Ji Lili, Shi Xiaojing, Wang Gaopin, Wu Huiping, Hu Zhansheng
Medical College of Soochow University, Suzhou, Jiangsu, People's Republic of China.
Department of Emergency Medicine, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning, People's Republic of China.
Folia Histochem Cytobiol. 2023;61(1):34-46. doi: 10.5603/FHC.a2022.0032. Epub 2022 Dec 30.
Acute lung injury (ALI) is a major cause of death in sepsis patients. The Six-transmembrane protein of prostate 2 (STAMP2) is a key regulator of inflammation, while its role in septic ALI remains unclear.
Male C57BL/6 mice were subjected to cecal ligation puncture (CLP) to induce experimental sepsis whereas lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were used as the models of septic ALI in vivo and in vitro, respectively. Overexpression of STAMP2 in mouse lungs and RAW264.7 cells was performed with an adenoviral vector. We measured histological lung injury, lung wet/dry weight (W/D) ratio, and pulmonary myeloperoxidase (MPO) activity to assess lung injury extent. Cell counts in bronchoalveolar lavage fluid (BALF) were measured using Giemsa staining. The concentration of inflammatory factors was detected by enzyme-linked immunosorbent assay. The polarization of macrophages was evaluated by inducible nitric oxide synthase (iNOS) and F4/80 staining. The activation of cell apoptosis and NF-κB pathway was evaluated using Western blot, TUNEL staining, immunofluorescence, and immunohistochemistry.
Overexpression of STAMP2 alleviated CLP-induced lung injury of mice with decreased W/D ratio of the lung, and MPO activity in lung tissue. STAMP2 overexpression reduced the lung infiltration of inflammatory cells, and the levels of TNF-a, IL-6, and macrophage chemoattractant protein-1 (MCP-1) in BALF. Overexpressed STAMP2 inhibited macrophage M1 polarization in lung tissues as indicated by F4/80 and iNOS stainings in lung tissue. STAMP2 overexpression inhibited RAW 264.7 cell apoptosis by increasing Bcl-2 and decreasing Bax and cleaved-caspase 3 expression. Besides, STAMP2 overexpression suppressed nuclear factor κB (NF-κB) p65 pathway activation, as evidenced by reduced phosphorylation of IκBα, and phosphorylation and translocation of NF-κB p65. In vitro study further proved that STAMP2 overexpression suppressed the NF-κB pathway (IκBα/p65) in macrophages and decreased macrophage M1 polarization and M1-associated inflammatory factor production (TNF-a, IL-6, and MCP-1).
Our study for the first time demonstrated that STAMP2 might be able to reduce inflammation in sepsis-induced ALI by inhibiting macrophage M1 polarization through repressing NF-κB signaling activation.
急性肺损伤（ALI）是脓毒症患者死亡的主要原因。前列腺六次跨膜蛋白2（STAMP2）是炎症的关键调节因子，但其在脓毒症相关性ALI中的作用尚不清楚。
雄性C57BL/6小鼠行盲肠结扎穿孔术（CLP）以诱导实验性脓毒症，而脂多糖（LPS）刺激的RAW 264.7细胞分别用作体内和体外脓毒症相关性ALI模型。用腺病毒载体在小鼠肺组织和RAW264.7细胞中过表达STAMP2。我们通过测量肺组织学损伤、肺湿/干重（W/D）比和肺组织髓过氧化物酶（MPO）活性来评估肺损伤程度。采用吉姆萨染色法检测支气管肺泡灌洗液（BALF）中的细胞计数。通过酶联免疫吸附测定法检测炎症因子的浓度。通过诱导型一氧化氮合酶（iNOS）和F4/80染色评估巨噬细胞的极化。使用蛋白质免疫印迹法、TUNEL染色、免疫荧光和免疫组织化学法评估细胞凋亡和NF-κB途径的激活情况。
STAMP2过表达减轻了CLP诱导的小鼠肺损伤，肺W/D比和肺组织MPO活性降低。STAMP2过表达减少了炎症细胞在肺组织中的浸润，以及BALF中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和巨噬细胞趋化蛋白-1（MCP-1）的水平。如肺组织中F4/80和iNOS染色所示，过表达的STAMP2抑制了肺组织中巨噬细胞的M1极化。STAMP2过表达通过增加Bcl-2表达并降低Bax和裂解的半胱天冬酶-3的表达来抑制RAW 264.7细胞凋亡。此外，STAMP2过表达抑制了核因子κB（NF-κB）p65途径的激活，这通过IκBα磷酸化以及NF-κB p65的磷酸化和转位减少得到证实。体外研究进一步证明，STAMP2过表达抑制了巨噬细胞中的NF-κB途径（IκBα/p65），并减少了巨噬细胞的M1极化以及M1相关炎症因子的产生（TNF-α、IL-6和MCP-1）。
我们的研究首次表明，STAMP2可能通过抑制NF-κB信号激活来抑制巨噬细胞M1极化，从而减轻脓毒症诱导的ALI中的炎症反应。
