脾气虚大鼠小肠组织Ca~(2+)/CaM信号通路关键分子的表达变化
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摘要
目的:观察脾气虚大鼠小肠组织Ca2+/Ca M信号通路中关键分子[Ca2+]i浓度以及Ca M、Ca MKⅡ、p-Ca MKⅡ蛋白表达水平的变化。方法:受试动物随机分为4组,即空白对照组,脾虚模型7d、14d、21d组,每组12只。除空白对照组外,其余受试动物采用复合法(苦寒破气法、游泳力竭法及饥饱失常法)成功建立脾气虚证大鼠模型,在观测各组大鼠一般生存状态、胃肠转运功能和小肠组织能量代谢相关酶活性的基础上,采用激光共聚焦技术检测小肠组织细胞内[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的表达变化。结果:与空白对照组比较,脾气虚大鼠随着造模时间的延长,胃残留率升高而小肠推进率下降(P<0.01);小肠组织乳酸脱氢酶(LDH)活性升高而琥珀酸脱氢酶(SDH)活性降低(P<0.05,P<0.01);小肠组织[Ca2+]i浓度和Ca M、Ca MKⅡ、p-Ca MKⅡ蛋白表达量显著升高(P<0.01);且脾虚模型7d、14d、21d组之间比较,以脾虚模型21d组变化更为显著(P<0.05,P<0.01)。结论:小肠组织Ca2+/Ca M信号通路关键分子的异常表达变化可能是脾气虚发生的病理机制之一。
Objective:To observe the changes in expression of the key molecules as CaM,CaMKII and p-CaMKⅡof Ca~(2+)/CaM signaling pathways in small intestine tissue of rats with syndrome of deficiency of spleen qi.Methods:Rats were randomly divided into 4 groups as control group,7d,14 d and 21 d model groups,and with 12 rats in each group.All the rats except rats in control group were used to establish the rat models with syndrome of deficiency of spleen qi by using a compound method,which included method of bitter-cold and removing stagnant qi,method of exhausting by swimming and method of either hunger or gorge.To test the concentration of[Ca~(2+)]i in small intestine tissue by using laser confocal microscopy based on the observation of general condition,gastrointestinal transshipment function and activity of enzymes related with energy metabolism in small intestine of rats.The changes in expression of proteins as CaM,CaMKⅡ,p-CaMKⅡ in small intestine were tested by using Western blot method.Results:Compared with control group,the stomach residue rate of rats with syndrome of deficiency of spleen qi was increased,and the small intestine propelling rate of rats in model groups was decreased with the prolongation of model establishing time(P<0.01).The activity of LDH in small intestine of rats in model groups was increased,and the activity of SDH was decreased(P<0.05,P<0.01).The concentration of[Ca~(2+)]i and the expression of CaM,CaMKⅡ and p-CaMKⅡ in small intestine tissue of rats in model groups were increased significantly(P<0.01),and these changes in rats of 21 d model group were the most remarkable compared with rats in 7d and 14 d model groups(P<0.05,P<0.01).Conclusion:The abnormal expression of the key molecules of Ca~(2+)/CaM signaling pathways in small intestine tissue might be one of the pathogenic mechanisms in the occurrence of syndrome of deficiency of spleen qi.
