芪麝丸对直立大鼠腰椎骨质增生的影响

Effects of Qishe Pill on vertebral hyperostosis induced by upright posture in rats

目的:观察芪麝丸对长期直立大鼠腰椎骨质增生的影响,探讨其可能的作用机制。方法:30只SD大鼠随机分为正常对照组、模型组和芪麝丸组。正常对照组不进行处理,普通饲养笼喂养。模型组和芪麝丸组大鼠行手术截断前肢配合强迫站立建立长期直立大鼠模型。芪麝丸组于术后8个月开始予芪麝丸5g/(kg·d)灌胃,连续灌胃1个月。所有大鼠术后9个月处死并取材,第5腰椎椎体行藏红-固绿染色、天狼星红染色观察病理学改变,免疫组织化学法检测Ⅰ型胶原、Ⅹ型胶原、血管内皮生长因子(vascular endothelial growth factor,VEGF)、转化生长因子β1(transforming growth factor β1,TGF-β1)表达;应用实时荧光定量逆转录聚合酶链反应技术检测第1～3腰椎生长板及椎间盘Ⅰ型胶原α2(typeⅠcollagenα2,Col1α2)、Ⅹ型胶原α1(typeⅩcollagenα1,Col10α1)、TGF-β1、VEGF和runt相关转录因子2(runt-related transcription factor 2,Runx2)基因的表达。结果:藏红-固绿染色显示模型组椎体边缘-椎间盘连接处非基质成分增加,发生骨质增生;正常对照组改变不明显;芪麝丸组与模型组比,相同部位非基质成分较少。天狼星红染色示正常对照组椎体边缘-椎间盘连接处胶原排列致密,模型组该处Ⅰ型和Ⅲ型胶原增加,芪麝丸组椎体边缘-椎间盘连接处胶原致密,以Ⅰ型胶原为主。免疫组织化学检测提示模型组骨质增生形成部位Ⅹ型胶原、VEGF和TGF-β1表达均较正常对照组增加,芪麝丸组Ⅹ型胶原和VEGF表达均较模型组减少,而TGF-β1表达无明显改变。实时荧光定量逆转录聚合酶链反应示芪麝丸组Col10α1和Runx2基因表达较模型组下降(P<0.01)。结论:芪麝丸能延缓腰椎边缘-椎间盘连接处骨质增生形成,其机制可能与减少增生骨质中细胞外基质Ⅹ型胶原的含量和椎间盘Ⅹ型胶原及Runx2基因表达有关。

Objective： To observe the effects of Qishe Pill, a compound traditional Chinese herbal medicine, on lumbar vertebral bone formation induced by long-time upright posture in rats and to investigate the potential mechanism. Methods： Thirty SD rats were randomly divided into normal control group, untreated group and Qishe Pill group. The rats in normal control group received no treatment and were raised in normal cages. The rats in untreated group and Qishe Pill group were cut off forelimbs by operation so as to be forced to adopt an upright posture for 8 months to induce hyperostosis. Rats in the Qishe Pill group were intragastrically administered with Qishe Pill at a dose of 5 g/（kg · d） for 1 month. All rats were sacrificed at the 9th month after surgery and all lumbar vertebrae were harvested for detection. Safranin O/fast green staining and picrosirius red staining were used to observe pathological changes. Expressions of type Ⅰ collagen （ColⅠ）, type Ⅹ collagen （ColⅩ）, vascular endothelial growth factor （VEGF） and transforming growth factor β1 （TGF-β1） in the 5th lumbar vertebra （L5） were detected by immunohistochemical method. Expressions of type Ⅰ collagen α2 （Col1α2）, type Ⅹ collagen α1 （Col10α1）, TGF-β1, and VEGF and runt-related transcription factor 2 （Runx2） mRNAs in L1-L3 were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction. Results： Safranin O/fast green staining showed that in the untreated group, non-matrix components increased at marginal lumbar vertebra and intervertebral disc junction, and hyperostosis appeared. However, no obvious change was observed in the normal control group. Non-matrix components decreased at the same location in Qishe Pill group as compared with the untreated group. Picrosirius red staining showed compact collagens at marginal lumbar vertebra and intervertebral disc junction in the normal control group, however, ColⅠ and ColⅩ increased at the same location in the untreated group. In the Qishe Pill group, it showed more compact collagens, especially ColⅠ. Compared with normal control group, expressions of ColⅩ, VEGF and TGF-β1 were increased at marginal lumbar vertebra and intervertebral disc junction in the untreated group. ColⅩ and VEGF expressions decreased in the Qishe Pill group as compared with the untreated group. Col10α1 and Runx2 mRNA expressions were down-regulated by Qishe Pill （P〈0.01）. Conclusion： Qishe Pill may delay hyperostosis at marginal lumbar vertebra and intervertebral disc junction, which may be related to reducing type Ⅹ collagen and Runx2 expressions.