不同刺灸法对功能性便秘大鼠结肠组织肠神经相关蛋白表达的影响

Effect of Manual Acupuncture,Electroacupuncture and Moxibustion on Intestinal Motility and Expression of Enteric Nervous Activity Related Proteins in Functional Constipation Rats

目的:比较手针、电针和艾灸对功能性便秘大鼠结肠组织肠神经活动相关蛋白降钙素基因相关肽(CGRP)、瞬时感受器电位香草素受体1(TRPV 1)、蛋白酶激活受体-4(PAR-4)表达的影响。方法:60只SD大鼠随机分为空白对照组(8只)、模型组(11只)、西沙必利组(8只)、手针组(11只)、电针组(11只)和艾灸组(11只)。以盐酸洛哌丁胺混悬液连续灌胃6d制备功能性便秘模型。西沙必利组和3个刺灸干预组分别于每日灌胃后1h给予相应干预:西沙必利组予西沙必利混悬液3mg/kg灌胃;其余3组分别采用手针、电针和艾条温和灸的方法对大鼠"天枢""上巨虚"穴干预15min,连续6d。计量第6天24h粪便量,采用炭餐指示剂进行小肠推进率实验,分别采用实时荧光定量PCR法和Western blot技术检测结肠组织CGRP、TRPV 1、PAR-4mRNA及其蛋白的表达。结果:与空白对照组比较,模型组24h粪便量、小肠推进率显著降低(P<0.01);与模型组比较,西沙必利组、手针组、电针组的粪便量和小肠推进率均升高(P<0.05,P<0.01),艾灸组小肠推进率也升高(P<0.01);与西沙必利组及电针组比较,手针、艾灸两组的小肠推进率均降低(P<0.01)。与空白对照组比较,模型组CGRP、TRPV 1、PAR-4蛋白及mRNA表达水平均升高(P<0.01);与模型组比较,各治疗组蛋白及mRNA表达水平除艾灸组CGRP外其余均降低(P<0.05,P<0.01);与西沙必利组比较,艾灸组3种蛋白表达水平均升高(P<0.05,P<0.01),3个刺灸干预组PAR-4mRNA表达水平均升高(P<0.01),手针组CGRP mRNA表达水平降低(P<0.05)、TRPV 1mRNA表达水平升高(P<0.05);3个刺灸干预组之间比较,手针、电针两组CGRP、TRPV 1蛋白及CGRP、PAR-4 mRNA表达均低于艾灸组(P<0.05,P<0.01),电针组TRPV 1mRNA表达低于手针组(P<0.05)。结论:3种刺灸方法对功能性便秘大鼠结肠组织CGRP、TRPV 1、PAR-4蛋白及mRNA表达均有不同程度的降低作用。

Objective To compare the effect of manual acupuncture （MA）, electroacupuncture （EA） and moxibustion interventions on intestinal motility and expression of enteric nervous activity related proteins calcitonin gene related peptide （CGRP）, transient receptor potential vanilloid 1 （TRPV 1） and proteinase activated receptor-4 （PAR-4） in colon tissue of rats with functional constipation （FC）, so as to select a better intervention method for FO. Methods Sixty male SD rats were ran- domly divided into blank control （ n = 8）, model （ n = 11 ）, medication （ n = 8）, MA （ n = 11 ）, EA （ n = 11 ）, and moxibustion （n = 11 ） groups. The FO model was established by gavage of suspension of Ioperamide hydrochlorid for 6 days. Rats of the medication group were treated by gavage of cisapride （3 mg/kg）, once daily for 6 days. MA, EA or moxibustion was separately applied to bilateral ＂Tianshu＇（ST 25） and ＂Shangjuxu＇（ST 37） for 15 min, once daily for 6 days. The stool weight within 24 h was measured every day, and the intestinal propulsive rate was calculated by feeding the rats with suspension fluid containing sodium carboxymethylcellulose and active carbon powder （3%） on the last day of the experiment. The expression levels of CGRP, TRPV 1 and PAR-4 proteins and genes in the colon tissue were measured by Western blot and quantitative real-time PCR, respectively. Results Compared with the control group, the stool weight within 24 h and intestinal propulsive rate were significantly decreased in the model group （ P〈0.01 ）. Compared with the model group, the two indexes considerably increased in the medication, MA and EA groups （P〈0.05, P〈0.01 ）, and the intestinal propulsive rate was also notably increased in the moxibustion group （P〈0.05）, suggesting an improvement of the intestinal motility after the treatment. The expression levels of intestinal CGRP, TRPV 1 and PAR-4 proteins and genes were significantly higher in the model group than in the control group （P〈0.01）, and considerably lower in the medication, MA, EA and moxibustion groups than in the model group （P〈0.05, P〈0.01, except the protein expression of OGRP in the moxibustion group）. No significant differences were found among the medication, MA, EA and moxibustion groups in the stool weight, between the medication and EA groups and between the EA and moxibustion groups in the intestinal propulsive rate, and among medication, EA and moxibustion groups in the expression levels of TRPV 1 mRNA, and among the medication, MA and EA groups in the expression levels of CGRP, TRPV 1 and PAR-4 proteins （P〉0.05）. Conclusion MA, EA and moxibustion interventions can promote the intestinal motility in FC rats, which may be related to their functions in down-regulating the expression of CGRP, TRPV 1 and PAR-4 proteins and genes （except CGRP in the moxibustion group）.