基于肠道菌群和代谢组学研究桦褐孔菌多糖对急性肺损伤小鼠的保护作用

Protective Effect of Polysaccharides from Inonotus obliquus on Acute Lung Injury in Mice Based on Gut Microbiota and Metabolomics

目的:探讨桦褐孔菌多糖(IOP)治疗脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)的保护作用。方法:40只雄性C57BL/6小鼠随机分为正常组、模型组、地塞米松组、IOP高、低剂量组,每组8只。IOP高、低剂量组分别按20、10 mg·kg^(-1)灌胃给药,正常组和模型组灌胃等体积生理盐水,地塞米松组腹腔注射磷酸地塞米松注射液30 mg·kg^(-1),连续给药21 d。构建LPS诱导的ALI小鼠模型,采用苏木素-伊红(HE)染色、免疫荧光染色和血常规检测肺组织病理损伤及血细胞含量。酶联免疫吸附测定法(ELISA)和实时荧光定量聚合酶链式反应(Real-time PCR)检测各炎症因子表达水平。16S rRNA测序和超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS)分别检测小鼠肠道菌群及血浆差异代谢物变化。结果:与模型组比较,IOP给药后明显改善ALI小鼠肺组织病变,其脾脏和胸腺指数明显升高(P<0.05,P<0.01),肺湿干重比明显下降(P<0.05,P<0.01),淋巴细胞数明显升高(P<0.05,P<0.01),中性粒细胞数显著下降(P<0.01),白细胞介素(IL)-6、IL-8、IL-1β、核转录因子-κB(NF-κB)、NOD样受体蛋白3(NLRP3)表达水平明显下降(P<0.05,P<0.01),白细胞介素-10(IL^(-1)0)水平显著增加(P<0.01)。16S rRNA测序结果显示,IOP能调节改善肠道微生物紊乱;UPLC-Q-TOF-MS结果表明,IOP治疗ALI小鼠可能涉及核苷酸糖代谢、组氨酸代谢、泛醌和其他萜类化合物-醌生物合成、淀粉和蔗糖代谢等与线粒体、糖、氨基酸代谢相关的通路。结论:IOP改善ALI小鼠肺组织病变、降低炎症反应,可能与维持肠道微生态平衡,调节线粒体电子氧化呼吸链及糖、氨基酸代谢途径,影响相关菌群代谢产物和三羧酸循环代谢产物的产生有关。

Objective:To explore the protective effect of polysaccharide from Inonotus obliquus(IOP)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.Method:A total of 40 male C57BL/6 mice were randomly divided into normal group,model group,dexamethasone group,and high-dose and low-dose IOP groups,with eight mice in each group.The high-dose and low-dose IOP groups were administered intragastrically with IOP at 20 and 10 mg·kg^(-1),respectively.The normal group and the model group were intragastrically administered with normal saline in equal volumes,and the dexamethasone group was intraperitoneally injected with dexamethasone phosphate injection of 30 mg·kg^(-1)for 21 days.An ALI mouse model induced by LPS was constructed,and hematoxylin-eosin(HE)staining,immunofluorescence staining,and blood routine were used to detect pathological damage of lung tissue and blood cell content.Enzyme-linked immunosorbent assay(ELISA)and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)were used to detect the expression levels of various inflammatory factors.Changes in gut microbiota and plasma differential metabolites in mice were detected using 16S rRNA sequencing and ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry(UPLC-Q-TOF-MS).Result:Compared with the model group,the lung tissue lesions of ALI mice were significantly improved after IOP administration,and the spleen and thymus index were dramatically increased(P<0.05,P<0.01).The ratio of wet-to-dry weight of lung tissue was sensibly decreased(P<0.05,P<0.01),and the number of lymphocytes was substantially increased(P<0.05,P<0.01).The number of neutrophils was markedly decreased(P<0.01).The expression level of interleukin-6(IL-6),interleukin-8(IL-8),interleukin-1β(IL-1β),nuclear factor-κB(NF-κB),and nucleotide-binding oligomerization domain-like receptor 3(NLRP3)decreased prominently(P<0.05,P<0.01)and the expression level of interleukin-10(IL-10)increased memorably(P<0.01).The 16S rRNA sequencing results show that IOP can regulate and improve intestinal microbial disorders.The UPLC-QTOF-MS results indicate that the treatment of ALI mice with IOP may involve pathways related to mitochondrial,sugar,and amino acid metabolism,such as nucleotide sugar metabolism,histidine metabolism,ubiquinone,and other terpenoid compound-quinone biosynthesis,as well as starch and sucrose metabolism.Conclusion:The improvement of lung tissue lesions and inflammatory response by IOP in ALI mice may be related to maintaining intestinal microbiota balance,regulating mitochondrial electron oxidation respiratory chain,as well as sugar and amino acid metabolism pathways,and affecting the production of related microbial metabolites and tricarboxylic acid cycle metabolites.