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.

References 37

1	Kishaba T. Evaluation and management of Idiopathic Pulmonary Fibrosis[J]. Respir Investig, 2019, 57(4): 300-11.
2	Shi J, Zhou LR, Wang XS, et al. KLF2 attenuates bleomycin-induced pulmonary fibrosis and inflammation with regulation of AP-1[J]. Biochem Biophys Res Commun, 2018, 495(1): 20-6.
3	Suzuki T, Tada, Gladson S, et al. Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition[J]. Respir Res, 2017, 18(1): 177.
4	Zhou Y, Li P, Duan JX, et al. Aucubin alleviates bleomycin-induced pulmonary fibrosis in a mouse model[J]. Inflammation, 2017, 40(6): 2062-73.
5	Cesta MF, Ryman-Rasmussen JP, Wallace DG, et al. Bacterial lipopolysaccharide enhances PDGF signaling and pulmonary fibrosis in rats exposed to carbon nanotubes[J]. Am J Respir Cell Mol Biol, 2010, 43(2): 142-51.
6	Wang DS, Zurek AA, Lecker I, et al. Memory deficits induced by inflammation are regulated by α5-subunit-containing GABAA receptors[J]. Cell Rep, 2012, 2(3): 488-96.
7	Yocum GT, Turner DL, Danielsson J, et al. GABA_A receptor α₄-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model[J]. Am J Physiol Lung Cell Mol Physiol, 2017, 313(2): L406-15.
8	Cui JJ, Zhou ZQ, Yang HY, et al. MST1 suppresses pancreatic cancer progression via ROS-induced pyroptosis[J]. Mol Cancer Res, 2019, 17(6): 1316-25.
9	Lee S, Suh GY, Ryter SW, et al. Regulation and function of the nucleotide binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 inflammasome in lung disease[J]. Am J Respir Cell Mol Biol, 2016, 54(2): 151-60.
10	Panganiban RA, Sun MY, Dahlin A, et al. A functional splice variant associated with decreased asthma risk abolishes the ability of gasdermin B to induce epithelial cell pyroptosis[J]. J Allergy Clin Immunol, 2018, 142(5): 1469-78. e2.
11	He DK, Chen JF, Shao YR, et al. Adenovirus-delivered angiopoietin-1 ameliorates phosgene-induced acute lung injury via inhibition of NLRP3 inflammasome activation[J]. Inhal Toxicol, 2018, 30(4/5): 187-94.
12	Hou L, Yang ZW, Wang ZK, et al. NLRP3/ASC-mediated alveolar macrophage pyroptosis enhances HMGB1 secretion in acute lung injury induced by cardiopulmonary bypass[J]. Lab Invest, 2018, 98(8): 1052-64.
13	Xu WJ, Wang XX, Jin JJ, et al. Inhibition of GGPPS1 attenuated LPS-induced acute lung injury and was associated with NLRP3 inflammasome suppression[J]. Am J Physiol Lung Cell Mol Physiol, 2019, 316(3): L567-L577.
14	Li ZY, Jia YF, Feng Y, et al. Methane alleviates sepsis-induced injury by inhibiting pyroptosis and apoptosis: in vivo and in vitro experiments[J]. Aging, 2019, 11(4): 1226-39.
15	Liu L, Sun BW. Neutrophil pyroptosis: new perspectives on sepsis[J]. Cell Mol Life Sci, 2019, 76(11): 2031-42.
16	Yokoyama S, Cai Y, Murata M, et al. A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death[J]. Elife, 2018, 7: e37854.
17	O’Brien J, Hayder H, Zayed Y, et al. Overview of microRNA biogenesis, mechanisms of actions, and circulation[J]. Front Endocrinol, 2018, 9: 402.
18	Ma YX, Shen N, Wicha MS, et al. The roles of the let-7 family of microRNAs in the regulation of cancer stemness[J]. Cells, 2021, 10(9): 2415.
