基于“脾肾相关”理论探讨补肾健脾活血方对大鼠废用性肌萎缩的作用及其机制

To investigate the effect and mechanism of Bushen Jianpi invigorating blood activating formula on disuse muscle atrophy in rats based on"spleen kidney related"theory

目的研究补肾健脾活血方对尾悬吊大鼠诱导的废用性肌萎缩的作用及其机制。方法40只雌性大鼠按体重随机平均分为5组:空白对照组、模型组、中药低剂量组、中药中剂量组、中药高剂量组。除空白对照组外,各组均给予尾悬吊造模处理。空白对照组和模型组蒸馏水灌胃,中药组分别以低、中、高3个剂量的补肾健脾活血方灌胃。称量及计算大鼠右后肢比目鱼肌肌肉湿重与体重比值;测量比目鱼肌平均横截面积;ELISA法检测大鼠血清内肌肉生长抑制素(MSTN)含量;Western Blot法检测大鼠比目鱼肌内AKT、mTOR、P70S6K、GSK-3β蛋白表达水平。结果与空白对照组比较,模型组肌肉湿重体重比、比目鱼肌平均横截面积明显下降,MSTN含量明显增多,AKT、mTOR、P70S6K蛋白表达明显降低,GSK-3β蛋白表达明显升高(P<0.01);与模型组相比,补肾健脾活血方各剂量组肌肉湿重体重比、比目鱼肌平均横截面积均有所增加(P<0.01),且AKT、P70S6K蛋白表达升高,中高剂量组MSTN含量有所下降(P<0.05),且GSK-3β蛋白表达中高剂量组明显降低(P<0.01),mTOR蛋白表达低剂量组明显降低(P<0.01),高剂量组表达量增高(P<0.01);与补肾健脾活血方低剂量组比较中高剂量组大鼠比目鱼肌肌肉湿重、比目鱼肌纤维横截面积明显增加(P<0.01),且AKT、mTOR、P70S6K蛋白表达明显升高(P<0.01),GKS-3β表达明显降低(P<0.01);与补肾健脾活血方中剂量组相比,高剂量组大鼠比目鱼肌肌肉湿重、比目鱼肌纤维横截面积明显增加(P<0.01),高剂量组AKT、mTOR、P70S6K蛋白表达升高(P<0.05),而GSK-3β蛋白表达降低(P<0.01)。结论补肾健脾活血方可有效防治尾悬吊诱导大鼠的废用性肌萎缩,其机制可能与调控AKT/GSK-3β和AKT/mTOR/P70S6K信号通路相关。

Objective To investigate the effect and mechanism of Bushen Jianpi invigorating blood activating formula on disuse muscle atrophy induced by tail suspension rats.Methods Forty female rats were randomly equally divided into five groups accord⁃ing to body weight:blank control group,model group,TCM low dosage group,TCM middle dosage group,and TCM high dosage group.Except for the normal group,each group was given tail suspension modeling treatment,the blank control group and the model group were gavaged with distilled water,and the Chinese medicine group was gavaged with Bushen Jianpi invigorating blood formula at three doses of low,medium and high,respectively.The ratio of wet weight to body weight of soleus muscle in right hind limb of rats was weighed and calculated;The mean soleus muscle cross-sectional area was measured;Measurement of myo⁃statin(MSTN)content in rat serum by ELISA;Western blot analysis of Akt,mTOR,P70S6K,GSK-3 in rat soleus muscleβProtein expression levels.Results Compared with the blank control group,the wet weight to body weight ratio of muscle and the average cross-sectional area of soleus muscle in the model group decreased significantly,MSTN content increased significantly,Akt,mTOR,P70S6K protein expression decreased significantly,GSK-3βProtein expression was significantly higher(P<0.01);Compared with the model group,the wet weight to body weight ratio of muscle and the average cross-sectional area of so⁃leus muscle in each dose of Bushen Jianpi invigorating blood circulation formula increased(P<0.01),and the protein expres⁃sions of Akt and P70S6K were increased,MSTN content was decreased(P<0.05),and GSK-3βProtein expression was sig⁃nificantly lower(P<0.01)in the medium-and high-dose groups,and mTOR protein expression was significantly lower(P<0.01)in the low-dose group and increased(P<0.01)in the high-dose group;Compared with the low-dose group using Bushen Jianpi invigorating blood circulation,the wet weight and cross-sectional area of soleus muscle fibers were significantly in⁃creased(P<0.01),and the protein expression of Akt,mTOR,P70S6K(P<0.01),gks-3βExpression was significantly lower(P<0.01);Compared with the medium dose group of Bushen Jianpi invigorating blood recipe,the wet weight and cross-sectional area of soleus muscle fibers were significantly increased in the high dose group(P<0.01),and the protein expressions of Akt,mTOR,P70S6K were increased in the high dose group(P<0.05),whereas GSK-3βProtein expression was reduced(P<0.01).Conclusion Bushen Jianpi invigorating blood recipe can effectively prevent disuse muscle atrophy induced by tail suspension in rats,and the mechanism may be related to the regulation of Akt/GSK-3βAnd Akt/mTOR/P70S6K signaling pathway related.