宏基因组联合UPLC-Q-TOF-MS/MS探讨地塞米松对大鼠肺炎的作用机制

Integrated Metagenomics and UPLC-Q-TOF-MS/MS to Explore the Mechanism of Dexamethasone on Pneumonia in Rats

[目的]基于多组学技术,探讨地塞米松(Dex)能否通过调节肠道菌群及代谢物治疗肺炎。[方法]将18只Sprague-Dawley大鼠随机均分为对照(Control)组、模型(Model)组和地塞米松(Dex)组,每组6只,除Control组外,余下两组持续使用4 mg/kg的脂多糖(LPS)腹腔注射7 d建立大鼠肺炎模型。然后Dex组给予2 mg/kg的Dex灌胃治疗12 d,余下两组均连续灌胃等量的无菌PBS缓冲液12 d。第19 d时收集大鼠的肺组织、血浆、粪便和肠道内容物。苏木精-伊红(H&E)染色和Bio-plex悬浮芯片系统评价Dex的药效。宏基因组测序和超高效液相色谱串联四极杆飞行时间高分辨率质谱(UPLC-Q-TOF-MS/MS)技术分别测定大鼠的肠道菌群和代谢物。[结果]H&E染色结果显示Model组大鼠肺组织炎症细胞浸润增加,肺泡间隔增大,肺泡出血,给予Dex治疗后肺组织病变得以缓解。与Control组相比,Model组中的3种炎症因子TNF-α(P<0.0001)、IL-1α(P=0.0096)和IL-6(P<0.0001)含量升高,Dex治疗后能降低3种炎症因子水平。综上所述,Dex给药能有效的缓解肺炎大鼠的症状。宏基因组分析表明3组大鼠的肠道菌群结构改变,与Model组相比,Dex组厚壁菌门的相对丰度升高,厚壁菌门/拟杆菌门比例增加,双歧杆菌科、毛螺菌科和乳酸杆菌科积累。多变量统计分析显示Model组和Dex组极大的分离,揭示代谢轮廓改变。此外,Model组与Dex组筛选出69个(P<0.05)差异代谢物,包括Model组中上调的38个代谢物和Dex组上调的31个代谢物,这些差异代谢物主要参与了亚油酸代谢、色氨酸代谢和初级胆汁酸生物合成这3条代谢通路。[结论]我们证明了Dex对肺炎大鼠的有益作用,与此同时,宏基因组学联用UPLC-Q-TOF-MS/MS非靶标代谢组学,揭示了Dex通过调节肠道菌群和宿主代谢物治疗LPS诱导的大鼠肺炎,本研究可能为Dex治疗大鼠肺炎的作用机制提供新见解。

[Objective]Using multi-omics technology,we conducted the present study to determine whether dexa⁃methasone has therapeutic effect on pneumonia rats through the regulation of intestinal flora and metabolites.[Methods]To⁃tally 18 Sprague-Dawley rats were randomly divided into 3 groups(n=6 each):Control group,Model group and Dexa⁃methasone(Dex)group.Lipopolysaccharide(LPS)was continuously injected intraperitoneally into rats at a dose of 4 mg/kg for 7 days to induce pneumonia except the Control group.Then the Dex group was given Dex at a dose of 2 mg/kg via oral gavage for 12 days,and both the other two groups received continuously equal volume of sterile PBS buffer for 12 days.On the 19th day,lung,plasma,feces and intestinal contents of rat were collected.Hematoxylin-eosin(H&E)staining and Bio-plex suspension chip system were applied to evaluate the effect of Dex on pneumonia.Furthermore,metagenomic sequencing and UPLC-Q-TOF-MS/MS technology were employed to determine the intestinal flora and metabolites of rats,respectively.[Results]H&E staining results showed that the lung tissue of the Model group was infiltrated with inflammato⁃ry cells,the alveolar septum was increased,alveolar hemorrhage,and histological lesions were less severe in Dex group than in the model group.The levels of 3 inflammatory cytokines including TNF-α(P<0.0001),IL-1α(P=0.0096)and IL-6(P<0.0001)in the Model group were increased compared with the Control group,while Dex treatment reduced the levels of the three inflammatory factors.Taken together,Dex treatment effectively reversed the features of pneumonia in rats.Metagenomic analysis revealed that the intestinal flora structure of the three groups of rats was changed.In contrast with the Model group,an increasing level of the Firmicutes and an elevated proportion of Firmicutes/Bacteroidetes were ob⁃served after Dex treatment.Dex-treated rats possessed notably enrichment of Bifidobacterium,Lachnospiraceae and Lacto⁃bacillus.Multivariate statistical analysis showed a great separation between Model group and Dex group,indicating meta⁃bolic profile changes.In addition,69 metabolites(P<0.05)were screened,including 38 up-regulated in the Model group and 31 elevated in the Dex group,all of which were mainly involved in 3 metabolic pathways:linoleic acid metabo⁃lism,tryptophan metabolism and primary bile acid biosynthesis.[Conclusions]In summary,we demonstrate the beneficial effects of Dex on the symptoms of pneumonia.Meanwhile,integrated microbiome-metabolome analysis reveals that Dex im⁃proves LPS-induced pneumonia in rats through regulating intestinal flora and host metabolites.This study may provide new insights into the mechanism of Dex treatment of pneumonia in rats.