补肾壮筋汤抑制椎间盘退变的机制研究

Mechanism Research of Bu Shen Zhuang Jin Decoction in Inhibiting Intervertebral Disc Degeneration

摘要: 目的：探讨补肾壮筋汤调控细胞外基质重塑延缓椎间盘退变的作用机制。方法：选取2月龄SPF级雄性SD大鼠共35只，随机分为即对照组(6只)和造模组(24只)。对照组行假手术处理，造模组采用椎间盘穿刺法建立动物模型，通过MRI扫描，验证椎间盘退变大鼠模型构建是否成功。将造模成功的大鼠随机分为：模型组、低剂量中药组、中剂量中药组、高剂量中药组，每组各6只。根据人体与动物药物等效剂量换算方法及前期实验研究，给药剂量为：低剂量组0.8 g/kg/天，中剂量组1.6 g/kg/天，高剂量组3.2 g/kg/天。从术后第2天开始，中药组分别给予不同浓度的补肾壮筋汤灌胃，对照组和模型组则予等量的生理盐水灌胃，1次/d，连续干预8周。HE染色观察各组组织形态结构的变化；qPCR、Western blot检测各组Aggrecan、Col2A1、MMP-13的mRNA转录水平和蛋白相对表达量。结果：① HE染色显示对照组椎间盘组织形态完整，结构清晰，髓核与纤维环间界限清晰，可见丰富的细胞外基质和大量髓核细胞；模型组椎间盘高度明显降低，髓核组织细胞外基质和髓核细胞明显减少，纤维软骨细胞排列紊乱；随着补肾壮筋汤组剂量的增加，椎间盘高度明显升高，髓核细胞和髓核中基质均增多，纤维软骨细胞排列趋于整齐。② qPCR、Western blot结果显示，椎间组织中Aggrecan、Col2A1的表达量，模型组显著低于对照组(P < 0.05, P < 0.01)，补肾壮筋汤组显著高于模型组(P < 0.05, P < 0.01)，MMP-13的表达模型组显著高于对照组(P < 0.01)，补肾壮筋汤组显著低于模型组(P < 0.01)。结论：补肾壮筋汤通过升高椎间盘组织中的Aggrecan、Col2A1的含量，降低椎间盘组织中MMP-13含量，从而延缓椎间盘的退变。

Abstract: Objective: To investigate the mechanism of Bushen Zhuangjin Decoction in inhibiting intervertebral disc degeneration. Methods: A total of 35 SPF male SD rats aged 2 months were selected and randomly divided into control group (6 rats) and modeling group (24 rats). The control group was treated with sham operation, and the model group was treated with intervertebral disc puncture to establish an animal model. The successful construction of the rat model of intervertebral disc degeneration was verified by MRI scanning. The rats were randomly divided into Model group, low-dose medicine group, medium-dose medicine group and high-dose medicine group, with 6 rats in each group. According to the equivalent dose conversion method of human and animal drugs and the preliminary experimental study, the dosage was as follows: low-dose group 0.8 g/kg/day, mid-dose group 1.6 g/kg/ day, high-dose group 3.2 g/kg/day. Starting from the second day after surgery, the medicine group was given different concentrations of Bushen Zhuangjin decoction by gavage, while the control group and the model group were given the same amount of normal saline by gavage, once a day, for continuous intervention for 8 weeks. The changes of tissue morphology and structure were observed by MRI and HE staining. The mRNA transcription levels and relative protein expression levels of Aggrecan, Col2A1 and MMP-13 in each group were detected by qPCR and Western blot. Results: ① HE staining showed that the intervertebral disc in the control group was complete, with clear structure, clear boundary between nucleus pulposus and annulus fibrosus, and abundant extracellular matrix and a large number of nucleus pulposus cells. In the model group, the height of intervertebral disc decreased significantly, the extracellular matrix and nucleus pulposus cells decreased significantly, and the arrangement of fibrochondrocytes was disordered. With the increase of dosage of Bushen Zhuangjin decoction group, the height of intervertebral disc increased significantly, the stroma in nucleus pulposus and nucleus pulposus increased, and the arrangement of fibrochondrocytes tended to be orderly. ② The results of qPCR and Western blot demonstrated that Aggrecan and Col2A1 expression was significantly decreased in the model group compared to the control group (P < 0.05, P < 0.01). The Bushen Zhuangjin decoction treatment led to a significant increase in the expression of both Aggrecan and Col2A1 compared to the model group (P < 0.05, P < 0.01). Conversely, the model group exhibited a significantly higher expression level of MMP-13 compared to the control group (P < 0.01). Bushen Zhuangjin decoction treatment resulted in a significant reduction in MMP-13 expression (P < 0.01). Conclusion: Bushen Zhuangjin decoction can increase the content of Aggrecan and Col2A1 in intervertebral disc tissue and decrease the content of MMP-13 in intervertebral disc tissue, thus delaying disc degeneration.