Ma Ke-Long, Han Zhi-Jun, Sun Juan, Tan Xiao-Fen, Wang Tian-Ming, Shao Jing, Yan Gui-Ming, Wang Chang-Zhong
College of Integrated Chinese and Western Medicine/College of Life Science,Anhui University of Chinese Medicine Hefei 230012,China Institute of Integrated Chinese and Western Medicine,Anhui Academy of Chinese Medicine Hefei 230012,China Anhui Province Key Laboratory of Traditional Chinese Medicinal Decoction Pieces of New Manufacturing Technology,Anhui University of Chinese Medicine Hefei 230012,China Key Laboratory of Xin'An Medicine,Ministry of Education,Anhui Academy of Chinese Medicine Hefei 230012,China.
College of Integrated Chinese and Western Medicine/College of Life Science,Anhui University of Chinese Medicine Hefei 230012,China.
Zhongguo Zhong Yao Za Zhi. 2021 Aug;46(15):3915-3925. doi: 10.19540/j.cnki.cjcmm.20210521.701.
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.
本研究通过高通量转录组测序，探讨三黄汤（SHD）对白色念珠菌（Ca）定植的葡聚糖硫酸钠（DSS）诱导的小鼠溃疡性结肠炎（UC）的治疗机制。具体而言，通过口服3.0% DSS 7天，随后灌胃1.0×10⁸个细胞的Ca悬液4天建立动物模型，然后用SHD灌肠治疗小鼠7天。之后，每天观察一般体征并记录疾病活动指数（DAI）。处死小鼠后，测定结肠长度和结肠黏膜损伤指数（CMDI），并用苏木精-伊红（HE）染色法观察组织形态学。用平板法检测粪便中的真菌载量。通过酶联免疫吸附测定（ELISA）检测血清中的抗酿酒酵母抗体（ASCA）和β-1,3-葡聚糖，以及血清和结肠中的肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）。采用高通量RNA测序方法鉴定对照组、模型组和SHD（15.0 g·kg⁻¹）组结肠组织的转录组。筛选各组间的差异表达基因（DEG），并对DEG进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。通过实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测与包含9个DEG的核苷酸结合寡聚化结构域样受体信号通路相关的NLRP3、凋亡相关斑点样蛋白（ASC）、半胱天冬酶-1（caspase-1）和IL-1β基因的表达水平。结果表明，SHD改善了一般体征，降低了DAI和粪便中的Ca载量，减轻了结肠水肿、糜烂和缩短，降低了小鼠血清中β-1,3-葡聚糖以及血清和结肠组织中TNF-α、IL-1β和IL-6的含量。转录组测序显示SHD组和模型组之间有383个DEG，主要参与免疫系统、对细菌的反应和先天免疫反应的生物学过程。它们主要富集在核苷酸结合寡聚化结构域样信号通路、细胞因子-细胞因子相互作用通路和视黄醇代谢通路。此外，SHD下调了NLRP3、caspase-1和IL-1β的mRNA和蛋白水平。总之，SHD改善了Ca定植的小鼠中DSS诱导的UC，这可能与其对核苷酸结合寡聚化结构域样受体信号通路的调节有关。
