miR-139-5p protects septic mice with acute lung injury by inhibiting Toll-like receptor 4/Myeloid differentiation factor 88/Nuclear factor-&mac_kgr;B signaling pathway

Abstract

Objectives: To investigate the role of miR-139-5p and the TLR4/MyD88/NF-κB signaling pathway in acute lung injury in septic mice.

Method: A total of 140 healthy male SPF C57BL/6 mice were divided into seven groups, i.e., Normal, Control, NC, miR-139-5p mimic, miR-139-5p inhibitor, TAK-242, and miR-139-5p inhibitor+TAK-242 groups. The levels of miR-139-5p, proteins related to the TLR4/MyD88/NF-κB signaling pathway (TLR4, MyD88, and p-NF-κB p50), and MPO, SOD, GSH, and MDA in lung tissue were measured. The lung tissue wet-to-dry mass ratio (W/D), arterial oxygen partial pressure (PaO2), and carbon dioxide partial pressure (PaCO2) were measured.

Results: A web-based bioinformatic tool predicted that MyD88 was a target of miR-139-5p, which was verified by a dual luciferase reporter assay. Compared with those in the Normal group, the levels of miR-139-5p, PaO2, SOD, and GSH were significantly lower, while those of TLR4, MyD88, p-NF-κB p50, W/D, PaCO2, IL-1β, TNF-α, IL-6, MPO, and MDA were higher in all other groups. Moreover, compared with their levels in the Control group, these indicators exhibited contrasting results in the miR-139-5p mimic and TAK-242 groups, but were similar in the miR-139-5p inhibitor group. In the miR-139-5p inhibitor+TAK-242 group, acute lung injury, aggravated by miR-139-5p inhibitor, was partially rescued by TAK-242.

Conclusion: miR-139-5p inhibits the TLR4/MyD88/NF-κB signaling pathway to alleviate acute lung injury in septic mice.

Figure 1. miR-139-5p targets MyD88 gene expression. The 3'-UTR region in which miR-139-5p binds to MyD88 (A); results of the dual luciferase reporter assay (B). The asterisk (*) indicates a significant difference compared with the NC group, where p<0.05. MyD88, myeloid differentiation factor 88.

Figure 2. Expression of miR-139-5p, TLR4, MyD88, and NF-κB p50 in the lung tissue of each group. The mRNA levels of miR-139-5p, TLR4, MyD88, and NF-κB p50 in mouse lung tissue (A); The protein expression levels of TLR4, MyD88, NF-κB p50 and p-NF-κB p50 in mouse lung tissue (B-C). Significant comparisons with each group, where p<0.05, are indicated by: “*” for the Normal group, “#” for the Control group, “%” for NC group, “&” for the miR-139-5p mimic group, “$” for the miR-139-5p inhibitor group, and “@” for the TAK-242 group. TLR4, toll-like receptor 4; MyD88, myeloid differentiation factor 88.

Figure 3. The W/D, PaO₂ and PaCO₂ of mice in each group. W/D of mouse lung tissue (A); PaO₂ in mice (B); PaCO₂ in mice (C). Significant comparisons with each group, where p<0.05, are indicated by: “*” for the Normal group, “#” for the Control group, “%” for NC group, “&” for the miR-139-5p mimic group, “$” for the miR-139-5p inhibitor group, and “@” for the TAK-242 g roup PaO₂, arterial oxygen partial pressure; W/D, lung tissue wet-to-dry mass ratio; PaCO₂, carbon dioxide partial pressure.

Figure 4. Serum levels of the inflammatory factors, IL-1β, IL-6, and TNF-α in mice of each group. IL-1β levels (A); IL-6 levels (B); TNF-α levels (C). Significant comparisons with each group, where p<0.05, are indicated by: “*” for the Normal group, “#” for the Control group, “%” for NC group, “&” for the miR-139-5p mimic group, “$” for the miR-139-5p inhibitor group, and “@” for the TAK-242 group.

Figure 5. Levels of MPO (A), SOD (B), GSH (C), MDA (D) in the lung tissue of mice in each group. Significant comparisons with each group, where p<0.05, are indicated by: “*” for the Normal group, “#” for the Control group, “%” for NC group, “&” for the miR-139-5p mimic group, “$” for the miR-139-5p inhibitor group, and “@” for the TAK-242 group. MPO, myeloperoxidase; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde.