Wang Ruijie, Song Wenliang, Xie Chengyuan, Zhong Wenhong, Xu Hui, Zhou Qiuping, Deng Yiyu, Hong Yimei, Li Xin, Fang Ming
Department of Intensive Care Unit, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, School of Medicine, South China University of Technology, Guangzhou, People's Republic of China.
Department of Emergency and Critical Care Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.
J Inflamm Res. 2021 May 17;14:1973-1989. doi: 10.2147/JIR.S303577. eCollection 2021.
Our previous study found that urinary trypsin inhibitor (ulinastatin, UTI) protected tight junctions (TJs) of lung endothelia via TNF-α inhibition, thereby alleviating pulmonary capillary permeability in septic rats. As the activated macrophage is the main source of TNF-α in sepsis, we speculate that UTI may exert the above effects by regulating the functions of macrophages.
Bone-marrow derived macrophages (BMDM) were divided into control, lipopolysaccharide (LPS), UTI+LPS and UTI groups. TNF-α, TGF-β, IL-10, CD86, CD206 and MCP-1 expression were assessed by Western blot. The phagocytosis and migration of BMDM were detected. Pulmonary microvascular endothelial cells (PMVECs) were cultured with the conditioned medium (CM) from each group of BMDM above. Sprague-Dawley rats were divided into sham, cecal ligation and puncture (CLP), and UTI+CLP groups. Western blot and immunofluorescence were used to detected zonula occludens-1 (ZO-1), occludin and claudin-5 expression in PMVECs, as well as TNF-α, TGF-β, iNOS, CD86 and CD206 expression in lungs. Pulmonary capillary permeability was assessed by extravasated Evans blue, lung injury score (LIS), wet-to-dry weight ratio and electron microscope.
TNF-α and CD86 expression were increased in LPS-treated BMDM, but were reversed by UTI pretreatment. TGF-β, IL-10 and CD206 expression were the opposite. UTI markedly decreased phagocytosis and migration of LPS-treated BMDM. ZO-1, occludin and claudin-5 expression were markedly decreased in PMVECs of the CM-LPS group, but significantly increased in the CM-UTI+LPS group. TNF-α, iNOS and CD86 expression were increased in the lungs of CLP-rats but decreased with UTI pretreatment, while TGF-β and CD206 expression were the opposite. UTI markedly ameliorated the lung EB leakage, improved LIS, reduced the wet-to-dry ratio and revised the damaged TJs of PMVECs in CLP-rats.
UTI effectively inhibits the conversion of M1 macrophage but increases M2, reduces the phagocytosis and migration, which helps to protect endothelia TJs and reduce pulmonary capillary permeability during sepsis.
我们之前的研究发现，尿胰蛋白酶抑制剂（乌司他丁，UTI）通过抑制肿瘤坏死因子-α（TNF-α）来保护肺内皮细胞的紧密连接（TJ），从而减轻脓毒症大鼠的肺毛细血管通透性。由于活化的巨噬细胞是脓毒症中TNF-α的主要来源，我们推测UTI可能通过调节巨噬细胞的功能发挥上述作用。
将骨髓来源的巨噬细胞（BMDM）分为对照组、脂多糖（LPS）组、UTI+LPS组和UTI组。通过蛋白质免疫印迹法评估TNF-α、转化生长因子-β（TGF-β）、白细胞介素-10（IL-10）、CD86、CD206和单核细胞趋化蛋白-1（MCP-1）的表达。检测BMDM的吞噬和迁移能力。用上述每组BMDM的条件培养基（CM）培养肺微血管内皮细胞（PMVEC）。将Sprague-Dawley大鼠分为假手术组、盲肠结扎穿孔（CLP）组和UTI+CLP组。采用蛋白质免疫印迹法和免疫荧光法检测PMVEC中小带闭合蛋白-1（ZO-1）、闭合蛋白和Claudin-5的表达，以及肺组织中TNF-α、TGF-β、诱导型一氧化氮合酶（iNOS）、CD86和CD206的表达。通过伊文思蓝外渗、肺损伤评分（LIS）、湿干重比和电子显微镜评估肺毛细血管通透性。
LPS处理的BMDM中TNF-α和CD86表达增加，但UTI预处理可使其逆转。TGF-β、IL-10和CD206的表达则相反。UTI显著降低LPS处理的BMDM的吞噬和迁移能力。CM-LPS组PMVEC中ZO-1、闭合蛋白和Claudin-5表达显著降低，但CM-UTI+LPS组显著增加。CLP大鼠肺组织中TNF-α、iNOS和CD86表达增加，但UTI预处理后降低，而TGF-β和CD206表达则相反。UTI显著改善CLP大鼠肺组织伊文思蓝渗漏，改善LIS，降低湿干比，并修复PMVEC受损的TJ。
UTI有效抑制M１巨噬细胞转化但增加M2巨噬细胞，降低吞噬和迁移能力，有助于在脓毒症期间保护内皮细胞TJ并降低肺毛细血管通透性。
