电针刺“足三里”对脾气虚大鼠下丘脑CaM/CaMKⅡ和NPY表达影响随机平行对照研究
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摘要
[目的]观测电针刺"足三里"对脾气虚大鼠下丘脑CaM/CaMKⅡ和NPY表达影响。[方法]使用随机平行对照方法,将32只SPF级SD大鼠随机数字表分为4组,空白对照组、脾气虚组、足三里组和非经非穴组,8只/组。参照劳倦过度和饮食不节复合因素复制脾气虚大鼠模型:实验第7d,先饱食1日,再禁食2日,3日为1个周期,每日负重游泳(水温35～37℃,5%体重保险丝绑于大鼠尾部)至力竭(即大鼠鼻尖没入水面10秒),连续5个周期;正常组每日自由饮水进食。实验第3d开始干预,1次/d,持续7d;足三里组:毫针(0.25×13mm)直刺双侧"足三里"5mm,接电针仪(SDZ-Ⅱ型),疏密波刺激(密波15Hz,疏波2Hz),电流0.5mA,连续刺激20min;非经非穴组:毫针(0.25×13mm)直刺双侧"非经非穴点"-双侧髂嵴上10～15mm、后正中线旁开20mm区段内一个固定对照点,平刺8～10mm,电针处理同足三里组;空白对照组和脾气虚组在操作台上固定20min,无处理。每日测量大鼠体质量,实验第30d,处死,取丘脑,荧光定量PCR法检测下丘脑组织中CaM/CaMKⅡ及NPY的mRNA表达。[结果]实验第1d至第8d,体质量空白对照组呈稳定增长趋势,脾气虚模型组、足三里组、非经非穴组轻度下降;实验第8d,脾气虚模型组、足三里组、非经非穴组均低于空白对照组,足三里组高于脾气虚模型组;非经非穴组低于足三里组,与脾气虚模型组无明显差异。实验第8d,大鼠下丘脑CAM/CAMKⅡ、NPY足三里组均高于空白对照组、脾气虚组、非经非穴组(P<0.01);非经非穴组、脾气虚组低于空白对照组(P<0.01);非经非穴组与脾气虚组无明显差异(P>0.05)。[结论]电针刺脾气虚大鼠"足三里"可提高下丘脑CaM/CaMKⅡ和NPY表达,提高体质量,明显改善神疲乏力、被毛稀疏无光泽、大便稀溏、体质量逐渐下降等"脾气虚"表现,提示针刺"足三里"对脾气虚良性调节作用可能是通过提高下丘脑CAM/CAMKⅡ、NPY表达水平来调控的。
[Objective] To observe the effects of Electroacupuncture on "Zusanli" on the expression of calcium/calmodulin kinase II(CaM/CaMK II) and neuropeptide Y(NPY) in the small intestine and hypothalamus of rats with spleen qi deficiency. [Methods] 32 SD rats were randomly divided into 4 groups, the normal group, the spleen Qi deficiency group, the Zusanli group and the non-acupoint group, with 8 rats in each group. Except the normal group, the other 3 groups were overfatigued and using irregular diet to establish the model of spleen Qi deficiency syndrome. After the success of the model, the treatment group was treated with electroacupuncture on the bilateral "Zusanli" point, non-acupoint was treated with electroacupuncture on non-acupoint point, with filiform needle stab 5 mm, using the density wave, 20 min/time, 1 time/d, which lasted for 7 days. The rats in the normal group and the spleen qi deficiency group were fixed on the operating table for 20 minutes, and no other treatment was given. After the end of treatment, take out the small intestine and hypothalamus, the detection of CaM/CaMKⅡ and NPY mRNA expression in rat's hypothalamus by fluorescence quantitative PCR. [Results] The CaM/CaMKⅡ and NPY expression levels in hypothalamus and small intestine tissue were significantly lower than those in normal group which in spleen deficiency group and non-acupoint group(P<0.05); CaM/CaMKⅡ and NPY expression levels in hypothalamus and small intestinetissue were significantly increased in Zusanli group, compared to the spleen deficiency group(P<0.05); No significant difference in non-acupoint group(P<0.05). [Conclusion] The signal pathway of CaM/CaMK II and NPY is the target of regulating gastrointestinal digestive function by electroacupuncture of "Zusanli" point,the function of regulating spleen and stomach and promoting digestion may be achieved by the signal pathway of CaM/CaMK Ⅱ and NPY.
