ABSTRACT
Objective:To explore the mechanism of mitochondrial apoptotic pathway in rat degenerative intervertebral disc cells in improving intervertebral disc degeneration under the action of Bushen Zhuangdu recipe. Method:The 100 SD male rats were randomly divided into blank group, model group, low, medium and high dose Bushen Zhuangdu recipe group (0.38,0.77,1.53 g·kg-1).Histopathological changes of rat intervertebral discs were observed by hematoxylin-eosin(HE) staining after 4 weeks of continuous administration of Chinese medicine. The apoptotic rate of nucleus pulposus cells in degenerative intervertebral discs was detected by TUNEL(terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), and the levels of active Cysteinyl aspartate-specific proteinase-3(active Caspase-3), B cell leukemia-2(Bcl-2), cytochrome C (cytC) and Bcl-2-associated X protein(Bax) protein in intervertebral discs were detected by Western blot. Result:Compared with blank group, the histopathological score of intervertebral disc in the model group was significantly increased, the apoptosis rate of nucleus pulposus was significantly increased (PPPPPPConclusion:Bushen Zhuangdu recipe may improve the degeneration of intervertebral disc by reducing the expression of active Caspase-3, cytC and Bax, increasing the expression of Bcl-2 and inhibiting the apoptotic pathway of mitochondria in a dose-dependent manner.
ABSTRACT
BACKGROUND: Numerous studies focus on animal models of intervertebral disc degeneration (IDD), but criteria for establishing the animal models of IDD have not been confirmed, and there is a lack of systematic comparison among models. OBJECTIVE: To compare the rat models of IDD established by puncturing at annulus, endplate injection and their combination, thus providing reference for IDD model selection. METHODS: Eighty Sprague-Dawley rats were equivalently randomized into four groups: puncturing group (puncturing at the annulus), endplate injection group (endplate injected with ethyl alcohol), combination group (puncturing at the L5-6annulus and endplate injection at the same segment) and sham operation group. Three rats in each group were taken at postoperative 4, 8, and 12 weeks for X-ray examination to measure the disc height; and the discs were removed for histological observation and immunohistochemical staining. RESULTS AND CONCLUSION: The results of X-ray examination, hematoxylin-eosin staining and immunohistochemical staining all showed that the IDD degree was gradually aggravated in all groups except the sham operation group. At postoperative 4 weeks, compared with the sham operation group, in the endplate injection and combination groups, the percent disc height was significantly decreased, the pathological scores were significantly increased and the average gray value of collagen type I was significantly reduced (P ＜ 0.05). At postoperative 8 and 12 weeks, compared with the sham operation group, the percent disc height in the other three groups were all significantly decreased, the pathological score was significantly increased, and the average gray value of collagen type I was significantly decreased (P ＜ 0.05). Compared with the puncturing and endplate injection group, in the combination group, the percent disc height at postoperative 8 weeks was significantly decreased, and the average gray value of collagen type I at postoperative 12 weeks was significantly decreased (P ＜ 0.05). These results suggest that the rat IDD model can be successfully constructed by above three methods. Puncturing at the annulus is easy to operate and control IDD progression, which can be used to study different stages of IDD. Endplate injection is suitable for the etiological study of IDD, and induces IDD earlier than puncturing, but the final results are similar. The combination method can significantly accelerate IDD aggravation, and thus is not time consuming.