Engineered exosome-mediated delivery of MicroRNA let-7i-5p targets toll-like receptor 4 to modulate inflammation and alleviate acute liver injury in septic mice.

Abstract

Engineered exosomes have emerged as a promising platform for the targeted delivery of therapeutic nucleic acids. The microRNA let-7i-5p is recognized for its anti-inflammatory properties, but its molecular targets remain incompletely defined. To elucidate its mechanism of action and facilitate its efficient delivery, we generated let-7i-5p-enriched engineered exosomes (EEXOs) by overexpressing let-7i-5p in genetically modified RAW264.7 cells. Droplet digital polymerase chain reaction confirmed that EEXO treatment significantly increased the intracellular level of let-7i-5p (3-fold increase; p&#xa0;<&#xa0;0.001). Bioinformatic analyses performed using four microRNA target prediction databases identified toll-like receptor 4 (TLR4) as a potential target. This finding was validated by a luciferase activity assay, which revealed direct binding of let-7i-5p to the 3' untranslated region of TLR4 mRNA, resulting in its suppression (p&#xa0;<&#xa0;0.001). In lipopolysaccharide (LPS)-stimulated macrophages, EEXO-delivered let-7i-5p downregulated TLR4 and its downstream signaling components, such as myeloid differentiation primary response 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), and nuclear factor-&#x3ba;B (NF-&#x3ba;B), thereby reducing the levels of proinflammatory cytokines such as tumor necrosis factor-&#x3b1; and interleukin-6 (all p&#xa0;<&#xa0;0.05). In vivo, systemic EEXO administration attenuated LPS-induced liver injury in adult male mice, as indicated by reduced histopathological damage, liver enzyme release, and tissue edema (all p&#xa0;<&#xa0;0.05). Furthermore, EEXOs suppressed the TLR4/MyD88/TRAF6/NF-&#x3ba;B pathway and proinflammatory cytokine production in liver tissues. In conclusion, engineered exosome-delivered let-7i-5p targets TLR4 and modulates MyD88/TRAF6/NF-&#x3ba;B signaling to alleviate inflammation and protect against LPS-induced liver injury.