Fig. 6: Exosomal APN promotes the lung tissue injury and inflammatory response in CLP model mice. | Communications Biology

Fig. 6: Exosomal APN promotes the lung tissue injury and inflammatory response in CLP model mice.

From: Macrophage-derived exosomal aminopeptidase N aggravates sepsis-induced acute lung injury by regulating necroptosis of lung epithelial cell

Fig. 6

The mice were subjected to sham or CLP and injected with LPS-exoshNC or LPS-exoshAPN (100 μg/mouse) or control PBS (same volume) immediately after CLP surgery. CLP + ExoshNC CLP mice were injected with LPS-exoshNC, CLP + ExoshAPN CLP mice were injected with LPS-exoshAPN. a Fluorescent images showed the accumulation of PKH67-labeled LPS-exo in the lung tissue of septic mice 6 h after injection. Scale bars, 50 µm. b Representative immunofluorescence images of APN (red) staining in lung tissues (n = 5). Scale bars, 75 µm. The mice were euthanized 24 h after injection. c RT-PCR analysis of APN in the lung of LPS-exoshAPN or LPS-exoshNC-treated CLP mice. d Kaplan–Meier survival analysis of LPS-exoshAPN or LPS-exoshNC-treated mice subjected to CLP as described in Materials and Methods for a 96 h survival study (n = 10, **P < 0.01 CLP + ExoshNC vs. CLP, #P < 0.05 CLP + ExoshNC vs. CLP + ExoshAPN) . e ELISA was used to measure the levels of pro-inflammatory cytokines IL-6, TNF-α and IL-1β in BALF. CLP + ExoshNC group showed significant upregulation of IL-6, TNF-α and IL-1β, while shAPN reversed the upregulation significantly (n = 5). f The H&E staining was used to detect lung pathology, (g) lung injury score was evaluated due to the pathology, and the levels of water content in the lung tissues were analyzed using the W/D method. h Lung mitochondrial structures were observed using an electron microscope (n = 5), Scale bars: 1 µm. i A bioluminescence-based assay was used to determine the ATP content of lung homogenates. The results are given in nmoles of ATP produced per gram of wet tissue. j Representative immunohistochemical images of TLR4, as well as p-p65, RIPK1, and RIPK3 in the lung tissue lesions of the Exo-treated CLP mice and the control groups (n = 5). Scale bars: 50 μm. k Western blotting analysis of the expression of necroptosis-related protein p-MLKL, MLKL, p-RIP1 and RIP1 in mice lung tissues. All results are based on three measurements. The data are presented as the mean ± SD (n = 3), *P < 0.05, **P < 0.01.

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