Abstract: Background　Intervertebral disc degeneration（IDD） is an essential cause of degenerative spine conditions. As endplate plays a vital role in mechanotransduction and nutrient transport in intervertebral discs，endplate degeneration will accelerate IDD. Compound preparations of Chinese Medicine have proven to be uniquely effective for IDD，but the specific mechanism of action is not completely clear. Objective　To observe the effect of Bushenhuoxue herbs on vertebral endplate of spinal motion segment in rabbits under pressure，to clarify the characteristics and mechanism of the medicine in regulating endplate chondrocytes to delay IDD progression. Methods　Forty healthy New Zealand rabbits were randomly divided into control group，herb group，herb inhibition group and herb activation group，with 10 rabbits in each. The model of IDD was created using an in vitro loading and culturing device for spinal motor segments with independent intellectual property rights. HE staining，immunohistochemistry，RT-PCR and Western blot were used for detecting relevant indicators，respectively. Including proteoglycan，typeⅡ collagen，Wnt-3α，β-catenin. Results　（1）Effect of Bushenhuoxue herbs on IDD：① The pathomorphology of the HE stained sample showed that herb group had better dispersion and layer arrangement，and more number of endplate chondrocytes than the control group on the 7th day of culture. The degeneration of endplate cartilage in herb group and control group worsened on the 14th day of culture，but the degree of degeneration of the control group was more obvious. ②Immunohistochemical staining in the sample tissue sections found that herb group had higher level of extracellular matrix proteoglycan expression than control group on the 1st and 3rd days of culture（P<0.05）. On the 7th and 14th days of culture，the level of extracellular matrix proteoglycan expression decreased significantly in herb group and control group compared to the baseline（P<0.05），but showed no significant intergroup differences（P>0.05）. Immunohistochemical staining of type Ⅱ collagen in the sample tissue sections found that herb group had higher level of type Ⅱ collagen expression in extracellular matrix than control group on the 1st and 3rd days of culture（P<0.05）. On the 7th and 14th days of culture，the level of type Ⅱ collagen expression in extracellular matrix declined obviously in herb group and control group compared to the baseline（P<0.05），but it was more reduced in the herb group（P<0.05）. ③The results of RT-PCR showed that compared to the baseline levels，Wnt-3α and β-catenin mRNA expression levels were much lower on the 1st，3rd，7th and 14th days of culture in herb group and control group（P<0.05），and they were more lower in herb group（P<0.05）. ④Western blot test indicated that significant changes were found in Wnt-3α and β-catenin protein expression levels in herb group and control group from baseline to 1，3，7，14 days post-culture（P<0.05）. Herb group demonstrated much higher Wnt-3α protein expression levels and notably lower β-catenin protein expression levels than control group on the 1st，3rd，7th and 14th days of culture（P<0.05）.  （2）Mechanism of Bushenhuoxue herbs delaying the progression of IDD：HE staining showed that herb activation group had better integrity and arrangement of endplate cartilage at the 3rd day of culture than herb inhibition group. ②Immunohistochemical staining in the sample tissue sections found that significant differences were found in expression levels of proteoglycan and type Ⅱ collagen in the sample tissue sections between herb group and herb activation group or herb inhibition group（P<0.05）. ③RT-PCR results showed that on the 3rd day of culture，Wnt-3α mRNA expression level showed no significant differences between herb group and herb activation group or herb inhibition group（P>0.05）；the β-catenin mRNA expression level in herb group was much higher than that of herb activation group，but was much lower than that of herb inhibition group（P<0.05）. ④Western blot test indicated that on the 3rd day of culture，the Wnt-3α and β-catenin protein expression levels in herb activation group were similar to those of herb group（P>0.05）. The Wnt-3α protein expression level in herb inhibition group was similar to that of herb group（P>0.05）. The β-catenin protein expression level in herb inhibition group was much higher than that of herb activation group（P<0.05）. Conclusion　Bushenhuoxue herbs could delay the progression of IDD by regulating endplate chondrocytes through Wnt/β-catenin signaling pathway.

