模型组连续性较好，纤维环可见裂隙，髓核部分皱缩，但中央透明空腔仍存在，表明通督活血汤在一定程度上可以延缓针刺诱导的椎间盘退变。 2.3 大鼠椎间盘组织免疫组化染色结果图2免疫组化染色结果显示，CD68 阳性和 CD206 阳性细胞均存在于模型组与通督活血汤组大鼠椎间盘组织中，呈黄褐色阳性表达。在髓核区域观察到假手术组大鼠椎间盘中Ⅱ型胶原高度表达，同时白细胞介素1β与白细胞介素10阳性染色较浅，反映了椎间盘组织内胶原结构正常，几乎不存在炎症浸润；同时，基质金属蛋白酶13的阳性着色区域较少，反映了大鼠椎间盘组织中分解代谢水平较低。模型组大鼠椎间盘中Ⅱ型胶原的染色平均吸光度值减少，基质金属蛋白酶13的染色平均吸光度值显著增加，表明椎间盘细胞外基质中胶原分解程度较高；同时可见白细胞介素1β染色区域扩大，提示退变椎间盘组织炎症。相较于模型组，通督活血汤组大鼠椎间盘中白细胞介素10、Ⅱ型胶原蛋白的表达增加，白细胞介素1β与基质金属蛋白酶13的表达减少，表明通督活血汤对椎间盘组织内巨噬细胞介导的炎症因子表达具有一定的调控作用。图3免疫组化定量分析结果显示，相较于假手术组，模型组椎间盘组织中CD68阳性染色平均吸光度值明显增加(P=0.003)；与模型组相比，通督活血汤组CD68阳性细胞染色吸光度值较低(P=0.013)。假手术组、模型组及通督活血汤组大鼠椎间盘中CD206阳性区平均吸光度值分别为(0.587±0.297)%、(6.983±0.615)%、(9.938±0.656)%，组间比较差异有显著性意义 (P < 0.001)；假手术组、模型组及通督活血汤组大鼠椎间盘组织中Ⅱ型胶原染色平均吸光度值分别为(54.832±11.761)%、(10.816±3.839)%、(26.565±2.276)%，组间差异有显著性意义(P < 0.001)。与模型组相比，通督活血汤组大鼠椎间盘中白细胞介素1β、基质金属蛋白酶13染色平均吸光度值相对较低(P=0.006，P=0.001)；而白细胞介素10与Ⅱ型胶原蛋白阳性表达则显著增加(P=0.024，P < 0.001)。 2.4 通督活血汤对巨噬细胞相关炎症相对基因表达量的影响如表2所示，与假手术组比较，模型组与通督活血汤组椎间盘组织中M1巨噬细胞标志物 CD68 mRNA表达量显著升高(P < 0.001，P=0.002)；与模型组相比，通督活血汤组CD68 mRNA表达明显下调(P=0.006)，CD206 mRNA表达显著升高(P=0.039)。相较于假手术组及通督活血汤组，模型组大鼠的椎间盘组织中白细胞介素1β、基质金属蛋白酶13 mRNA表达量明显增加，各组间差异有显著性意义 (P < 0.001)；与模型组相比，通督活血汤组大鼠椎间盘组织中白细胞介素1β与基质金属蛋白酶13 mRNA表达量相对下降(P=0.033，P=0.046)。3组之间白细胞介素1β与基质金属蛋白酶13 mRNA转录水平差异有显著性意义(P < 0.001，P=0.007)。与假手术组相比，模型组大鼠的椎间盘组织中白细胞介素10 mRNA转录水平上升(P=0.032)，而Ⅱ型胶原转录水平则相对下降(P=0.015)。"

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