白术对慢传输型便秘小鼠短链脂肪酸和肠道屏障的影响

Effect of Atractylodis Macrocephalae Rhizoma on Short-chain Fatty Acids and Intestinal Barrier in Mice with Slow-transit Constipation

目的:通过观察生白术对慢传输型便秘(STC)小鼠的短链脂肪酸和肠道屏障的影响,探究其治疗STC的作用机制。方法:将48只雄性KM小鼠随机分为空白组、模型组、生白术低、中、高剂量组(2.5、5、10g·kg^(-1))及莫沙必利组(2.5 mg·kg^(-1))。除空白组外,其余各组均灌胃洛哌丁胺混悬液(5 mg·kg^(-1)),2次/d,连续灌胃14 d,构建STC小鼠模型。同时各给药组给予相应的药物灌胃治疗,连续14 d,空白组和模型组灌胃等体积蒸馏水。观察生白术治疗对小鼠体质量、排便频率、粪便含水率、肠道推进率的影响;苏木素-伊红(HE)和过碘酸-雪夫(PAS)染色观察小鼠结肠的病理变化;酶联免疫吸附测定法(ELISA)检测小鼠血清中胃泌素(GAS)和胃动素(MTL)的含量;气相色谱质谱联用法(GC-MS)检测小鼠粪便中短链脂肪酸(SCFAs)的含量;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测小鼠结肠中闭锁小带蛋白-1(ZO-1)、咬合蛋白(Occludin)和闭合蛋白-1(Claudin-1)的mRNA和蛋白表达水平。结果:与空白组比较,模型组小鼠的体质量、排便频率、粪便含水率、肠道推进率均明显下降(P<0.05,P<0.01);结肠组织排列紊乱,杯状细胞数量减少;血清中GAS、MTL的含量均显著下降(P<0.01);粪便中SCFAs的含量均有下降趋势,其中乙酸、丙酸、丁酸、异丁酸、戊酸的含量明显下降(P<0.05,P<0.01);结肠组织中ZO-1、Occludin和Claudin-1的mRNA和蛋白表达水平均显著降低(P<0.01)。与模型组比较,生白术低、中、高剂量组小鼠体质量、排便频率、粪便含水率、肠道推进率均明显上升(P<0.05,P<0.01);结肠组织黏膜层结构完整,无明显损伤,杯状细胞的数量增多;血清中GAS、MTL的含量均显著上升(P<0.01);粪便中短链脂肪酸的含量均有上升趋势,其中乙酸、丙酸、丁酸、异丁酸的含量明显上升(P<0.05,P<0.01);结肠组织中ZO-1、Occludin、Claudin-1的mRNA和蛋白表达水平均明显上升(P<0.05,P<0.01)。结论:生白术能够改善STC小鼠的便秘症状,其作用机制可能与提高肠道中SCFAs含量及促进结肠中ZO-1、Occludin和Claudin-1的mRNA及蛋白水平表达有关。

Objective:To investigate the mechanism of Atractylodis Macrocephalae Rhizoma(AMR)in the treatment of slow-transmission constipation(STC)by observing the effects of AMR on short-chain fatty acids and intestinal barries in STC mice.Method:Forty-eight male KM mice were randomly divided into blank group,model group,AMR low-,medium-,high-dose groups(2.5,5,10 g·kg^(-1))and mosapride group(2.5 mg·kg^(-1)).Except for the blank group,all groups were gavaged with loperamide suspension(5 mg·kg^(-1))twice daily for 14 d to construct the STC mouse model.At the same time,each drug administration group was given the corresponding drug by gavage for consecutive 14 d,the blank and model groups were gavaged with equal volume of distilled water.The effects of the treatment of AMR on body mass,defecation frequency,fecal water content and intestinal propulsion rate of mice were observed,the pathological changes of mouse colon were observed by hematoxylin-eosin(HE)staining and periodic acid-Schiff(PAS)staining,the levels of gastrin(GAS)and motilin(MTL)in serum were detected by enzyme-linked immunosorbent assay(ELISA),gas chromatography-mass spectrometry(GC-MS)was used to detect the contents of short-chain fatty acids(SCFAs)in mouse feces,real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to determine the mRNA and protein expression levels of zonula occludens-1(ZO-1),Occludin,and Claudin-1 in the colon of mice.Result:Compared with the blank group,the body mass,defecation frequency,fecal water content and intestinal propulsion rate of mice in the model group were significantly decreased(P<0.05,P<0.01),the arrangement of colonic tissues was disordered,and the number of goblet cells was reduced,the levels of GAS and MTL in serum were significantly decreased(P<0.01),and the levels of SCFAs in the feces were on a decreasing trend,with the contents of acetic acid,propionic acid,butyric acid,isobutyric acid and valeric acid were significantly decreased(P<0.05,P<0.01),the mRNA and protein expression levels of ZO-1,Occludin and Claudin-1 in the colonic tissues were significantly decreased(P<0.01).The above results suggested that STC mouse model was successfully constructed.Compared with the model group,the body mass,defecation frequency,fecal water content and intestinal propulsion rate of mice in AMP administration groups all increased significantly(P<0.05,P<0.01),the mucosal layer of the colonic tissues was structurally intact without obvious damage,and the number of goblet cells increased,serum levels of GAS and MTL were significantly increased(P<0.01),the contents of SCFAs in the feces were all on a rising trend,with the contents of acetic,propionic,butyric and isobutyric acids rising significantly(P<0.05,P<0.01),the mRNA and protein expression levels of ZO-1,Occludin and Claudin-1 in the colonic tissues were significantly increased(P<0.05,P<0.01).Conclusion:AMR is able to improve the constipation symptoms in STC mice,and its mechanism may be related to increasing the contents of SCFAs in the intestine as well as promoting the mRNA and protein expression levels of ZO-1,Occludin and Claudin-1 in the colon.