Key words: Intervertebral disc degeneration；Bushenhuoxue herbs；Wnt/&beta, -catenin signaling pathway；Endplate；Chondrocytes

摘要： 背景　椎间盘退行性变是导致脊柱退行性疾病发生的根本因素，终板是椎间盘力学传导与营养传输的主要结构，终板退变会加速椎间盘退变的发生，中药复方对延缓椎间盘退变具有独特的疗效，但具体作用机制尚不完全明确。目的　观察补肾活血中药对压力下离体兔脊柱运动节段椎体终板的影响，阐明其调控终板软骨细胞、延缓椎间盘退行性变的效应特点及作用机制。方法　2019年将40只健康新西兰兔采用随机数字表法分为对照组、中药组、中药抑制剂组、中药激动剂组，每组10只。采用具有自主知识产权的脊柱运动节段离体加载和培养装置进行造模，并通过HE染色病理形态学方法、免疫组化法、荧光定量聚合酶链式反应法（RT-PCR）和Western blot检测方法检测相关指标，包括蛋白多糖、Ⅱ型胶原及Wnt-3α、β-catenin mRNA和蛋白的表达水平。结果　（1）药物对椎间盘退变的影响：①HE染色显示，培养第7天时中药组软骨细胞分散程度、数目及软骨层排列均较对照组完整；培养第14天时对照组、中药组终板软骨组织退变程度均加重，对照组退变程度较中药组更加明显。②免疫组化法检测结果显示，中药组培养第1、3天时细胞外基质蛋白多糖、Ⅱ型胶原表达量均较对照组升高（P<0.05）；中药组及对照组培养第7、14天细胞外基质蛋白多糖、Ⅱ型胶原表达量均较培养前降低（P<0.05）；中药组细胞外基质蛋白多糖表达量与对照组比较，差异无统计学意义（P>0.05），而对照组Ⅱ型胶原表达量较中药组升高（P<0.05）。③RT-PCR检测结果显示，中药组及对照组培养第1、3、7、14天时Wnt-3α和β-catenin mRNA表达水平均较培养前降低（P<0.05），中药组培养第1、3、7、14天时Wnt-3α、β-catenin mRNA表达水平均较对照组降低（P<0.05）。④Western blot检测结果显示，中药组及对照组培养第1、3、7、14天时Wnt-3α、β-catenin蛋白表达水平与培养前比较，差异有统计学意义（P<0.05）；中药组培养第1、3、7、14天时Wnt-3α蛋白表达水平较对照组升高（P<0.05），但β-catenin蛋白表达水平较对照组降低（P<0.05）。（2）药物延缓椎间盘退变机制：①HE染色显示，中药激动剂组较中药抑制剂组培养第3天时终板软骨组织结构的完整性、软骨层排列较好。②免疫组化法检测结果显示，中药激动剂组、中药抑制剂组蛋白多糖、Ⅱ型胶原表达量分别与中药组比较，差异均有统计学意义（P<0.05）。③RT-PCR检测结果显示，中药激动剂组、中药抑制剂组第3天时Wnt-3α mRNA表达水平分别与中药组比较，差异均无统计学意义（P>0.05）；中药激动剂组β-catenin mRNA表达水平较中药组降低（P<0.05），中药抑制剂组β-catenin mRNA表达水平较中药组升高（P<0.05）。④Western blot检测结果显示，中药激动剂组第3天时Wnt-3α和β-catenin蛋白表达水平以及中药抑制剂组Wnt-3α蛋白表达水平分别与中药组比较，差异均无统计学意义（P>0.05）；而中药抑制剂组β-catenin蛋白表达水平较中药组升高（P<0.05）。结论　补肾活血中药可以通过介导Wnt/β-catenin信号通路调控终板软骨细胞延缓椎间盘退变进程。

关键词: 椎间盘退行性变；补肾活血中药；Wnt/&beta, -catenin信号通路；终板；软骨细胞