理气通便方对功能性便秘气滞证大鼠脑肠肽的影响

Effects of Liqi Tongbian Formula on brain-gut peptides in rats with functional constipation of qi stagnation pattern

目的探讨理气通便方治疗功能性便秘的机制。方法采用复方地芬诺酯灌胃及夹尾激怒法构建功能性便秘气滞证大鼠模型。按照随机数字表法将48只雌雄各半大鼠分为空白组、模型组、莫沙必利组(2 mg/kg)、理气通便方低剂量组(5.15 g/kg)、理气通便方中剂量组(10.3 g/kg)、理气通便方高剂量组(20.6 g/kg),每组8只,连续给药14 d。观察各组大鼠一般情况、灌胃墨汁后首粒黑便排出时间、粪便干湿重变化。ELISA法检测血清5-羟色胺(5-HT)、血管活性肠肽(VIP)、去甲肾上腺素(NE)的含量。Real-time PCR法检测结肠组织中P物质(SP)、促肾上腺皮质激素释放因子(CRF)mRNA表达。Western blot法测定结肠组织中SP、CRF蛋白表达。结果治疗前,与空白组比较,模型组及各给药组大鼠首粒黑便排出时间延长(P<0.01),粪便含水率降低(P<0.01)。经过7 d治疗后,与模型组比较,各给药组大鼠首粒黑便排出时间均缩短(P<0.05,P<0.01),理气通便方高剂量组大鼠粪便含水率升高(P<0.05)。治疗14 d后,与模型组比较,各给药组大鼠首粒黑便排出时间均缩短(P<0.01),莫沙必利组及理气通便方中、高剂量组大鼠粪便含水率升高(P<0.05,P<0.01)。与莫沙必利组比较,理气通便方高剂量组大鼠首粒黑便排出时间缩短(P<0.05)。经过14 d治疗后,与空白组比较,模型组、莫沙必利组和理气通便方各剂量组5-HT含量降低(P<0.05,P<0.01),模型组、莫沙必利组和理气通便方低剂量组NE含量升高(P<0.01),理气通便方高剂量组NE含量降低(P<0.01),模型组、莫沙必利组、理气通便方各剂量组VIP含量升高(P<0.05,P<0.01),模型组、莫沙必利组和理气通便方低剂量组SP、CRF mRNA及SP蛋白表达水平降低(P<0.05,P<0.01),理气通便方中、高剂量组SP蛋白表达水平升高(P<0.05,P<0.01),模型组、莫沙必利组及理气通便方各剂量组CRF蛋白表达水平均降低(P<0.05,P<0.01)。与模型组比较,莫沙必利组及理气通便方各剂量组5-HT含量升高(P<0.01);莫沙必利组和理气通便方中、高剂量组NE含量降低(P<0.01);莫沙必利组及理气通便方各剂量组VIP含量降低(P<0.01);理气通便方中、高剂量组SP mRNA及SP蛋白表达水平升高(P<0.01),理气通便方各剂量组CRF mRNA及CRF蛋白表达水平升高(P<0.05,P<0.01),理气通便低剂量组大鼠SP蛋白表达水平降低(P<0.01)。与莫沙必利组比较,理气通便方低剂量组NE、VIP含量升高(P<0.01),理气通便方中剂量组NE含量降低(P<0.01),VIP含量升高(P<0.05),理气通便方高剂量组NE、VIP含量均降低(P<0.05,P<0.01),理气通便方中、高剂量组5-HT含量、SP mRNA及SP蛋白表达水平升高(P<0.01),理气通便方各剂量组CRF mRNA及CRF蛋白表达水平升高(P<0.05,P<0.01)。结论理气通便方通过改善大鼠胃肠动力和肠道水液代谢达到治疗功能性便秘的作用,其机制可能与调节脑肠肽的分泌有关。

Objective To explore the mechanism of Liqi Tongbian Formula(Qi-regulating Bowel-opening Decoction,LQTBF)in the treatment of functional constipation(FC).Methods Rat FC models with qi stagnation pattern were established by using intragastric gavage of compound diphenoxylate and tail-clamping irritation.The rat models were randomly divided(Random Number Table)into blank group,model group,mosapride group(2 mg/kg),low-dose LQTBF(5.15 g/kg),mid-dose LQTBF group(10.3 g/kg),and high-dose LQTBF group(20.6 g/kg)(n=8 in each group)and received intervention for 14 continuous days.The general condition,time to first black bowel movement after intragastric gavage of ink,and the dry and wet weight of feces in each group of rats were evaluated.The levels of 5-HT,VIP and NE in the serum were detected by using ELISA.The mRNA expression of SP and CRF in rat colon tissue was measured with real time PCR.The expression of SP and CRF protein in colon tissue was measured with Western blot assay.Results Before treatment,compared with the blank group,time to first black bowel movement in the model group and interventional groups was prolonged(P<0.01),and the fecal moisture content was lowered(P<0.01).After 7 days of treatment,compared with the model group,time to the first black bowel movement in each treatment group was shortened(P<0.05,P<0.01),and the fecal moisture content in the high-dose LQTBF group was increased(P<0.05).After 14 days of treatment,compared with the model group,time to first black bowel movement in each treatment group was shortened(P<0.01);The fecal water content of rats in mosapride group and mid-dose,high-dose LQTBF groups increased(P<0.05,P<0.01);Compared with Mosapride group,time to first black bowel movement in the high-dose group was shortened(P<0.05).After 14 days of treatment,compared with the blank group,the content of 5-HT in the model group,mosapride group and LQTBF groups were decreased(P<0.05,P<0.01).The content of NE in model group,mosapride group and low-dose LQTBF group increased(P<0.01),while the content of NE in high-dose LQTBF group decreased(P<0.01).The content of VIP in model group,mosapride group and LQTBF groups increased(P<0.05,P<0.01).The expression levels of SP,CRF mRNA and SP protein in model group,mosapride group and low-dose LQTBF group were decreased(P<0.05,P<0.01),the expression levels of SP protein in mid-dose and high-dose LQTBF groups were increased(P<0.05,P<0.01),and the expression levels of CRF protein in model group,mosapride group and LQTBF groups were decreased(P<0.05,P<0.01);Compared with the model group,the content of 5-HT in mosapride group and each dose group of LQTBF formula was increased(P<0.01).the content of NE in mosapride group and middle and high dose groups of LQTBF formula was decreased(P<0.01),and the content of VIP in mosapride group and LQTBF groups was decreased(P<0.01).The expression levels of SP mRNA and SP protein were increased in mid-dose and high-dose LQTBF groups(P<0.05,P<0.01),and the expression levels of CRF mRNA and CRF protein were increased in all LQTBF groups(P<0.05,P<0.01).The expression of SP protein decreased in low-dose LQTBF group(P<0.01);Compared with mosapride group,the contents of NE and VIP in low-dose LQTBF group were increased(P<0.01).In the middle dose LQTBF group,the content of NE were decreased(P<0.01),and the content of VIP were increased(P<0.05).The contents of NE and VIP in high-dose LQTBF group were decreased(P<0.05,P<0.01).the content of 5-HT,the expression levels of SP mRNA and SP protein in middle and high-dose LQTBF groups were increased(P<0.01).the expression levels of CRF mRNA and protein were increased(P<0.05,P<0.01).Conclusion LQTBF could improve gastrointestinal motility and intestinal water metabolism in rats.Its mechanism may be related to regulating the secretion of brain-gut peptides.