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

doi:10.12122/j.issn.1673-4254.2024.11.05

Abstract

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 over-expression. 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.

Key words: diazepam, let-7a-5p, MYD88, pyroptosis, pulmonary fibrosis

Duanyi SONG, Yun LI, Xuefang TANG, Hua LI, Kang TAO. Diazepam alleviates pulmonary fibrosis in mice by inhibiting LPS-induced pyroptosis and inflammation via the let-7a-5p/MYD88 axis[J]. Journal of Southern Medical University, 2024, 44(11): 2092-2101.

Figures/Tables 5

Tab.1 Sequences of primers for RT-qPCR

Target	Sequence
let-7a-5p	F: 5'-TGAGGTAGTAGGTTGTATAGTT-3'
	R: 5'-AGTGCAGGGTCCGAGGTATT-3'
MYD88	F: 5'-GGCTGCTCTCAACATGCGA-3'
MYD88	R: 5'-CTGTGTCCGCACGTTCAAGA-3'
U6	F: 5'-CTCGCTTCGGCAGCACA-3'
U6	R: 5'-AACGCTTCACGAATTTGCGT-3'
GAPDH	F: 5'-GAAGGTGAAGGTCGGAGTC-3'
GAPDH	R: 5'-GAAGATGGTGATGGGATTTC-3'

Fig.1 LPS induces pyroptosis and inflammation and causes pulmonary fibrosis. A: Effects of different concentrations of LPS on proliferation of MRC-5 cells. B: Detection of the levels of inflammation-related factors in MRC-5 cells by ELISA. C: Detection of the levels of inflammatory factors in mouse lung tissues by ELISA. D, E: Detection of the expressions of fibrosis- and pyroptosis-related proteins in MRC-5 cells by Western blotting. F, G: Detection of the expressions of fibrosis- and pyroptosis-related proteins in mouse lung tissues by Western blotting. H, I: Immunofluorescence staining of α-SMA in MRC-5 cells and mouse lung tissues (Scale bar=100 μm). J: Masson staining of mouse lung tissues (Scale bar=100 μm). *P<0.05, **P<0.01, ***P<0.001.

Fig.2 Verification of expressions of let-7a-5p and MYD88 in mouse lung tissues and their targeting relationship. A: Detection of let-7a-5p expression in lung tissues by RT-qPCR. B: Detection of MYD88 expression in mouse lung tissues by RT-qPCR. C: Target-binding sequences of let-7a- 5p and MYD88. D: Verification of the targeting relationship between let-7a-5p and MYD88 by dual-luciferase assay. E: Confirmation of the transfection efficiency of let-7a-5p mimic. F: Detection of the expression of MYD88 by RT-qPCR. *P<0.05, **P<0.01.

Fig.4 Diazepam inhibits LPS-induced pyroptosis and inflammatory response in MRC-5 cells through the let-7a-5p/MYD88 axis. A: Expression of let-7a-5p in MRC-5 cells. B: Expression of MYD88 in MRC-5 cells. C: Levels of inflammatory factors in MRC-5 cells detected by ELISA. D: Expressions of fibrosis-related proteins in MRC-5 cells detected by Western blotting. E: Expressions of pyroptosis-related proteins in MRC-5 cells detected by Western blotting. F: Immunofluorescence staining of α-SMA in MRC-5 cells (Scale bar=100 μm).*P<0.05, **P<0.01, ***P<0.001.

Fig.5 Diazepam inhibits LPS-induced pyroptosis and inflammatory response and alleviates pulmonary fibrosis in mice through the let-7a-5p/MYD88 axis. A, B: Expression of let-7a-5p and MYD88 in mouse lung tissues. C: Levels of inflammatory factors in mouse lung tissues detected by ELISA. D, E: Expressions of fibrosis- and pyroptosis-related proteins in mouse lung tissues detected by Western blotting. F: Immunofluorescence staining of α-SMA in mouse lung tissues (Scale bar=100 μm). G: Masson staining of mouse lung tissues (Scale bar=100 μm).*P<0.05, **P<0.01, ***P<0.001.

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