[Objective] To observe the effects of Electroacupuncture on "Zusanli" on the expression of calcium/calmodulin kinase II(CaM/CaMK II) and neuropeptide Y(NPY) in the small intestine and hypothalamus of rats with spleen qi deficiency. [Methods] 32 SD rats were randomly divided into 4 groups, the normal group, the spleen Qi deficiency group, the Zusanli group and the non-acupoint group, with 8 rats in each group. Except the normal group, the other 3 groups were overfatigued and using irregular diet to establish the model of spleen Qi deficiency syndrome. After the success of the model, the treatment group was treated with electroacupuncture on the bilateral "Zusanli" point, non-acupoint was treated with electroacupuncture on non-acupoint point, with filiform needle stab 5 mm, using the density wave, 20 min/time, 1 time/d, which lasted for 7 days. The rats in the normal group and the spleen qi deficiency group were fixed on the operating table for 20 minutes, and no other treatment was given. After the end of treatment, take out the small intestine and hypothalamus, the detection of CaM/CaMKⅡ and NPY mRNA expression in rat's hypothalamus by fluorescence quantitative PCR. [Results] The CaM/CaMKⅡ and NPY expression levels in hypothalamus and small intestine tissue were significantly lower than those in normal group which in spleen deficiency group and non-acupoint group(P<0.05); CaM/CaMKⅡ and NPY expression levels in hypothalamus and small intestinetissue were significantly increased in Zusanli group, compared to the spleen deficiency group(P<0.05); No significant difference in non-acupoint group(P<0.05). [Conclusion] The signal pathway of CaM/CaMK II and NPY is the target of regulating gastrointestinal digestive function by electroacupuncture of "Zusanli" point,the function of regulating spleen and stomach and promoting digestion may be achieved by the signal pathway of CaM/CaMK Ⅱ and NPY.
引文
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[2]宋庆峰.中医四大名著[M].沈阳:辽海出版社,2016.
[3]成映霞,段永强,杜娟,等.脾气虚大鼠小肠组织钙调蛋白信号通路中钙调蛋白、钙调素依赖蛋白激酶Ⅱ基因表达规律的研究[J].中医研究,2014,27(12):51-56.
[4]吴天石,张会永,张哲,等.脾虚证动物模型造模方法述评[J].中医杂志,2015,56(11):978-983.
[5]李忠仁.实验针灸学[M].2版.北京:中国中医药出版社,2007:263-264.
[6]王晓建,杨旭,宋晓东,等.实时荧光定量PCR法检测转基因小鼠拷贝数[J].中国实验动物学报,2007,15(3):170-174.
[7]王冰订补.黄帝内经素问[M].北京:人民卫生出版社,1963:399-400.
[8]吕林,唐旭东,王凤云,等.从“脾主运化”理论探讨论治功能性胃肠病[J].时珍国医国药,2016,27(1):160-162.
[9]施茵,吴焕淦.足三里穴在免疫功能调节中的应用[J].现代中医药,2003(3):3-6.
[10]段永强,成映霞,梁玉杰,等.益气健脾中药对脾虚大鼠骨骼肌组织中Ca2+/CaMKⅡ通路关键分子的干预作用[J].中药材,2015,38(3):562-566.
[11]姚巧玲,帕尔哈提·阿不都热依木,尹改珍,等.针刺对肥胖大鼠Leptin-下丘脑NPY轴的影响[J].新疆医科大学学报,2008,31(1):28-30.
[12]刘志诚,周群,王沂争,等.针灸对单纯性肥胖病并发高脂血症患者的脂质、TXB2、6-Keto-PGF1α的影响[J].针刺研究,1996(4):17-21.