活血补肾方对大鼠退变腰椎间盘mTOR通路依赖性自噬的影响

Effect of Huoxue Bushen Decoction on Autophagy Mediated by mTOR Pathway in Lumbar Intervertebral Disc Degeneration of Rats

目的观察活血补肾方对大鼠退变腰椎间盘mTOR通路依赖性自噬的影响,探讨该方延缓腰椎间盘退变的作用机制。方法将60只SD大鼠随机分为对照组、模型组、雷帕霉素组、活血补肾方组和雷帕霉素+活血补肾方组(简称联合组)5组。除对照组外,其余各组均采用去前肢诱导动静力失衡法复制腰椎间盘退变动物模型。建模成功后,分别给予相应处置。给药6周后,取L5/6椎间盘分别行HE和阿尔新兰-核固红染色,镜下观察腰椎间盘退变的形态学和基质变化,并检测mTOR通路下游蛋白p70S6K、4E-BP1和自噬相关蛋白beclin-1、lc-3b的表达情况。结果①腰椎间盘退变的程度(组织形态学观察):模型组最严重,对照组最轻,雷帕霉素组、活血补肾方组和联合组三组相似,介于前两者之间;②细胞外基质中蛋白聚糖含量:对照组最高,联合组较对照组略少,雷帕霉素组和活血补肾方组次之,且两者差异不明显,模型组最低;③mTOR信号通路下游蛋白:各实验组均与模型组含量存在显著差异(P<0.05),p70S6K表达水平:模型组>雷帕霉素组>活血补肾方组>联合组;4E-BP1表达水平为:模型组>活血补肾方组>雷帕霉素组>联合组;④自噬相关蛋白:beclin-1及lc-3b表达水平:联合组>活血补肾方组>雷帕霉素>模型组>对照组,并且各实验组含量均与模型组差异存在显著性(P<0.05)。结论活血补肾方具有延缓大鼠腰椎间盘退变的作用,其作用机制与其抑制mTOR信号通路,提高腰椎间盘内自噬水平有关。

Objective To observe the effect of Huoxue Bushen Decoction on autophagy mediated by mTOR signaling pathway in lumbar intervertebral disc degeneration of rats and to explore the mechanism of the decoction to delay the degeneration of lumbar intervertebral disc.Methods A total of 60 SD rats were randomly divided into 5 groups,which were the Control group,Model group,Rapamycin group,Huoxue Bushen Decoction group and Huoxue Bushen decoction+Rapamycin group(Combination group).In addition to the control group,the other 4 groups were amputated both forelimbs to prolong upright posture to induce lumbar intervertebral disc degeneration rats model.After successful modeling,each group was treated accordingly.After 6 weeks of administration,L5/6 intervertebral discs were taken as samples.The samples were taken HE staining and alcian blue-nuclear fast red staining separately for microscopic observation of morphological and matrix changes in lumbar intervertebral disc,and to detect the situation of protein expression of mTOR signaling pathway downstream proteins 4E-BP1,p70S6K,and autophagy related proteins becline-1,lc-3b;Results①Morphological observation of the degree of lumbar intervertebral disc degeneration showed that the Model group had the most degenerative change,and the Control group had the least degenerative change.The Rapamycin group,Huoxue Bushen Decoction group and the Combination group were in similar level,between the other 2 groups;②The content of proteoglycan in the extracellular matrix showed that the Control group was the most.The Combination group was the second.And then came the Rapamycin group and Huoxue Bushen Decoction group which in similar level.The Model group content was the least;③The downstream protein detection of mTOR signaling pathway showed that there were significant differences between the experimental groups and the Model group(P<0.05).For the expression level of p70S6K:the Model group was higher than Rapamycin group and Huoxue Bushen Decoction group,and the Combination group was the least.The expression level of 4E-BP1was that,the model group was higher than Huoxue Bushen decoction group and Rapamycin group,and the Combination group was the least;④Autophagy-related protein detection showed:the expression level of beclin-1 and lc-3b:the Combination group was the highest,and the Huoxue Bushen decoction group was the second,Rapamycin group was the third,Model group was the forth,Control group was the least,and there were significant differences between the experimental groups and the Model group(P<0.05).Conclusion Huoxue Bushen decoction could delayed the degeneration of rat lumbar intervertebral disc via inhibited the activity of mTOR signaling pathway in lumbar intervertebral disc and increased the level of autophagy in lumbar intervertebral disc.