Knockdown of EPSTI1 alleviates lipopolysaccharide-induced inflammatory injury through regulation of NF-κB signaling in a cellular pneumonia model

Main Article Content

Adilijiang Kari
Zhihua M
Zhayidan Aili
Ayinuerguli Adili
Nadire Hairula
Abulaiti Abuduhaer

Keywords

epithelial-stromal interaction 1, inflammation, nuclear factor κ-light-chain-enhancer of activated B cells, pneumonia

Abstract

Background: Although early diagnosis, antibiotic therapies, corticosteroid application, and health care services are conventional managements for pneumonia, antibiotic resistance and adverse reactions remain as limitations for pneumonia treatment.


Objectives: The study attempted to evaluate the potential role of EPSTI1 against pneumonia and reveal its underlying mechanism.


Methods: Lipopolysaccharide (LPS) (5, 10, and 20 μg/mL) was applied in WI-38 cells to establish the in vitro pneumonia model. Knockdown of epithelial-stromal interaction 1 (EPSTI1) was performed by transfection with EPSTI1 siRNA (siEPSTI1) into LPS-treated cells. Cell Counting Kit-8 assays were implemented to measure cell viability, and apoptotic cells were detected using flow cytometry. Interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were quantified using enzyme-linked immunosorbent assay (ELISA). Immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to quantify EPSTI1 expression, and proteins related to nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) signaling, including p-p65, p65, p-IκBα, and IκBα.


Results: EPSTI1 was highly expressed in LPS-treated WI-38 cells. Cell apoptosis was promoted, and cell viability was inhibited after being exposed to LPS. Besides, IL-1β, IL-6, and TNF-α were dramatically upregulated. Knockdown of EPSTI1 restored cell viability, inhibited cell apoptosis, and attenuated expressions of proinflammatory factors. Additionally, knockdown of EPSTI1 visibly decreased the increased ratios of p-p65/p65 and p-IκBα/IκBα induced by LPS. Knockdown of EPSTI1 alleviated inflammatory injury through the inactivation of the NF-κB pathway.


Conclusions: These results provided promising management in preventing pneumonia in patients.

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References

1. Prina E, Ceccato A, Torres A. New aspects in the management of pneumonia. Crit care. 2016;20(1):1–9. 10.1186/s13054-016-1442-y

2. Liu F, Wen Z, Wei J, Xue H, Chen Y, Gao W, et al. Epidemiology, microbiology and treatment implications in adult patients hospitalized with pneumonia in different regions of China: a retrospective study. J Thorac Dis. 2017;9(10):3875. 10.21037/jtd.2017.09.18

3. Tramper-Stranders GA. Childhood community-acquired pneumonia: a review of etiology-and antimicrobial treatment studies. Paediatr Respir Res. 2018;26:41–8. 10.1016/j.prrv.2017.06.013

4. Nielsen HL, Rønnov-Jessen L, Villadsen R, Petersen OW. Identification of EPSTI1, a novel gene induced by epithelial–stromal interaction in human breast cancer. Genomics. 2002;79(5):703–10. 10.1006/geno.2002.6755

5. Greenaway J, Moorehead R, Shaw P, Petrik J. Epithelial–stromal interaction increases cell proliferation, survival and tumorigenicity in a mouse model of human epithelial ovarian cancer. Gynecol Oncol. 2008;108(2):385–94. 10.1016/j.ygyno.2007.10.035

6. Fan M, Arai M, Tawada A, Chiba T, Fukushima R, Uzawa K, et al. Contrasting functions of the epithelial-stromal interaction 1 gene, in human oral and lung squamous cell cancers. Oncol Rep. 2022;47(1):1–10. 10.3892/or.2021.8216

7. Barclay WW, Woodruff RD, Hall MC, Cramer SD. A system for studying epithelial-stromal interactions reveals distinct inductive abilities of stromal cells from benign prostatic hyperplasia and prostate cancer. Endocrinology. 2005;146(1):13–8. 10.1210/en.2004-1123

8. Fujino N, Ota C, Takahashi T, Suzuki T, Suzuki S, Yamada M, et al. Gene expression profiles of alveolar type II cells of chronic obstructive pulmonary disease: a case–control study. BMJ Open. 2012;2(6):e001553. 10.1136/bmjopen-2012-001553

