[Diazepam alleviates pulmonary fibrosis in mice by inhibiting LPS-induced pyroptosis and inflammation via the let-7a-5p/MYD88 axis].

Abstract

Objective: To explore the mechanism by which diazepam alleviates lipopolysaccharide (LPS) -induced pyroptosis and inflammation to delay the progression of pulmonary fibrosis.

Methods: MRC-5 cells challenged with LPS were treated with diazepam and transfected with a let-7a-5p mimic alone or co-transfected with pc-DNA-MYD88. The changes in cellular expressions of inflammatory factors were analyzed with ELISA, and the expressions of fibrosis- and pyroptosis-related proteins were detected using Western blotting. In the animal experiment, C57BL/6 mice were randomized for treatment with LPS, LPS+diazepam, LPS+diazepam+let-7a-5p mimic, LPS+diazepam+ST2825 (a MYD88 inhibitor), or LPS+diazepam+let-7a-5p mimic+pc-DNA-MYD88, and pulmonary fibrosis and pulmonary expression of α-SMA were examined using Masson staining and immunofluorescence staining, respectively.

Results: LPS exposure of MRC-5 cells significantly downregulated let-7a-5p expression, up-regulated MYD88 expression, increased the levels of IL-4, IL-6, TGF-β and TNF- α, and enhanced the expressions of fibrosis-related proteins (Col-Ⅰ, Col-Ⅲ, and α-SMA) and pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD-N). Diazepam treatment of LPS-stimulated cells effectively inhibited the expressions of inflammation-related factors and the fibrosis- and pyroptosis-related proteins. In C57BL/6 mice, diazepam treatment obviously alleviated LPS-induced pulmonary fibrosis and reduced and pulmonary expression of α -SMA, and these effects were further enhanced by treatment with let-7a-5p mimic or ST2825, but the effect of let-7a-5p mimic was significantly attenuated by MYD88 overexpression.

Conclusion: Diazepam can negatively regulate MYD88 by upregulating the expression of let-7a-5p to inhibit LPS-induced pyroptosis and inflammatory response, thereby alleviating lung fibrosis in mice.