基于16S rDNA测序技术分析薏苡附子败酱散对急性溃疡性结肠炎小鼠肠道菌群的影响

Effect of Yiyi Fuzi Baijiang powder on intestinal flora of acute ulcerative colitis mice based on 16S rDNA sequencing

目的从肠道菌群的角度探索薏苡附子败酱散治疗溃疡性结肠炎(UC)的作用机制。方法将24只雄性SPF级C75BL/6J小鼠随机分为空白组、模型组、阳性药对照组、中药组4个组别,通过自由饮用3%DSS溶液的方法制作急性UC小鼠模型。造模成功后,中药组予以1.048 g/mL的薏苡附子败酱散溶液,阳性药对照组予以0.031 g/mL的嗜酸乳杆菌(益君康)溶液,空白组和模型组予以同等体积的生理盐水,7 d的治疗结束后处死所有小鼠,取小鼠结肠组织和肠内容物。实验过程中监测小鼠的体质量、精神等一般情况,进行DAI评分,HE染色法在光镜下观察小鼠肠道病理变化,进行HS评分。提取肠道菌群的DNA,采用高通量测序技术对其16S rDNA V4区进行测序分析,并采用PCR荧光定量技术对肠道菌群中的乳杆菌进行定量检测。结果模型组小鼠DAI评分明显高于空白组(t=4.137 0,P=0.000 4),药物干预后,中药组和阳性药对照组DAI评分明显下降(t=2.240 3,P=0.035 0;t=2.081 6,P=0.048 6),差异均有统计学意义;病理HS评分显示,与模型组比较,其余组别小鼠HS评分均明显降低(空白组:t=5.452 8,P=0.000 2;中药组:t=3.117 1,P=0.009 8;阳性药对照组:t=3.306 2,P=0.007 0),差异均有统计学意义;测序结果显示模型组小鼠肠道菌群物种多样性和丰度均明显降低,药物干预后菌群多样性和丰度提高,模型组致病菌丰度较高,药物治疗后可增加有益菌数量,减少致病菌数量。PCR荧光定量检测乳杆菌结果显示,与模型组相比,其余组别小鼠肠道中乳杆菌含量均明显增加(空白组:t=3.871 8,P=0.002 6;中药组:t=2.224 3,P=0.048 0;阳性药对照组:t=2.976 4,P=0.012 6),差异均有统计学意义,而阳性药对照组和中药组之间未见明显区别(t=2.007 0,P=0.070 0)。结论薏苡附子败酱散能够调节肠道菌群的结构,增加物种多样性,改善和恢复有益菌和有害菌的丰度比例,从而发挥对急性UC的治疗作用,其中乳杆菌发挥了明显作用。

Objective To explore the mechanism of Yiyi Fuzi Baijiang powder in the treatment of ulcerative colitis(UC) from the perspective of intestinal flora. Methods Acute UC mouse model was established by giving free drinking of 3% DSS solution. After successful modeling, the traditional Chinese medicine(TCM) group was given 1.048 g/mL solution of Yiyi Fuzi Baijiang powder, the positive control group was given 0.031 g/mL Lactobacillus(Yijunkang) solution, and the blank control group and model group were given the same volume of normal saline. After 7 days of treatment, all the mice were killed, and the colon tissue and intestinal contents were collected. During the experiment, the weight, spirit and other general conditions of mice were monitored. DAI score and HE staining method were used to observe the pathological changes of intestinal tract under light microscope. HS score was used to extract the DNA of intestinal flora, 16 S rDNA V4 region was sequenced using high-throughput sequencing technology, and Lactobacillus in intestinal flora was quantitatively detected with PCR fluorescence quantitative technology. Results The DAI score in the model group was significantly higher than that in the blank control group. After drug intervention, the DAI scores in the TCM group and the positive control group decreased significantly(P<0.05);pathological HS scoring showed that compared with the model group, the HS scores in the other groups decreased significantly. The results of sequencing showed that the species diversity and abundance of intestinal flora in the model group significantly decreased, which increased after drug intervention. The abundance of pathogenic bacteria in the model group was higher, which reduced after drug treatment while beneficial bacteria increased. The results of PCR fluorescence quantitative detection showed that compared with the model group, the count of Lactobacillus in the intestinal tract of mice in other groups significantly increased(P<0.05), and that in the blank control group was the highest, followed by the positive control group. Conclusion Yiyi Fuzi Baijiang powder can regulate the structure of intestinal flora, increase species diversity, improve and restore the abundance ratio of beneficial bacteria to harmful bacteria, playing a role in the treatment of acute UC;Lactobacillus plays a significant role in the mechanism.