9. Shaath H, Vishnubalaji R, Elkord E, Alajez NM. Single-cell transcriptome analysis highlights a role for neutrophils and inflammatory macrophages in the pathogenesis of severe COVID-19. Cells. 2020;9(11):2374. 10.3390/cells9112374

10. Gray J, Zhao J. Implications of epithelial–stromal interaction 1 in diseases associated with inflammatory signaling. Cell Commun Insights. 2016;(8). 10.4137/CCI.S33397

11. Sun J-l, Zhang H-Z, Liu S-Y, Lian C-F, Chen Z-l, Shao T-H, et al. Elevated EPSTI1 promote B cell hyperactivation through NF-κB signalling in patients with primary Sjögren’s syndrome. Ann Rheum Dis. 2020;79(4):518–24. 10.1136/annrheumdis-2019-216428

12. Gasparini C, Feldmann M. NF-κB as a target for modulating inflammatory responses. Curr Pharm Des. 2012;18(35):5735–45. 10.2174/138161212803530763

13. Dai J, Gu L, Su Y, Wang Q, Zhao Y, Chen X, et al. Inhibition of curcumin on influenza A virus infection and influenzal pneumonia via oxidative stress, TLR2/4, p38/JNK MAPK and NF-κB pathways. Int Immunopharmacol. 2018;54:177–87. 10.1016/j.intimp.2017.11.009

14. Zhang L, Dong L, Tang Y, Li M, Zhang M. miR-146b protects against the inflammation injury in pediatric pneumonia through MyD88/NF-κB signaling pathway. Infect Dis. 2020;52(1):23–32. 10.1080/23744235.2019.1671987

15. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25(4):402–8. 10.1006/meth.2001.1262

16. Song Q, Xu B-P, Shen K-L. Effects of bacterial and viral co-infections of mycoplasma pneumoniae pneumonia in children: analysis report from Beijing Children’s Hospital between 2010 and 2014. Int J Clin Exp Med. 2015;8(9):15666.

17. Zhang Y, Zhu Y, Gao G, Zhou Z. Knockdown XIST alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via targeting miR-370-3p/TLR4 in acute pneumonia. Cell Biochem Funct. 2019;37(5):348–58. 10.1002/cbf.3392

18. Antunes G, Evans S, Lordan J, Frew A. Systemic cytokine levels in community-acquired pneumonia and their association with disease severity. Eur Respir J. 2002;20(4):990–5. 10.1183/09031936.02.00295102

19. Vasconcellos ÂG, Clarêncio J, Andrade D, Cardoso M-RA, Barral A, Nascimento-Carvalho CM. Systemic cytokines and chemokines on admission of children hospitalized with community-acquired pneumonia. Cytokine. 2018;107:1–8. 10.1016/j.cyto.2017.11.005

20. Chen J, Li X, Huang C, Lin Y, Dai Q. Change of serum inflammatory cytokines levels in patients with chronic obstructive pulmonary disease, pneumonia and lung cancer. Technol Cancer Res Treat 2020;19:1533033820951807. 10.1177/1533033820951807

21. Frisoni P, Neri M, D’Errico S, Alfieri L, Bonuccelli D, Cingolani M, et al. Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1β, IL-6, IL-15 and TNF-α. Forensic Sci Med Pathol. 2022;18:4–19. 10.1007/s12024-021-00414-9

22. Shafiek HK, El Lateef HMA, Boraey NF, Nashat M, Abd-Elrehim GA, Abouzeid H, et al. Cytokine profile in Egyptian children and adolescents with COVID-19 pneumonia: a multicenter study. Pediatr Pulmonol. 2021;56(12):3924–33. 10.1002/ppul.25679

23. Zhang Y, Crawford HC, Pasca di Magliano M. Epithelial-stromal interactions in pancreatic cancer. Annu Rev Physiol. 2019;81:211–33. 10.1146/annurev-physiol-020518-114515

24. Kim Y-H, Lee J-R, Hahn M-J. Regulation of inflammatory gene expression in macrophages by epithelial-stromal interaction 1 (Epsti1). Biochem Biophys Res Commun. 2018;496(2):778–83. 10.1016/j.bbrc.2017.12.014

25. Li T, Lu H, Shen C, Lahiri SK, Wason MS, Mukherjee D, et al. Identification of epithelial stromal interaction 1 as a novel effector downstream of Krüppel-like factor 8 in breast cancer invasion and metastasis. Oncogene. 2014;33(39):4746–55. 10.1038/onc.2013.415