理气通便方对气滞证慢传输型便秘大鼠肠道动力及肠道菌群的影响

Effects of Liqi Tongbian Decoction on intestinal motility and intestinal flora in rats with slow transit constipation with Qi stagnation pattern

目的:观察理气通便方对气滞证慢传输型便秘(slow transit constipation,STC)大鼠肠道动力及肠道菌群的影响。方法:以36只Wistar大鼠为研究对象,根据随机数字表法选取6只为正常组,其余大鼠采用“洛哌丁胺混悬液+夹尾刺激”复制气滞证STC大鼠模型。造模成功后,将大鼠随机分为气滞证STC模型组、琥珀酸普芦卡必利片组和理气通便方低、中、高剂量组,每组6只。正常组与气滞证STC模型组给予等容积的蒸馏水灌胃,琥珀酸普芦卡必利片组给予0.18mg/(kg·d)混悬液灌胃,理气通便方低、中、高剂量组分别用5.15、10.30和20.60g/kg药液灌胃。通过粪便数量、粪便含水量、小肠推进率评价理气通便方的治疗效果。采用免疫组化方法检测大鼠结肠组织中Cajal间质细胞(Cajal interstitial cell,ICC)、5-HT、5-HT3受体(5-HT3R)及5-HT4受体(5-HT4R)蛋白表达水平。采用气相色谱-质谱联用法测定短链脂肪酸(short-chain fatty acids,SCFAs)含量的变化。采用16SrDNA高通量测序分析肠道菌群的变化。结果:与正常组比较,气滞证STC模型组大鼠的粪便数量减少、粪便含水量和小肠推进率均下降(P<0.05),结肠组织中ICC、5-HT、5-HT3R及5-HT4R蛋白表达水平均降低(P<0.05),肠道菌群的丰度和多样性显著下降(P<0.05),毛螺菌属、梭菌属丰度下降(P<0.05),粪球菌属、克雷伯菌属丰度增加(P<0.05),粪便中SCFAs含量降低(P<0.05);与气滞证STC模型组比较,给药组大鼠粪便数量及含水率均增加(P<0.05),小肠推进率除理气通便方低剂量组差异无统计学意义外,其他均差异有统计学意义(P<0.05);结肠组织中ICC、5-HT、5-HT3R及5-HT4R蛋白表达水平升高,其中除了理气通便方低剂量组5-HT蛋白表达及琥珀酸普芦卡必利片组ICC、5-HT、5-HT3R蛋白表达水平差异无统计学意义外,其余均差异有统计学意义(P<0.05);琥珀酸普芦卡必利片组与理气通便方中、高剂量组能够显著提高SCFAs的总含量(P<0.05),其中显著增加了丁酸的含量;理气通便方各剂量组可提高菌群的丰度和多样性,显著增加毛螺菌属、梭菌属丰度,降低粪球菌属丰度(P<0.05)。结论:理气通便方可能通过增加ICC、5-HT及其受体的表达、调整肠道菌群结构及SCFAs的含量,从而起到治疗气滞证STC大鼠的作用。

Objective:To investigate the effects of Liqi Tongbian Decoction on intestinal motility and intestinal flora in rats with slow transit constipation(STC)with Qi stagnation pattern.Methods:Thirty-six Wistar rats were selected according to the random number table method,and 6rats were selected as the normal group,and the rest of the rats were used to replicate the STC rat model of Qi stagnation pattern by using"loperamide suspension and tail clamping stimulation".After successful modelling,the rats were randomly divided into the STC model group,the prucalopride succinate tablet group,and the low,medium and high dose groups of the formula,with 6rats in each group.The normal group and the STC model group were given equal volumes of distilled water,the prucalopride succinate tablet group was given 0.18mg/(kg·d)suspension by gavage,and the low,medium and high dose groups of the formula were given 5.15g/kg,10.30g/kg and 20.60g/kg by gavage,respectively.The therapeutic efficacy of the formula was evaluated by the water content of faeces and the propulsion rate of small intestine,and the protein expression levels of murine Cajal interstitial cells(ICC),serotonin(5-HT),serotonin 3receptor(5-HT3R)and serotonin 4receptor(5-HT4R)were detected by immunohistochemistry in colon tissues,and the content of short-chain fatty acids(SCFAs)was measured by gas chromatography-mass spectrometry(GCMS).SCFAs content changes,and 16SrDNA high-throughput sequencing was used to analyse the changes in intestinal flora.Results:Compared with the normal control group,rats in the STC model group with Qi stagnation pattern showed a decrease in the number of faeces,a significant decrease in the water content of faeces and the rate of small intestinal propulsion(P<0.05),a significant decrease in the levels of expression of the 5-HT,5-HT3R and 5-HT4Rproteins in the colonic tissues(P<0.05),and a significant decrease in abundance and diversity of intestinal flora(P<0.05),with a increase in the abundance of Trichoderma spp,Clostridium spp.decreased in abundance(P<0.05),Coccidioides faecalis spp.and Klebsiella spp.increased in abundance(P<0.05),and the content of short-chain fatty acids in the faeces was significantly reduced(P<0.05);when compared with the STC model group of Qi stagnation pattern,the number of faeces and water content of rats in the administered group increased(P<0.05),and the rate of propulsion of the small intestine was statistically(P<0.05),except for no statistical difference in the low dose group of Liqi Tongbian Decoction;the middle and high dose groups of Prucalopride Succinate Tablets and Liqi Tongbian Decoction were able to increase the total content of SCFAs(P<0.05),including a significant increase in the content of butyric acid;the dose groups of Liqi Tongbian Decoction could increase the abundance and diversity of bacterial flora,significantly increase the abundance of Lachnospiraceae_NK4A136spp.and Clostridium spp.and decrease the abundance of Coccidioides faecalis spp.(P<0.05).Conclusion:The Liqi Tongbian Decoction may play a role in the treatment of STC with Qi stagnation pattern by increasing the expression of ICC,5-HT and their receptors,adjusting the structure of intestinal flora and the content of SCFAs.