尿素气管内滴注促进肺脏炎症及肠道特定微生物群变化诱导肉鸡肺动脉高压

Inflammationin lungs and specific gut microbiota changes promoted by intratracheal instillation of urea induces pulmonary arterial hypertension in broiler chicken

18日龄AA肉鸡随机分为试验组和对照组2组,每组9只。气管滴注尿素100μL/只(1.2g/L),每3d滴注1次,共滴注3次,对照组肉鸡滴注等量的PBS溶液。在最后1次气管滴注后3d,采集肺脏进行病理形态学分析;采用免疫组化法检测内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)和Ⅱ型精氨酸酶(arginase 2,ARG2)的表达;采用Western blot法检测炎症因子的表达;采用丙二醛(malondialdehyde,MDA)检测试剂盒检测肺组织中MDA水平;采用体内基质胶试验观察尿素对内皮祖细胞(endothelial progenitor cells,EPCs)血管生成能力的影响;无菌收集空肠内容物,采用16SrRNA测序鉴定肠道菌群。结果显示,气管内滴注尿素显著促进肺血管重构和丛样病变形成,上调肺血管内皮ARG2的表达,抑制eNOS生成,促进MDA和炎症因子生成;尿素处理显著抑制EPCs血管生成能力;与对照组相比,尿素处理组肉鸡肠道中与促炎反应和氧化应激相关的菌属(Tyzzerella_3)丰度升高,与抗炎有关的产短链脂肪酸的菌属(Virvallis,Papillibacter和Barnesiella)丰度降低。结果表明,肺脏局部尿素生成增多可诱导炎症反应和氧化应激,引起肺血管内皮和EPCs功能障碍,促进肺动脉高压(pulmonary arterial hypertension,PAH)形成;肠道微生物群变化可能在PAH进程中发挥作用。

AA broilers at 18-day old were intratracheally instilled with 100μL urea(1.2g/L)per broiler for three times at 3-day intervals,while the control group received phosphate-buffered saline(PBS)treatment.Lung samples were collected 3days after the final dose for morphological analysis.Immunohistochemistry was performed to detect the expression of endothelial nitric oxide synthase(eNOS)and arginase 2(ARG2).Western blot was conducted to detect the expression of inflammatory factors.Lipid oxidation levels was determined by malondialdehyde(MDA)detection kit.The angiogenic ability of urea-treated endothelial progenitor cells(EPCs)was evaluated by in vivo Matrigel plug assay.The contents of jejunum were collected aseptically and the gut microbiota was identified by 16SrRNA sequencing.Intratracheal instillation of urea led to pulmonary vascular remodeling and plexiform lesion formation,accompanied by increased ARG2and reduced eNOS expression in the endothelium.Urea exposure also induced elevated MDA production and pro-inflammatory factor synthesis in the lung tissues.Additionally,urea-treated EPCs displayed impaired angiogenic ability.Furthermore,16SrRNA sequencing revealed alterations in gut microbiota,with an increase in Tyzzerella_3,apopulation associated with proinflammatory responses and oxidative stress,in urea-treated broilers.Conversely,the abundance of intestinal Virvallis,Papillibacter,and Barnesiella,known to produce short-chain fatty acids related to inflammation inhibition,was reduced in urea-treated broilers.Overall,these findings suggest that elevated urea levels in the lung can trigger inflammatory reactions and oxidative stress,leading to endothelial and EPC dysfunction and promoting pulmonary arterial hypertension(PAH)formation.Alterations in gut microbiota may play a role in this process.