19	Duan SY, Li JX, Tian JQ, et al. Crosstalk between let-7a-5p and BCL-xL in the Initiation of Toxic Autophagy in Lung Cancer[J]. Mol Ther Oncolytics, 2019, 15: 69-78.
20	Chen SY, Chen YL, Li PC, et al. Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury[J]. J Biomed Sci, 2024, 31(1): 30.
21	Deguine J, Barton GM. MyD88: a central player in innate immune signaling[J]. F1000Prime Rep, 2014, 6: 97.
22	Oda K, Kitano H. A comprehensive map of the toll-like receptor signaling network[J]. Mol Syst Biol, 2006, 2: 2006.0015.
23	Akira S. Toll-like receptor signaling[J]. J Biol Chem, 2003, 278(40): 38105-8.
24	Li Y, Song DY, Bo FY, et al. Diazepam inhibited lipopolysaccharide (LPS)‑induced pyroptotic cell death and alleviated pulmonary fibrosis in mice by specifically activating GABA_A receptor α4-subunit[J]. Biomed Pharmacother, 2019, 118: 109239.
25	Gu NN, Xing SP, Chen SH, et al. Lipopolysaccharide induced the proliferation of mouse lung fibroblasts by suppressing FoxO3a/p27 pathway[J]. Cell Biol Int, 2018, 42(10): 1311-20.
26	Dong ZW, Yuan YF. Juglanin suppresses fibrosis and inflammation response caused by LPS in acute lung injury[J]. Int J Mol Med, 2018, 41(6): 3353-65.
27	de Lima CB, Sakai M, Latorre AO, et al. Effects of different doses and schedules of diazepam treatment on lymphocyte parameters in rats[J]. Int Immunopharmacol, 2010, 10(11): 1335-43.
28	Nicolás FH, Cristian F, Lucía R, et al. Diazepam effects on immune cells actively involved during the development of experimental autoimmune encephalomyelitis[J]. Front Immunol, 2015, 6: 224.
29	Fernández Hurst N, Zanetti SR, Báez NS, et al. Diazepam treatment reduces inflammatory cells and mediators in the central nervous system of rats with experimental autoimmune encephalomyelitis[J]. J Neuroimmunol, 2017, 313: 145-51.
30	Sanders RD, Grover V, Goulding J, et al. Immune cell expression of GABAA receptors and the effects of diazepam on influenza infection[J]. J Neuroimmunol, 2015, 282: 97-103.
31	Huang Y, Xu W, Zhou RB. NLRP3 inflammasome activation and cell death[J]. Cell Mol Immunol, 2021, 18(9): 2114-27.
32	Song C, He LJ, Zhang J, et al. Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL-1β/IL-1R1/MyD88/NF‑κB pathway[J]. J Cell Mol Med, 2016, 20(11): 2064-77.
33	Wang T, Zhu H, Yang SF, et al. Let-7a-5p may participate in the pathogenesis of diabetic nephropathy through targeting HMGA2[J]. Mol Med Rep, 2019, 19(5): 4229-37.
34	Zhang L, Hao CF, Zhai RN, et al. Downregulation of exosomal let-7a-5p in dust exposed-workers contributes to lung cancer development[J]. Respir Res, 2018, 19(1): 235.
35	Di Padova F, Quesniaux VFJ, Ryffel B. MyD88 as a therapeutic target for inflammatory lung diseases[J]. Expert Opin Ther Targets, 2018, 22(5): 401-8.
36	Liu L, Zhou L, Wang LL, et al. MUC1 attenuates neutrophilic airway inflammation in asthma by reducing NLRP3 inflammasome-mediated pyroptosis through the inhibition of the TLR4/MyD88/NF-κB pathway[J]. Respir Res, 2023, 24(1): 255.
37	Li CC, Yu Y, Li WJ, et al. Phycocyanin attenuates pulmonary fibrosis via the TLR2-MyD88-NF‑κB signaling pathway[J]. Sci Rep, 2017, 7(1): 5843.

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