目的 探索麻杏石甘汤（Maxing Shigan Decoction）通过影响小鼠肠道菌群组成及趋化因子产生而防治流感病毒感染的潜在机制。方法 通过鼻腔接种法建立A型流感病毒感染小鼠模型，经ig给药3 d和7 d后，观察小鼠体质量等一般情况；HE染色检测结肠组织病理变化；免疫组化检测结肠组织趋化因子CCL5、CXCL10的表达；酶联免疫法检测结肠组织中CCL5、CXCL10含量，并取小鼠肠内容物进行16S rRNA基因V3～V4可变区测序、物种注释及聚类，进行Alpha多样性和Beta多样性分析，线性判别式分析[linear discriminant analysis（LDA）effect size，LEfSe]筛选组间差异物种，冗余分析及Spearman等级相关系数分析肠道菌群与CCL5、CXCL10的关联性。结果 给药3 d后，模型组小鼠体质量下降（P<0.01），肠黏膜固有层中炎性细胞明显浸润且高表达CCL5和CXCL10（P<0.01），结肠组织匀浆中CCL5和CXCL10含量增加（P<0.01）；与模型组比较，各药物组小鼠体质量明显增加，炎性细胞浸润减少，CCL5、CXCL10阳性表达细胞减少，肠组织匀浆中CCL5、CXCL10含量降低，以奥司他韦组和麻杏石甘汤组差异显著（P<0.05、0.01）。菌群多样性分析和物种差异分析结果显示，给药3 d后，模型组小鼠肠道菌群组成与对照组及各药物组存在明显差异。模型组小鼠变形菌门、埃希菌属相对丰度较对照组明显增加（P<0.05），厚壁菌门、乳杆菌属、粪球菌属相对丰度明显降低（P<0.05、0.01）；与模型组比较，各药物组变形菌门、埃希菌属相对丰度降低（P<0.05），奥司他韦组和麻杏石甘汤组厚壁菌门相对丰度显著增加（P<0.01），抗病毒颗粒组颤螺旋菌属相对丰度增加（P<0.05），麻杏石甘汤组乳杆菌属、粪球菌属相对丰度增加（P<0.05、0.01）。给药7 d后，模型组小鼠结肠组织病变减轻，趋化因子表达下降，变形菌门、埃希菌属相对丰度较给药3 d后明显降低（P<0.01）；与模型组比较，各药物组疣微菌门相对丰度明显降低（P<0.05、0.01），拟杆菌门相对丰度明显升高（P<0.05）。关联性分析显示，小鼠结肠组织中CCL5、CXCL10含量与埃希菌属、克雷伯菌属、梭菌属、粪球菌属等相对丰度显著相关（P<0.05、0.01）。结论 A型流感病毒感染可引起小鼠肠道菌群结构紊乱及免疫功能失衡，麻杏石甘汤通过调节肠道菌群结构并影响趋化因子的产生，对流感病毒引起的肠道免疫损伤有一定的保护作用。

Objective To investigate the potential mechanism of Maxing Shigan Decoction (MXSGD, 麻杏石甘汤) by affecting the intestinal flora and chemokines in mice in order to prevent and treat the influenza virus infection. Methods The infected mice model of influenza A virus was established by intranasal inoculation. After 3 and 7 d of gavage administration or saline, the state of mice was observed. Colon tissue was detected by HE staining. The expressions and contents of chemokines CCL5 and CXCL10 were detected by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The bacteria in colonic contents was sequenced, annotated and clustered by using the V3—V4 variable region of 16S rRNA. The Alpha diversity, Beta diversity, and the species difference among groups were analyzed by linear discriminant analysis (LDA) effect size, LEfSe. Redundancy analysis and Spearman’s rank correlation coefficient were used for correlation analysis between intestinal flora and CCL5, CXCL10. Results Compared with the normal group, the weight of mice in the model control group was decreased (P<0.01), the inflammatory cell infiltration in the lamina propria of intestinal mucosa was obvious and the expression of CCL5 and CXCL10 was upregulated (P<0.01), the levels of CCL5 and CXCL10 in the colon tissue were both increased (P<0.01) after 3 d treatment. Compared with the model control group, the weight of mice was increased in each treatment group. Inflammatory cell infiltration, CCL5 and CXCL10 positive cells, the contents of CCL5 and CXCL10 in intestinal mucosa were all decreased, especially in oseltamivir group and MXSGD group (P<0.01, P<0.05). There were significant differences in the composition of the gut microbiota among groups according to the analysis of flora diversity and the species differences. The composition of intestinal flora in the model control group was significantly different from the normal control group and each drug group. The relative abundances of Proteobacteria and Escherichia in the model control group were significantly higher than the normal control group (P<0.05), while Firmicutes, Lactobacillus and Coprococcus were significantly lower (P<0.05). Compared with the model control group, the relative abundances of Proteobacteria and Escherichia in each drug group were significantly lower (P<0.05), the relative abundance of Firmicutes in oseltamivir group and MXSGD group were increased significantly (P<0.01), the relative abundance of Oscillosporia in antiviral granule group was increased (P<0.05), and the relative abundance of Lactobacillus and Coprococcus in MXSGD group was increased significantly (P<0.01, P<0.05). After 7 d, pathological changes of colon tissue, the expression of chemokines and the abundance of Proteobacteria and Escherichia species (P<0.01) in the model control group were all decreased. Compared with the model control group, the relative abundances of Verrucomicrobia in each drug group were significantly lower (P<0.01, P<0.05) and the relative abundance of Bacteroidetes were increased significantly (P<0.05). Correlation analysis showed that CCL5 and CXCL10 contents in the colon of mice were significantly correlated with the abundance of Escherichia, Klebsiella, Clostridium and Coprococcus (P<0.01, P<0.05). Conclusion The infection of influenza A virus can make the intestinal flora structure disorganized and immune function in mice unbalanced. MXSGD may regulate the intestinal flora structure and then affect the production of chemokines, which has a certain protective effect on the intestinal immune damage caused by influenza virus.

R285

国家自然科学基金资助项目（81774126）；国家自然科学基金资助项目（82074250）；湖南省自然科学基金项目（2020JJ4063）；湖南省教育厅创新平台开放基金项目（17K067）；湖南中医药大学中西医结合一流学科开放基金（2018ZXYJH11）；湖南中医药大学研究生创新课题立项项目（2019CX05）；湖南中医药大学研究生创新课题立项项目（2019CX22）；贵州省科技创新人才团队（黔科合平台人才[2020]5010）