A型流感病毒对小鼠肺部菌群及趋化因子CCL5和CXCL10的影响及麻杏石甘汤干预作用研究

目的 探索麻杏石甘汤（Maxing Shigan Decoction，MSD）通过影响小鼠肺部菌群及趋化因子CCL5和CXCL10表达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给药或生理盐水3、7 d后，计算肺指数和肺指数抑制率，HE染色检测肺组织病理变化，应用免疫组化、酶联免疫吸附试验（ELISA）、实时荧光定量PCR（RT-PCR）法检测肺组织中CCL5和CXCL10蛋白和mRNA表达变化，并取小鼠肺组织进行16S rRNA基因V3~V4可变区测序、物种注释及聚类，分析Alpha多样性和Beta多样性及组间差异物种，分析CCL5、CXCL10表达与肺部菌群变化的关系。结果 给药3 d后，模型组肺指数明显高于对照组（P<0.01）和药物组（P<0.05、0.01），肺部炎性细胞浸润明显。麻杏石甘汤组肺部炎性细胞浸润明显减轻，肺指数抑制率与奥司他韦组相似，肺损伤的组织学定量评价指标（IQA）值较模型组明显降低（P<0.01）。模型组CCL5和CXCL10表达明显高于对照组（P<0.01），奥司他韦组和麻杏石甘汤组CCL5和CXCL10表达明显低于模型组（P<0.05、0.01）。16S rRNA基因测序结果显示，模型组拟杆菌属、埃希菌属、变形杆菌属相对丰度增加，粪球菌属相对丰度降低。奥司他韦和麻杏石甘汤能显著降低拟杆菌属、埃希菌属、变形杆菌属相对丰度，增加粪球菌属相对丰度；Alpha多样性分析结果显示各组ACE指数、Chao1指数、Shannon指数比较P>0.05，组间丰富度和多样性无差异。Beta多样性分析结果显示，给药3 d后，组间样品点无交集，组间肺菌群构成有差异。各组间有显著性差异物种。Spearman相关性分析显示，CCL5和CXCL10表达与埃希菌属、变形杆菌属、拟杆菌属丰度呈正相关，与粪球菌属丰度呈负相关。给药7 d后，各组间肺部菌群结构组成及CCL5、CXCL10表达无显著差异。结论 麻杏石甘汤可能通过促进肺部有益菌生长改善肺部微生态环境及免疫微环境，对流感病毒引起的肺损伤有一定的保护作用。

Effect of influenza A virus infection on pulmonary flora and chemokines CCL5 and CXCL10 in mice and intervention of Maxing Shigan Decoction

Objective To investigate the potential mechanism of Maxing Shigan Decoction (MSD) in the prevention and treatment of influenza virus infection by influencing pulmonary flora and expression of chemokines CCL5 and CXCL10 of mice. Method The infected mice model of influenza A virus was tested by intranasal inoculation. After 3 and 7 d of gavage or saline, the lung index and lung index inhibition rate were calculated. Pathological changes of lung tissue were detected by HE staining. The expression of CCL5 and CXCL10 in the lung tissue of mice was detected by immunohistochemistry and ELISA. The expression of CCL5 mRNA and CXCL10 mRNA in lung tissue of mice was detected by real-time fluorescence quantitative PCR (RT-PCR). The bacteria in lung tissue was sequenced by using the V3-V4 variable region of 16S rRNA, annotated and clustered. The alpha diversity, beta diversity and the species difference among groups were analyzed. The correlation of the expression of CCL5 and CXCL10 with the change of intestinal flora was also analyzed. Results After 3 d of administration, the lung index of model group was significantly higher than normal group (P<0.01) and drug group (P<0.05, 0.01). Pulmonary inflammatory cell infiltration was obvious. The infiltration of pulmonary inflammatory cells in MSD group was significantly reduced, and the inhibition rate of lung index was similar to that in oseltamivir group. The value of IQA in lung injury was decreased significantly (P<0.01). The expressions of CCL5 and CXCL10 in the lung tissue of the model control group were significantly higher than those of the normal control group (P<0.01), and the expressions of CCL5 and CXCL10 in the oseltamivir group and the MSD were significantly lower than those in the model control group (P<0.05, 0.01). The results of 16S rRNA gene sequencing showed the relative abundances of Bacteroides, Escherichia, and Proteus were increased, while that of Coprococcus was decreased in the model control group. In oseltamivir group and MSD group, the relative abundances of Bacteroides, Escherichia, and Proteus were significantly decreased, while the relative abundance of Coprococcus was increased. The results of alpha diversity showed that the ace index, Chao1 index, and Shannon index of each group were all higher than 0.05, and there was no difference in richness and diversity among groups. The results of beta diversity showed that there was no intersection of sample points among groups and difference in the composition of pulmonary flora among groups. Species among groups were significant differences. Spearman correlation analysis showed that the expression of CCL5 and CXCL10 was positively correlated with the abundance of Escherichia, Proteus, and Bacteroides, and negatively correlated with the abundance of Coprococcus. After 7 d of administration, there was no significant difference in the composition of pulmonary flora and the expression of CCL5 and CXCL10. Conclusion MSD may improve the micro-ecological environment and immune microenvironment of the lung by promoting the growth of beneficial bacteria, and has a certain protective effect on the lung injury caused by influenza virus.