补肾壮督方对大鼠退变椎间盘细胞线粒体凋亡通路的影响

目的:观察补肾壮督方对大鼠退变椎间盘细胞凋亡的影响,探讨其改善椎间盘退变的机制。方法:100只SD雄性大鼠,采用纤维环全层针刺造模,随机分为空白组,模型组,补肾壮督方低、中、高剂量组(0. 38,0. 77,1. 53 g·kg-1),连续中药灌胃4周后,苏木素-伊红(HE)染色观察大鼠椎间盘组织病理学变化;原位末端转移酶标记法(TUNEL)检测退变椎间盘中髓核细胞凋亡率,蛋白免疫印迹法(Western blot)检测椎间盘组织中活化半胱氨酸蛋白酶-3(active Caspase-3),B淋巴细胞瘤-2(Bcl-2),细胞色素C(cyt C)及Bcl-2相关X蛋白(Bax)蛋白水平。结果:与空白组比较,模型组大鼠椎间盘组织病理学评分显著增加,髓核细胞凋亡率明显增加(P 0. 05),active Caspase-3,cyt C和Bax表达量均明显增加(P 0. 05),Bcl-2表达量明显下降(P 0. 05)。与模型组比较,补肾壮督方低、中、高剂量组组织病理学评分明显降低(P 0. 05),髓核细胞凋亡率明显减少(P 0. 05),active Caspase-3,cyt C及Bax表达量均明显下降(P 0. 05),Bcl-2表达量明显增加(P 0. 05)。结论:补肾壮督方可能通过减少active Caspase-3,cyt C和Bax蛋白表达,增加Bcl-2蛋白表达,抑制线粒体凋亡通路,并存在一定剂量依赖性,从而改善椎间盘退变。

Effect of Bushen Zhuangdu Recipe on Mitochondrial Apoptotic Pathway in Degenerative Intervertebral Disc Cells in Rats

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 (P<0.05), the expression levels of active Caspase-3, cytC and Bax were significantly increased (P<0.05), and the expression levels of Bcl-2 were significantly decreased (P<0.05).After the intervention of Bushen Zhuangdu recipe.Compared with model group, the histopathological score of Bushen Zhuangdu recipe low, medium and high dose groups were significantly reduced(P<0.05), the expression levels of active Caspase-3, cytC and Bax were significantly decreased (P<0.05), and the expression levels of Bcl-2 were significantly increased (P<0.05). Conclusion: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.