Objective:To observe the changes in expression of the key molecules as CaM,CaMKII and p-CaMKⅡof Ca~(2+)/CaM signaling pathways in small intestine tissue of rats with syndrome of deficiency of spleen qi.Methods:Rats were randomly divided into 4 groups as control group,7d,14 d and 21 d model groups,and with 12 rats in each group.All the rats except rats in control group were used to establish the rat models with syndrome of deficiency of spleen qi by using a compound method,which included method of bitter-cold and removing stagnant qi,method of exhausting by swimming and method of either hunger or gorge.To test the concentration of[Ca~(2+)]i in small intestine tissue by using laser confocal microscopy based on the observation of general condition,gastrointestinal transshipment function and activity of enzymes related with energy metabolism in small intestine of rats.The changes in expression of proteins as CaM,CaMKⅡ,p-CaMKⅡ in small intestine were tested by using Western blot method.Results:Compared with control group,the stomach residue rate of rats with syndrome of deficiency of spleen qi was increased,and the small intestine propelling rate of rats in model groups was decreased with the prolongation of model establishing time(P<0.01).The activity of LDH in small intestine of rats in model groups was increased,and the activity of SDH was decreased(P<0.05,P<0.01).The concentration of[Ca~(2+)]i and the expression of CaM,CaMKⅡ and p-CaMKⅡ in small intestine tissue of rats in model groups were increased significantly(P<0.01),and these changes in rats of 21 d model group were the most remarkable compared with rats in 7d and 14 d model groups(P<0.05,P<0.01).Conclusion:The abnormal expression of the key molecules of Ca~(2+)/CaM signaling pathways in small intestine tissue might be one of the pathogenic mechanisms in the occurrence of syndrome of deficiency of spleen qi.
引文
[1]隋峰,王汝俊,王建华.补中益气汤、党参、白术含药血清对脾虚大鼠壁细胞胞内Ca2+/Ca M-PKⅡ活性的影响.中药药理与临床,2005,21(5):4-5
[2]隋峰,王汝俊,王建华.补中益气汤、党参、白术含药血清对脾虚大鼠壁细胞胞内Ca2+/CaM活性的影响.中药药理与临床,2005,21(4):1-2
[3]修宗昌,李德新.脾虚证Ca2+-CaM信号系统的实验研究.中国医药学报,2002,17(12):758-759
[4]Corcoran E E,Means A R.Defining Ca2+/calmodulin-dependent protein kinase cascades in transcriptional regulation.J Biol Chem,2001,276(4):2975-2978
[5]Soderling T R,Chang B,Brickey D.Cellular signaling through multifunctional Ca2+/calmodulin-dependent protein kinase.Biol Chem,2001,276(5):3719-3722
[6]陈小野,邹世洁,张智.大鼠长期脾虚造模的实验研究.中国中医基础医学杂志,1995,1(1):37-41
