生、制枳实换用的枳实芍药散对便秘型肠易激综合征大鼠“脑-肠-菌”轴功能的影响

Effect of Raw and Processed Aurantii Fructus Immaturus Switched to Zhishi Shaoyaosan on Function of Brain-Gut-Microbiota Axis in Rats with Constipation-predominant Irritable Bowel Syndrome

目的:从“脑-肠-菌”轴的角度探讨生、制枳实换用的枳实芍药散改善便秘型肠易激综合征(C-IBS)大鼠的作用机制。方法:将80只大鼠随机分为空白组、模型组、匹维溴铵组(15.625 mg·kg^(-1))、生枳实芍药散组、清炒枳实芍药散组、麸炒枳实芍药散组、炒炭枳实芍药散组、成品枳实芍药散组(3.75 g·kg^(-1)),每组10只。除空白组灌胃常温0.9%氯化钠溶液外,其余各组均灌胃0~4℃冰0.9%氯化钠溶液(2 mL·d^(-1),共14 d)建立C-IBS大鼠模型。连续给药14 d后检测大鼠粪便含水量、小肠推进率;酶联免疫吸附测定法(ELISA)检测5-羟色胺(5-HT)、血管活性肠肽(VIP)、神经肽Y(NPY)、降钙素基因相关肽(CGRP)、P物质(SP)、二胺氧化酶(DAO)、D-乳酸(D-LA)水平;苏木素-伊红(HE)染色观察结肠组织病理损伤变化;实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织环磷酸腺苷(cAMP)、蛋白激酶A(PKA)、水通道蛋白3(AQP3)mRNA的表达及蛋白免疫印迹法(Western blot)检测结肠组织VIP和AQP3蛋白的表达;采用气质联用色谱法(GC-MS)检测短链脂肪酸(SCFAs)含量的变化。结果:与空白组比较,模型组大鼠粪便含水量、小肠推进率显著降低(P<0.01);血清中5-HT、VIP、CGRP、SP水平显著上升,NPY水平显著下降(P<0.01);血浆中DAO和D-LA水平显著升高(P<0.01);结肠组织黏膜上皮轻度破损,杯状细胞减少且腔内颗粒明显减少;结肠组织中AQP3、cAMP、PKA mRNA表达水平和AQP3、VIP蛋白表达水平明显下降(P<0.05,P<0.01);粪便中SCFAs总量呈明显下降趋势,其中,乙酸、异丁酸、异戊酸、戊酸、己酸含量显著减少,丙酸、丁酸含量明显上升(P<0.05,P<0.01)。与模型组比较,各给药组均明显提高C-IBS大鼠小肠推进率,改善肠黏膜屏障功能,调整模型大鼠血清中脑肠肽水平(P<0.05,P<0.01),明显升高大鼠结肠组织中AQP3、cAMP、PKA mRNA及VIP、AQP3蛋白的表达水平(P<0.05,P<0.01),除异丁酸外各给药组对大鼠粪便中短链脂肪酸含量均具有显著回调作用,并且麸炒枳实入药的枳实芍药散的作用稍强于其他炮制品枳实入药的枳实芍药散。结论:生、制枳实换用的枳实芍药散可通过对“脑-肠-菌”轴功能的调节来改善C-IBS,并且使用麸炒枳实入药的枳实芍药散效果更佳。

Objective:To research the mechanism underlying the effect of raw and processed Aurantii Fructus Immaturus switched to Zhishi Shaoyaosan(ZSS)on constipation-predominant irritable bowel syndrome(C-IBS)rats via the brain-gut-microbiota axis.M ethod:Eighty rats were randomly divided into the blank,model,positive drug(pinaverium bromide,15.625 mg·kg^(-1)),raw ZSS,stir-fried ZSS,bran-fried ZSS,charcoal-fried ZSS and finished ZSS groups(3.75 g·kg^(-1)),with 10 rats in each group.Except for the blank group,which received intragastric administration of 0.9%sodium chloride solution at room temperature,all other groups were administered the ice solution at 0 to 4°C(2 mL·d^(-1),for a total of 14 d)to establish the C-IBS rat model.The fecal water content and the propulsion rate of small intestine were detected after 14 d of continuous drug administration.The levels of 5-hydroxytryptamine(5-HT),vasoactive intestinal peptide(VIP),neuro-peptide Y(NPY),calcitonin gene-related peptide(CGRP),substance P(SP),diamine oxidase(DAO)and D-lactic acid(D-LA)were detected by enzyme linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)staining was used to observe the changes in colonic pathological injury in each group.The expression levels of cyclic adenosine monophosphate(cAMP),protein kinase A(PKA)and aquaporin-3(AQP3)mRNA in colon tissues were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and the protein expressions of VIP and AQP3 in colon tissues were detected by Western blot.The content of short chain fatty acids(SCFAs)was determined by gas chromatography-mass spectrometry.Result:Compared with the blank group,the fecal water content and intestinal propulsion rate of rat in the model group were significantly decreased(P<0.01),and the levels of 5-HT,VIP,CGRP and SP in serum were significantly increased.Simultaneously,the NPY levels significantly decreased(P<0.01),the levels of DAO and D-LA in plasma were significantly increased(P<0.01),and the mucosal epithelium of colon tissue was slightly damaged,with reduced goblet cells and significantly reduced luminal granules.The mRNA expression levels of AQP3,cAMP and PKA and the protein expression levels of AQP3 and VIP in colon tissue were significantly decreased(P<0.05,P<0.01).The total amount of SCFAs in feces showed an obvious decreasing trend,with the contents of acetic acid,isobutyric acid,isovaleric acid,valeric acid and caproic acid decreased significantly,while the contents of propionic acid and butyric acid increased significantly(P<0.05,P<0.01).Compared with the model group,the treatment groups increased the intestinal propulsion rate,improved the intestinal mucosal barrier function,and adjusted the level of serum brain-gut peptide in C-IBS rats(P<0.05,P<0.01).The expression levels of AQP3,cAMP,PKA mRNA and VIP,AQP3 protein in colon tissue of rats in all treatment groups were increased.All the treatment groups had a significant downregulation of the content of SCFAs except for isobutyric acid in rat feces,and the effect of ZSS prepared by the bran-fried Aurantii Fructus Immaturus was superior than that of other ZSS.Conclusion:The raw and processed Aurantii Fructus Immaturus switched to ZSS could influence the brain-gut-microbiota axis to treat C-IBS rats and it is more reasonable to use bran-fried Aurantii Fructus Immaturus in ZSS.