Peripheral blood exosomal Hsa-miR-335-3p alleviates burn-induced acute lung injury by targeting TLR4 and the...

Due to the incessant advancements in burn treatment technology, the cure rate among patients with severe burns has significantly improved. However, acute lung injury/acute respiratory distress syndrome secondary to severe burns remains one of the challenges in the treatment of severe burns. Exosomes play a role in information and material exchange by transporting lipids, miRNA, proteins, mtDNA, and other substances to target cells. This study primarily explores the role and molecular mechanism of exosomal miRNA in peripheral blood after burn injury in acute lung injury.

From September 2021 to August 2023, data on severely burned patients treated in our hospital were collected. Based on the new global criteria for ARDS, these severely burned patients were divided into the ARDS group and the non-ARDS group. Exosomes from peripheral blood of severely burned patients collected 4-7 days post-injury were extracted using ultracentrifugation; Exosomes were identified using transmission electron microscopy, NTA, and exosome marker proteins CD63 and CD9. The Hsa-miR-335-3p in the two groups of exosomes were analyzed using RT-qPCR. After labeling exosomes with PKH 67 dye and co-culturing them with BEAS-2B cells, the uptake of exosomes by the cells was observed using confocal microscopy. The dual-luciferase gene reporter assay verified the specific binding of Hsa-miR-335-3p to the 3'-UTR region of TLR4 mRNA. The expression levels of proteins such as TLR4, Bcl-2, BAX, and C-caspase 3 were detected using Western blot. After Annexin V/PI double staining, flow cytometry was used to measure the apoptosis rate of cells in each group. TNF-α, IL-1β, and IL-6 in the cell supernatant were determined by ELISA.

A positive correlation between TBSA, III°, and BI and Hsa-miR-335-3p. A strong correlation between inhalation injury and mechanical ventilation and Hsa-miR-335-3p. Furthermore, a negative correlation between PaO2/FiO2 and Hsa-miR-335-3p. Among ARDS patients undergoing mechanical ventilation, PaO2/FiO2 still showed a negative correlation with Hsa-miR-335-3p. The results of ROC curve analysis showed that Hsa-miR-335-3p had high sensitivity and specificity (0.944 and 0.819) for the diagnosis of post-burn ARDS, with an area under the curve of 0.907. Exosomes could be uptake by BEAS-2B cells. Exosomes delivered Hsa-miR-335-3p, which inhibited the expression of TLR4 protein in BEAS-2B cells. The results of the dual-luciferase gene reporter assay suggest that Hsa-miR-335-3p specifically binds to the 3'-UTR region of TLR4 mRNA, promoting the degradation of TLR4 mRNA and inhibiting its expression. In the burn microenvironment, Hsa-miR-335-3p protects against BEAS-2B cells apoptosis and mitigates inflammatory responses. Modulating TLR4 affects Hsa-miR-335-3p in protecting against cell apoptosis and reducing inflammatory responses. Hsa-miR-335-3p protects against burn lung injury by through the PI3K/AKT/NF-κB pathway.

Hsa-miR-335-3p in peripheral blood exosomes from severely burned patients is positively correlated with the severity of burns and negatively correlated with PaO2/FiO2. It exhibits high sensitivity and specificity in the diagnosis of ARDS. Exosomes deliver Hsa-miR-335-3p to lung epithelial cells, negatively regulating the expression of TLR4. Through the PI3K/AKT/NF-κB pathway, it inhibits apoptosis and reduces inflammatory responses, thereby mitigating lung epithelial cell damage caused by severe burns.