基于高通量转录组测序研究三黄汤缓解白念珠菌定植下DSS诱导小鼠溃疡性结肠炎的作用机制

Mechanism of Sanhuang Decoction in alleviating dextran sulfate sodium induced ulcerative colitis in mice with Candida albicans colonization:based on high-throughput transcriptome sequencing

利用高通量转录组测序技术探讨三黄汤(Sanhuang Decoction,SHD)治疗白念珠菌(Candida albicans,Ca)定植小鼠溃疡性结肠炎(ulcerative colitis,UC)的作用机制。采用葡聚糖硫酸钠(dextran sulfate sodium,DSS)联合Ca灌胃方式构建Ca定植下DSS诱导的小鼠UC模型,并予以SHD灌肠治疗;观察小鼠一般状态、疾病活动指数(disease activity index,DAI)、结肠长度和结肠黏膜损伤指数(colon mucosa damage index,CMDI)变化;HE染色法进行组织病理学分析;平板培养法检测小鼠肠道Ca载荷;ELISA法检测小鼠血清抗酿酒酵母抗体(anti-saccharomces cerevisiae antibody,ASCA)和β-1,3-葡聚糖的含量,以及血清和肠组织TNF-α、IL-1β和IL-6表达水平;高通量转录组测序技术分析对照组、模型组和三黄汤组的差异表达基因,并对差异表达基因进行GO和KEGG pathway富集分析;qRT-PCR和Western blot检测差异基因显著富集的NOD样信号通路相关基因NLRP3、ASC、caspase-1和IL-1β的表达水平。结果显示,SHD灌肠治疗可改善UC小鼠的一般状态,降低DAI评分;SHD能减少肠道Ca载荷,减轻肠道充血、糜烂和结肠缩短程度,并且降低血清β-1,3-葡聚糖含量,下调血清和肠道组织TNF-α、IL-1β和IL-6水平;SHD组vs模型组有383个差异表达基因,显著富集于免疫系统过程、细菌防御反应和固有免疫反应等生物学过程,KEGG pathway分析提示差异基因显著富集于NOD样受体信号通路、细胞因子-细胞因子受体相互作用和视黄醇代谢等信号通路;SHD下调UC小鼠结肠组织NLRP3、caspase-1和IL-1β的mRNA和蛋白表达水平。以上结果表明,SHD可缓解Ca定植的小鼠UC,其作用机制可能与其对NOD样受体信号通路调控有关。

This study explored the mechanism of Sanhuang Decoction(SHD) in treating dextran sulfate sodium(DSS)-induced ulcerative colitis(UC) in mice with Candida albicans(Ca) colonization via high-throughput transcriptome sequencing. Specifically, the animal model was established by oral administration of 3.0% DSS for 7 days followed by intragastrical administration of Ca suspension at 1.0 × 108 cells for 4 days and then the mice were treated with SHD enema for 7 days. Afterwards, the general signs were observed and the disease activity index(DAI) was recorded every day. After mice were sacrificed, colon length and colon mucosa damage index(CMDI) were determined and the histomorphology was observed with the HE staining method. The fungal loads of feces were detected with the plate method. Anti-saccharomyces cerevisiae antibody(ASCA) and β-1,3-glucan in serum, and TNF-α, IL-1β, and IL-6 in serum and colon were detected by ELISA. High-throughput RNA sequencing method was adopted to identify transcriptome of colon tissues from the control, model and SHD(15.0 g·kg-1) groups. Differentially expressed genes(DEGs) among groups were screened and the GO and KEGG pathway enrichment analysis of the DEGs was performed. The expression levels of NLRP3, ASC, caspase-1, and IL-1β genes related to the NOD-like receptor signaling pathway which involved 9 DEGs, were examined by qRT-PCR and Western blot. The results demonstrated that SHD improved the general signs, decreased DAI and Ca loads of feaces, alleviated colon edema, erosion, and shortening, and lowered the content of β-1,3-glucan in serum and TNF-α, IL-1β, and IL-6 in serum and colon tissues of mice. Transcriptome sequencing revealed 383 DEGs between SHD and model groups, which were mainly involved in the biological processes of immune system, response to bacterium, and innate immune response. They were mainly enriched in the NOD-like signaling pathway, cytokine-cytokine interaction pathway, and retinol metabolism pathway. Moreover, SHD down-regulated the mRNA and protein levels of NLRP3, caspase-1, and IL-1β. In a word, SHD ameliorates DSS-induced UC in mice colonized with Ca, which probably relates to its regulation of NOD-like receptor signaling pathway.