脾气虚证模型大鼠股四头肌线粒体未折叠蛋白反应及CHOP蛋白表达的研究

Study on UPR^(mt) and CHOP expressions in quadriceps femoris of rat with syndrome of spleen qi deficiency

目的:通过观察脾气虚证模型大鼠股四头肌线粒体应激反应元件(CHOP)及线粒体未折叠蛋白反应(UPRmt)的变化,从UPRmt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)、过氧化氢(H2O2)水平及呼吸链复合体(CⅠ~CⅣ)酶活性;免疫印迹(Western blot)法检测CⅠ~CⅣ、热休克蛋白70/60(HSP70/60)、Lon蛋白酶(LonP)/Clp蛋白酶(ClpP)及CHOP蛋白表达。结果:比色结果显示,与对照组比较,模型组股四头肌ATP含量下降(P<0.05)、H2O2含量升高(P<0.05);CⅡ、CⅣ酶活性下降(P<0.05,P<0.01)。Westernblot结果显示,与对照组比较,模型组股四头肌CⅠ蛋白表达升高(P<0.01),CⅡ蛋白表达下降(P<0.01),HSP60蛋白表达下降(P<0.01),ClpP蛋白表达升高(P<0.01);CHOP蛋白表达下降(P<0.05)。结论:"脾气虚四肢不用"可能与CHOP抑制所致UPRmt紊乱有关。

Objective:To investigate changes of mitochondrial unfolded protein response(UPR^(mt))and C/EBP homologous protein(CHOP)expressions in quadriceps femoris of rat with syndrome of spleen qi deficiency,explore the mechanism of‘spleen qi deficiency limb disuse’from the perspective of mitochondria.Methods:Rats were randomly divided into the control group and spleen qi deficiency group(model group),eight in each group.Quadriceps femoris were taken out after fasting combined with fatigue modeling method,then adenosine triphosphate(ATP),hydrogen peroxide(H_(2)O_(2))and respiratory chain complexes I~IV(CI~CIV)contents were detected by colorimetry method;The relative expression of heat shock protein 70/60(HSP70/60),Lon Protease(LonP),ATP-dependent Clp proteolytic subunit 1(ClpP)and CHOP were detected by Western blot method.Results:The colorimetric results showed that compared with control group,the ATP content of model group was decreased(P<0.05),H2O2 level elevated(P<0.05),the enzymatic activity of CII and CIV were both decreased(P<0.05,P<0.01).Western blot results showed that compared with control group,the relative expression of CI elevated(P<0.01),CII decreased(P<0.01).The relative expression of HSP60 decreased(P<0.01),ClpP elevated(P<0.01);and the relative expression of CHOP decreased(P<0.05).Conclusion:Spleen qi deficiency limb disuse might be related to the disorder of UPR^(mt) induced by the suppression of CHOP expression.