[7]钱泽南,钱会南.脾虚大鼠脑内TGF-β1、TNF-α的变化以及益气扶正中药干预研究.辽宁中医杂志,2012,39(10):2078-2079
[8]陈奇.中药药理研究方法.北京:人民卫生出版社,1993:332
[9]叶仁群,张光奇.二金汤镇痛作用和对胃肠运动功能影响的实验研究.中南药学,2004,2(1):39-40
[10]苗明三.实验动物和动物实验技术.北京:中国中医药出版社,1997:17-31
[11]方喜业.医学实验动物学.北京:人民卫生出版社,1995:21-40
[12]陈小野.实用中医证候动物模型学.北京:中国协和医科大学联合出版社,1993:35-61
[13]尚冰,丛培玮,王蕊芳,等.对脾虚大鼠模型建立模糊数学评价体系的研究.辽宁中医药大学学报,2013,15(3):64-66
[14]王晓明,易杰,廖世新,等.脾虚证动物模型的客观评估.中华中医药杂志,2006,21(7):406-408
[15]吴敬中.胃动力障碍中虚证辨证施治.中国中西医脾胃杂志,1999,7(1):35-36
[16]李志,肖国辉,徐州,等.香砂六君颗粒对脾虚患者及大鼠胃肠运动的调节作用.世界华人消化杂志,2009,17(5):512-515
[17]廖志航,郑凯,李春林,等.胃康颗粒对实验性脾虚动物胃肠功能的作用.成都中医药大学学报,2004,27(3):44-46
[18]隋波,姜萍.跑台训练队大鼠糖酵解、有氧氧化功能系统限速酶影响.山东体育学院学报,2009,25(8):54-57
[19]李洁,汪浩.不同强度急性疲劳运动对大鼠心肌线粒体电子传递链酶复合体活性的影响.中国运动医学杂志,2007,26(3):304-312
[20]张宽朝,姬惠惠.琥珀酸脱氢酶的提取与活力测定的研究进展.中国中医药杂志,2007,5(10):32-33
[21]田振军,石磊,刘小杰,等.过度训练对大鼠血清CK、LDH、SOD、SDH活性及UMb含量影响的研究.中国运动医学杂志,2000,19(1):49-50
[22]李燕舞,王汝俊,王建华,等.补脾方药对脾虚大鼠壁细胞胃泌素受体及细胞内[Ca2+]i作用的观察.中国中医基础医学杂志,2006,12(3):180-182
[23]聂克,王汝俊,王建华.脾虚大鼠胃壁细胞胞浆游离钙离子浓度及黄芪对其调节作用的研究.中药药理与临床,2001,17(2):15-17
[2]隋峰,王汝俊,王建华.补中益气汤、党参、白术含药血清对脾虚大鼠壁细胞胞内Ca2+/CaM活性的影响.中药药理与临床,2005,21(4):1-2
[3]修宗昌,李德新.脾虚证Ca2+-CaM信号系统的实验研究.中国医药学报,2002,17(12):758-759
[4]Corcoran E E,Means A R.Defining Ca2+/calmodulin-dependent protein kinase cascades in transcriptional regulation.J Biol Chem,2001,276(4):2975-2978
[5]Soderling T R,Chang B,Brickey D.Cellular signaling through multifunctional Ca2+/calmodulin-dependent protein kinase.Biol Chem,2001,276(5):3719-3722
[6]陈小野,邹世洁,张智.大鼠长期脾虚造模的实验研究.中国中医基础医学杂志,1995,1(1):37-41
[7]钱泽南,钱会南.脾虚大鼠脑内TGF-β1、TNF-α的变化以及益气扶正中药干预研究.辽宁中医杂志,2012,39(10):2078-2079
[8]陈奇.中药药理研究方法.北京:人民卫生出版社,1993:332
[9]叶仁群,张光奇.二金汤镇痛作用和对胃肠运动功能影响的实验研究.中南药学,2004,2(1):39-40
[10]苗明三.实验动物和动物实验技术.北京:中国中医药出版社,1997:17-31
[11]方喜业.医学实验动物学.北京:人民卫生出版社,1995:21-40
[12]陈小野.实用中医证候动物模型学.北京:中国协和医科大学联合出版社,1993:35-61
[13]尚冰,丛培玮,王蕊芳,等.对脾虚大鼠模型建立模糊数学评价体系的研究.辽宁中医药大学学报,2013,15(3):64-66
[14]王晓明,易杰,廖世新,等.脾虚证动物模型的客观评估.中华中医药杂志,2006,21(7):406-408
[15]吴敬中.胃动力障碍中虚证辨证施治.中国中西医脾胃杂志,1999,7(1):35-36
[16]李志,肖国辉,徐州,等.香砂六君颗粒对脾虚患者及大鼠胃肠运动的调节作用.世界华人消化杂志,2009,17(5):512-515
[17]廖志航,郑凯,李春林,等.胃康颗粒对实验性脾虚动物胃肠功能的作用.成都中医药大学学报,2004,27(3):44-46
[18]隋波,姜萍.跑台训练队大鼠糖酵解、有氧氧化功能系统限速酶影响.山东体育学院学报,2009,25(8):54-57
[19]李洁,汪浩.不同强度急性疲劳运动对大鼠心肌线粒体电子传递链酶复合体活性的影响.中国运动医学杂志,2007,26(3):304-312
[20]张宽朝,姬惠惠.琥珀酸脱氢酶的提取与活力测定的研究进展.中国中医药杂志,2007,5(10):32-33
[21]田振军,石磊,刘小杰,等.过度训练对大鼠血清CK、LDH、SOD、SDH活性及UMb含量影响的研究.中国运动医学杂志,2000,19(1):49-50
[22]李燕舞,王汝俊,王建华,等.补脾方药对脾虚大鼠壁细胞胃泌素受体及细胞内[Ca2+]i作用的观察.中国中医基础医学杂志,2006,12(3):180-182
[23]聂克,王汝俊,王建华.脾虚大鼠胃壁细胞胞浆游离钙离子浓度及黄芪对其调节作用的研究.中药药理与临床,2001,17(2):15-17