脾气虚证大鼠股四头肌线粒体自噬水平及AMPK/ULK1途径变化的研究  

A study on mitophagy and AMPK/ULK1 pathway changes in quadriceps femoris in rats with spleen qi deficiency

作　　者：刘文俊[1] 李振钰 许欣竹 冷雪[1] 陈文娜[1] 穆靖洲[2] 单德红[1] Liu Wenjun;Li Zhenyu;Xu Xinzhu;Leng Xue;Chen Wenna;Mu Jingzhou;Shan Dehong(Liaoning University of Traditional Chinese Medicine,Liaoning 110847;Dalian Medical University,Liaoning 116044)

机构地区：[1]辽宁中医药大学,辽宁110847 [2]大连医科大学

出　　处：《北京中医药大学学报》2019年第9期760-765,共6页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金青年项目(No.81803986)~~

摘　　要：目的观察脾气虚证大鼠股四头肌线粒体自噬水平及腺苷酸活化蛋白激酶(AMPK)/UNC-51-类似自噬激活激酶1(ULK1)途径的变化,从线粒体方面研究"脾气虚四肢不用"的机制。方法运用饮食失节+劳倦法复制脾气虚证大鼠模型,随机分为对照组和脾气虚模型组(模型组),造模成功后取股四头肌,比色法检测三磷酸腺苷(ATP)含量;JC-1法检测线粒体膜电位(MMP);免疫荧光及印迹法(Western blot)法检测微管相关蛋白1轻链3-B (LC3 B)、选择性自噬接头蛋白(p62)表达水平及与线粒体共定位的量;Western blot法检测AMPKα及ULK1蛋白表达。结果与对照组比较,模型组股四头肌ATP、MMP下降(P<0.05,P<0.01);LC3B-II蛋白表达及与线粒体共定位增加(均P<0.01)、p62蛋白表达及与线粒体共定位减少(均P<0.01);p-AMPKα/AMPKα及p-ULK1/ULK1比值升高(均P<0.01)。结论 "脾气虚四肢不用"可能与AMPK/ULK1途径激活所致的线粒体自噬水平提高不足有关。Objective To investigate changes in mitophagy and Amp-activated protein kinase(AMPK)/UNC-51-like kinase 1(ULK1) pathways in quadriceps femoris in rats with spleen qi deficiency and to explore the pathogenesis of "disuse of the four limbs due to spleen qi deficiency" from the perspective of mitochondria. Methods Rats were randomly divided into the control group and spleen qi deficiency group(model group). The models were established by irregular diet and exertion and then quadriceps femoris were taken out. Adenosine triphosphate(ATP) content was detected by colorimetry and mitochondrial membrane potential(MMP) by JC-1. The expressions of light chain 3-B(LC3 B), selective autophagy adaptor protein(p62) and their co-localization with mitochondria were detected by Western blot and immunofluorescence. Finally, the expressions of AMPKα/ULK1 pathway proteins were detected by Western blot. Results Compared with the control group, ATP and MMP levels of quadriceps femoris were decreased in the model group(P<0.05, P<0.01). LC3 B-II protein expression and its co-localization with mitochondria were increased(all P<0.01) but p62 and its co-localization with mitochondria were decreased(all P<0.01). Both p-AMPKα/AMPKα and p-ULK1/ULK1 ratios were increased(all P<0.01). Conclusion "Limb disuse due to spleen qi deficiency" might be related to the insufficient increase in mitochondrial autophagy induced by the activation of AMPK/ULK1 pathway.

关 键 词：脾气虚 股四头肌 线粒体自噬 微管相关蛋白1轻链3 选择性自噬接头蛋白 腺苷酸活化蛋白激酶 UNC-51-类似自噬激活激酶1 

分 类 号：R223.1[医药卫生—中医基础理论]