基于16S rDNA测序技术探索白头翁汤灌肠对湿热型溃疡性结肠炎大鼠肠道菌群的影响

Effect of Enema with Pulsatilla Decoction on Intestinal Flora in Rats with Damp-Heat Ulcerative Colitis:An Analysis Based on 16S rDNA Sequencing

目的采用不同剂量白头翁汤对比美沙拉嗪干预治疗湿热型溃疡性结肠炎(ulcerative colitis,UC)大鼠,观察肠道菌群是否参与了UC的发病过程及药物治疗前后肠道菌群的变化情况。方法随机将62只SD健康雄性大鼠分为正常组,模型组,白头翁汤低、中、高剂量组和美沙拉嗪组,其中正常组12只,其余各组10只。采用三硝基苯磺酸/乙醇复合法复制UC大鼠模型。模型复制成功后,白头翁汤低、中、高剂量组分别以白头翁汤每日2.15、4.31、8.62 g/kg灌肠,美沙拉嗪组予以美沙拉嗪灌肠液每日0.35 g/kg灌肠,正常组和模型组予以生理盐水灌肠,连续给药14 d。治疗结束后观察各组大鼠一般情况和疾病活动指数,光镜下观察结肠病理变化,并收集大鼠结肠新鲜的粪便,提取粪便微生物DNA,测序及分析PCR扩增基因组DNA,采用Miseq平台测序。结果①与模型组相比,白头翁汤各剂量组和美沙拉嗪组体质量逐渐回升,便血及腹泻次数减少。②与模型组比较,白头翁汤各剂量组、美沙拉嗪组疾病活动指数评分明显降低,差异均有统计学意义(P<0.05)。③模型组大鼠菌群丰度、多样性均降低,白头翁汤及美沙拉嗪治疗后菌群丰度及多样性均有提升。④模型组大鼠厚壁菌门与拟杆菌门比值较正常组升高,白头翁汤和美沙拉嗪治疗后二者比值回调。⑤在菌群构成方面,UC大鼠较正常大鼠具有较低的普氏菌属、乳杆菌属生物丰度,白头翁汤和美沙拉嗪治疗后可回调普氏菌属、乳杆菌属益生菌丰度。结论肠道菌群参与了UC的发病过程,白头翁汤和美沙拉嗪可能通过调节肠道益生菌而起到对于疾病的治疗作用。

Objective To investigate whether intestinal flora participates in the pathogenesis of ulcerative colitis(UC)and the change in intestinal flora after drug treatment by comparing the clinical effect of Pulsatilla Decoction at different doses versus mesalazine in the treatment of damp-heat UC.Methods A total of 62 healthy male Sprague-Dawley rats were randomly divided into normal group,model group,low-,middle-,and high-dose Pulsatilla Decoction groups,and mesalazine group,with 12 rats in the normal group and 10 in each of the other groups.A rat model of UC was established by the trinitrobenzenesulfonic acid/ethanol method.After the model was successfully established,the rats in the low-,middle-,and high-dose Pulsatilla Decoction groups were given enema with Pulsatilla Decoction(2.15,4.31,and 8.62 g/kg)once a day,those in the mesalazine group were given enema with mesalazine 0.35 g/(kg·d)once a day,and those in the normal group and the model group were given enema with normal saline.The course of treatment was 14 consecutive days for all groups.General status and disease activity index(DAI)were observed after treatment,and the pathological changes of the colon were observed under a light microscope.Fresh fecal samples were collected from the colon,fecal microbial DNA was extracted,and the Miseq platform was used for the sequencing and analysis of PCR-amplified genomic DNA.Results Compared with the model group,the Pulsatilla Decoction group and the mesalazine group had a gradual increase in body weight and a reduction in the frequency of hematochezia and diarrhea.Compared with the model group,the Pulsatilla Decoction group and the mesalazine group had a significant reduction in DAI(P<0.05).The model group had reductions in the abundance and diversity of intestinal flora,and there were increases in the abundance and diversity of intestinal flora after the treatment with Pulsatilla Decoction and mesalazine.The model group had an increase in Firmicutes/Bacteroidetes ratio compared with the normal group,while there was a reduction in this ratio after the treatment with Pulsatilla Decoction and mesalazine.As for the composition of intestinal flora,the rats with UC had lower biological abundance of Prevotella and Lactobacillus than the normal rats,while Pulsatilla Decoction and mesalazine improved the abundance of the probiotics Prevotella and Lactobacillus after treatment.Conclusion Intestinal flora is involved in the pathogenesis of UC.Pulsatilla Decoction and mesalazine may exert a therapeutic effect on UC by regulating intestinal probiotics.