Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, PR China; Department of Infectious Diseases,The People's Hospital of Jiulongpo District, Chongqing,China.
Department of Gastroenterology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, PR China.
Int Immunopharmacol. 2024 Oct 25;140:112835. doi: 10.1016/j.intimp.2024.112835. Epub 2024 Jul 31.
In recent years, researchers have focused on studying the mechanism of sepsis-induced immunosuppression, but there is still a lack of suitable animal models that accurately reflect the process of sepsis-induced immunosuppression. The aim of this study was to evaluate the immune status at various stages in a model of sepsis-induced secondary pneumonia and to demonstrate whether pyroptosis is one of the modes of immune cell death in sepsis. Firstly, we established a sepsis model in C57BL/6J mice using cecal ligation and puncture (CLP). The surviving mice were treated with a 40 μL suspension of P.aeruginosa (Pa) under anesthesia on day 4 post-CLP to establish a sepsis-induced secondary pneumonia model. Secondly, routine blood tests, serum ALT and PCT levels, gross lung specimens, and H&E staining of the lung and liver tissues were used to assess the successful establishment of this model. Serum levels of TNF-α and IL-6, the CD4+/CD8+ratio in blood, H&E staining of the spleen, and immunohistochemistry of CD4 and CD8 in the spleen were detected to evaluate the immune status of the model mice. Finally, the expression levels of pyroptosis-related proteins in the spleen were detected by Western blot. The expression of GSDMD was assessed using immunohistochemistry, and pyroptosis was directly observed through transmission electron microscopy. The experimental results above confirmed the successful construction of the model for sepsis-induced secondary pneumonia, demonstrating its ability to reflect sepsis-induced immunosuppression. Moreover, the expression of pyroptosis-related proteins, immunohistochemical GSDMD, and transmission electron microscopy of the spleen showed that pyroptosis was one of the modes of immune cell death in sepsis.
近年来，研究人员专注于研究脓毒症引起的免疫抑制的机制，但仍然缺乏能够准确反映脓毒症引起的免疫抑制过程的合适动物模型。本研究旨在评估脓毒症继发肺炎模型中不同阶段的免疫状态，并证明细胞焦亡是否是脓毒症中免疫细胞死亡的模式之一。首先，我们使用盲肠结扎和穿孔术（CLP）在 C57BL/6J 小鼠中建立脓毒症模型。在 CLP 后第 4 天，在麻醉下用 40μL 铜绿假单胞菌（Pa）悬浮液处理存活的小鼠，建立脓毒症继发肺炎模型。其次，通过常规血液检查、血清 ALT 和 PCT 水平、大体肺标本以及肺和肝组织的 H&E 染色来评估该模型的成功建立。检测血清 TNF-α和 IL-6 水平、血液中 CD4+/CD8+比值、脾的 H&E 染色以及脾中 CD4 和 CD8 的免疫组化，评估模型小鼠的免疫状态。最后，通过 Western blot 检测脾中细胞焦亡相关蛋白的表达水平。用免疫组化评估 GSDMD 的表达，并通过透射电子显微镜直接观察细胞焦亡。上述实验结果证实了脓毒症继发肺炎模型的成功构建，表明其能够反映脓毒症引起的免疫抑制。此外，脾中细胞焦亡相关蛋白的表达、免疫组化 GSDMD 和透射电子显微镜显示，细胞焦亡是脓毒症中免疫细胞死亡的模式之一。
