三黄汤对溃疡性结肠炎合并白假丝酵母菌感染的小鼠肠道真菌菌群的影响

Effect of Sanhuang Decotion enema on intestinal fungal flora of ulcerative colitis mice with Candida albicans infection

目的 基于内源转录间隔区(ITS)rRNA的高通量测序技术探讨三黄汤(SHD)对溃疡性结肠炎(UC)合并白假丝酵母菌感染的小鼠肠道真菌菌群的影响。方法 50只SPF级昆明小鼠随机分为正常对照组(Control组)、模型组(Model组)、 SHD低剂量组(SHD-L组)、 SHD高剂量组(SHD-H组)和柳氮磺胺吡啶(SASP)组,每组10只。采用葡聚糖硫酸钠联合白假丝酵母菌灌胃法构建UC合并白假丝酵母菌感染的小鼠模型。SHD-L组和SHD-H组分别给予3.75 g/kg、15.00 g/kg的SHD灌肠治疗,SASP组给予0.72 g/kg的SASP灌肠治疗,Control组和Model组小鼠采用蒸馏水处理。观察测定小鼠体质量、大便性状和便血情况,计算小鼠的疾病活动指数(DAI)。苏木精-伊红(HE)染色检测结肠组织病理变化。高通量测序技术检测各组小鼠肠道真菌菌群组成和结构特征。结果 与Control组相比,Model组小鼠的体重减轻,DAI显著升高,结肠明显缩短(p<0.01)。SHD组小鼠结肠炎症状和粘膜损伤程度减轻,SHD-H组的DAI小于Model组,结肠长度大于Model组(p<0.05)。真菌菌群多样性分析发现SHD-H组Chao1和Observed_species指数明显大于Model组(p<0.05),SHD-H组与Model组真菌菌群的结构聚类位置分离,且与Control组有明显差异(p<0.05)。物种组成分析显示SHD-H组的假丝酵母属占16.0%,与Model组(39.6%)相比有显著差异(p<0.05)。SHD-H组有90个差异菌种,Control和Model组各有8个差异菌种。菌群功能分析表明,SHD治疗UC合并白假丝酵母菌感染的小鼠的作用机制涉及呼吸、酶辅助因子的合成,以及脂肪酸和脂质的合成和分解等生物学途径。结论 SHD灌肠可改善UC合并白假丝酵母菌感染小鼠症状,降低肠道白假丝酵母菌的丰度,改变肠道的真菌菌群多样性。

Objective To explore the effect of Sanhuang Decoction(SHD) on the fungal flora of mice with ulcerative colitis(UC) and Candida albicans infection via internal transcribed spacer(ITS) rRNA high-throughput sequencing.Methods Fifty SPF Kunming mice were randomly divided into five groups:control group,model group,SHD low dose(SHD-L) group,SHD high dose(SHD-H) group,and sulfasalazine(SASP) group,with 10 mice in each group.A mouse model was established through oral administration of C.albicans combined with dextran sulphate sodium.The mice in the SHD-L group and the SHD-H group were treated with 3.75 g/kg and 15.00 g/kg SHD,respectively,while the SASP group was administered 0.72 g/kg SASP.The mice in both the control group and model group were treated with the same volume of distilled water.The body weights,stool consistencies and rectal bleeding of the mice were observed,and the disease activity index(DAI) score was calculated.Pathological changes in colon tissue were observed through staining with hematoxylin and eosin.High-throughput sequencing technology was used to analyze the composition and characteristics of gut fungal communities in each group of mice.Results Compared with the control group,the model group showed weight loss,significantly increased DAI,and a shortened colon(P<0.01).The DAI in the SHD-H group was lower,while the colon length was greater than in the model group respectively(P<0.05).Analysis of fungal diversity revealed that the Chao1 and Observed_species indices in the SHD-H group were significantly higher than those in the model group(P<0.05),and the structure of fungal community was different from that in the model group and significantly different from that in the control group(P<0.05).Species composition analysis showed that the Candida in the SHD-H group accounted for 16.0%,significantly different from the model group(39.6%)(P<0.05).Differential species analysis showed that the SHD-H group had 90 differential fungal species,while the control and model groups each had 8 differential fungal species.Microbial functional analysis showed that the mechanism of action of SHD in treating UC combined with C.albicans infection in mice involved biological pathways such as respiration,synthesis of enzyme cofactors,as well as synthesis and breakdown of fatty acids and lipids.Conclusion SHD enema can improve the symptoms of UC combined with C.albicans infection in mice,reduce the abundance of C.albicans in the intestine,and alter the diversity and structure of the fungal flora.SHD enema can effectively alleviate the symptoms of colitis in mice and reduce the degree of mucosal damage.