四君子汤对脾虚大鼠骨骼肌细胞色素c氧化酶Ⅰ、ⅣmRNA及蛋白表达的影响

Effect of Sijunzi Decoction on Skeletal Muscle Cytochrome c OxidaseⅠ,Ⅳ mRNA and Protein Expression in Rats with Spleen Deficiency

【目的】探讨脾虚证中线粒体能量代谢障碍机制及健脾益气方药的干预作用。【方法】采用皮下注射利血平法构建脾虚大鼠模型,将造模成功后的大鼠分为脾虚模型组和四君子汤组,每组各7只,另取正常组7只。四君子汤组给予四君子汤1.0 g·mL^(-1)·kg^(-1)灌胃,脾虚模型组和正常组灌胃等体积生理盐水,每日1次,持续21 d。给药期间记录大鼠一般情况。给药结束后,采用对溴苯胺法测定尿D-木糖排泄率,采用苏木素-伊红(HE)染色法观察骨骼肌病理改变,采用聚合酶链反应(PCR)法检测大鼠骨骼肌细胞色素c氧化酶(COX)Ⅰ、COXⅣmRNA表达,采用免疫组织化学法、蛋白免疫印迹法检测大鼠骨骼肌COXⅠ、COXⅣ蛋白表达。【结果】与正常组比较,脾虚模型组大鼠骨骼肌肌纤维排列紊乱、间隙增大,细胞核分布不均,胞质染色不匀;与脾虚模型组比较,四君子汤组骨骼肌纤维排列较整齐。脾虚模型组大鼠骨骼肌COXⅠ、COXⅣmRNA和蛋白表达水平较正常组降低(P<0.05或P<0.01);四君子汤组大鼠骨骼肌COXⅠ、COXⅣmRNA和蛋白表达水平较脾虚模型组升高(P<0.05或P<0.01)。【结论】脾虚大鼠存在骨骼肌病理形态损伤,骨骼肌COXⅠ、COXⅣmRNA和蛋白表达均降低。四君子汤改善脾虚大鼠骨骼肌病理结构,提高COXⅠ、COXⅣm RNA和蛋白表达可能是其修复脾虚证能量代谢障碍的机制之一。

Objective To explore the mechanism of mitochondrial energy metabolism disorder in spleen deficiency syndrome and the intervention of formulas for invigorating spleen and replenishing qi on it.Methods The rat model of spleen deficiency was constructed by subcutaneous injection of reserpine.The successful modeled rats were divided into spleen deficiency model group and Sijunzi Decoction group,with 7 rats in each group,another7 healthy rats were set up as the normal group.Sijunzi Decoction group was intragastrically given 1.0 g·mL^(-1)·kg^(-1)of Sijunzi Decoction,and the spleen deficiency model group and the normal group were intragastrically given the same volume of normal saline.Both administrations placed once a day for 21 consecutive days.During medication period,the general condition of the rats was recorded.After medication,the urinary D-xylose excretion rate was measured by p-bromoaniline method,the pathological changes of skeletal muscle were observed by hematoxylineosin(HE)staining,the m RNA expression of cytochrome c oxidase(COX)I and COXⅣin rat skeletal muscle was detected by polymerase chain reaction(PCR),and the protien expression of COXⅠand COXⅣin rat skeletal muscle was determined by immunohistochemical and Western Blotting method.Results Compared with the normal group,the skeletal muscle fibers in the spleen deficiency model group were disordered,with enlarged gaps,uneven distribution of nuclei and uneven cytoplasmic staining;compared with the spleen deficiency model group,the skeletal muscle fibers in the Sijunzi Decoction group were more neatly arranged.The mRNA and protein expression levels of COXⅠand COXⅣin skeletal muscle of rats in the spleen deficiency model group were decreased compared with those in the normal group(P<0.05 or P<0.01);the mRNA and protein expression levels of COXⅠand COXⅣin skeletal muscle of rats in the Sijunzi Decoction group were increased compared with those in the spleen deficiency model group(P<0.05 or P<0.01).Conclusion Spleen deficiency rats had pathological morphological damage in skeletal muscle,and mRNA and protein expression of COXⅠand COXⅣin skeletal muscle were reduced.The improvement of the pathological structure of skeletal muscle and the increase of COXⅠand COXⅣmRNA and protein expression in spleen deficiency rats by Sijunzi Decoction may be one of the mechanisms to repair the impaired energy metabolism disorder in spleen deficiency syndrome.