益气健脾中药对脾虚大鼠骨骼肌组织中Ca^(2+)/CaMKⅡ通路关键分子的干预作用

Intervention of Qi-activating and Spleen-strengthening Herbs on Ca^(2+)/ CaMKⅡ Signaling Pathways Key Factors in Skeletal Muscle Tissue of Rats with Spleen-qi Deficiency

目的:观察脾气虚大鼠骨骼肌Ca2+/Ca MKⅡ信号通路关键分子[Ca2+]i以及Ca M、Ca MKⅡ、p-Ca MKⅡ蛋白表达及益气健脾中药四君子汤和红芪提取物的干预作用。方法:动物随机分为正常对照组、脾气虚模型组、四君子汤组和红芪提取物组,每组10只。除正常对照组外,其余动物采用复合法建立脾气虚证大鼠模型,四君子汤组给予四君子汤煎剂20 g/(kg·d)灌胃,红芪提取物组给予红芪提取物6 g/(kg·d)灌胃,连续干预21 d后,观测各组大鼠一般生存状态、胃肠激素GAS、MOT含量和骨骼肌组织ATP酶活性,同时采用激光共聚焦技术检测骨骼肌组织[Ca2+]i浓度,蛋白免疫印迹技术检测小肠组织Ca M、Ca MKⅡ和p-Ca MKⅡ表达变化。结果:与正常对照组比较,脾气虚模型大鼠一般生存状态较差,胃肠激素GAS、MOT含量显著降低(P<0.01);骨骼肌组织Na+-K+-ATPase和Ca2+-Mg2+-ATPase活性、[Ca2+]i浓度及Ca M、Ca MKⅡ、p-Ca MKⅡ蛋白表达量显著降低(P<0.01);与模型组比较,四君子汤组和红芪提取物组大鼠一般生存状态明显改善,小肠组织胃肠激素MOT含量升高(P<0.05),四君子汤组小肠组织GAS含量显著升高(P<0.05);各给药组骨骼肌组织Na+-K+-ATPase和Ca2+-Mg2+-ATPase活性及[Ca2+]i浓度和Ca M、Ca MKⅡ蛋白表达量显著升高(P<0.05);四君子汤组骨骼肌组织p-Ca MKⅡ蛋白表达显著升高(P<0.05)。结论:四君子汤组和红芪提取物能调节脾气虚大鼠胃肠激素GAS、MOT分泌,提高骨骼肌组织Na+-K+-ATPase和Ca2+-Mg2+-ATPase活性,并能上调Ca2+/Ca M信号通路关键分子表达,且以四君子汤作用显著。

Objective： To observe changes of [Ca^2＋]i concentration and Ca M,Ca MKⅡ and p-Ca MKⅡ of Ca^2＋/ Ca MKⅡ signaling pathways in skeletal muscle tissue of rats with spleen-qi deficiency and intervention of Sijunzi decoction and extract of Hedysarum polybotrys. Methods： Rats were randomized into four groups： normal control group,spleen-qi deficient model group,extract from Hedysarum polybotrys group and Sijunzi decoction group,ten rats in each group. After the spleen-qi deficient models were built by comprehensive application of rhubarb,exhaustive and hungry methods,and treatment groups were treated with extract from Hedysarum polybotrys at6 g /（ kg·d） or Sijunzi decoction at 20 g /（ kg·d） for 21 d. Then,general existence,gastrointestinal hormones GAS and MOT levels,and activities of Na^＋-K^＋-ATPase and Ca^2＋-Mg^2＋-ATPase of skeletal muscle were evaluated. Also,confocal laser technology was used to test cellular[Ca^2＋]i concentrations in skeletal muscle and Western blotting technique was used to test Ca M,Ca MKⅡ and p-Ca MKⅡ expression in intestinal tissue of spleen-qi deficient model rats. Results： Compared with normal group,general condition was poor,levels of GAS and MOT decreased（ P〈0. 01）,activities of Na^＋-K^＋-ATPase and Ca^2＋-Mg^2＋-ATPase,[Ca^2＋]i concentration as well as expression of Ca M,Ca MK Ⅱ and p-Ca MK Ⅱ in skeletal muscle decreased significantly（ P〈0. 01） in spleen-qi deficienct model rats. Compared with model group,general condition improved significantly,as well as level of MOT in intestinal increased（ P〈0. 05） in the rats of extract from Hedysarum polybotrys group and Sijunzi decoction group,while level of GAS increased in intestinal（ P〈0. 05） in the rats of Sijunzi decoction group; Moreover,activities of Na^＋-K^＋-ATPase and Ca^2＋-Mg^2＋-ATPase as well as [Ca^2＋]i concentration and expression of Ca M and Ca MKⅡ in skeletal muscle tissue increased（ P〈0. 05） in the rats of extract from Hedysarum polybotrys group and Sijunzi decoction group,while p-Ca MK Ⅱ in skeletal muscle tissue increased in the rats of Sijunzi decoction group（ P〈0. 05）. Conclusion： Sijunzi decoction and extract of Hedysarum polybotrys can be applied to treat spleen-qi deficiency syndrome through the mechanism of regulating GAS and MOT secretion and raising expression of Ca^2＋/ Ca M signaling pathways key factors in skeletal muscle tissue. Sijunzi decoction has the better effect.