Knockdown of PHLDA1 alleviates sepsis-induced acute lung injury by downregulating NLRP3 inflammasome activation

Main Article Content

Lijun Meng
Tijun Gu
Jinhai Wang
He Zhang
Chao Nan

Keywords

lung injury, NLRP3, PHLDA1, sepsis

Abstract

Objective: To investigate the regulatory mechanism of pleckstrin homology-like domain, family A, member 1 (PHLDA1) in sepsis-induced acute lung injury (ALI).


Method: Mice model of sepsis were established by cecal ligation and puncture (CLP). The expression of PHLDA1 was reduced by injecting short hairpin RNA (shRNA)–PHLDA1 into the tail vein. The levels of PHLDA1, pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1β, IL-18, super-oxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), molecular mechanism related to pyroptosis, such as caspase 1, adaptor apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin D (GSDMD)-N, and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) were tested by Western blot analysis, quantitative real-time polymerase chain reaction, and enzyme-linked-immunosorbent serologic assay. Pathological changes in lung tissues were examined by hematoxylin and eosin staining. Wet–dry weight ratio of lung tissues was observed.


Results: The expression of PHLDA1 was up-regulated in lung tissues from CLP-induced septic mice. Knockdown of PHLDA1 could reduce lung injury and wet–dry weight ratio in mice with sepsis-induced ALI. Moreover, silencing of PHLDA1 decreased the expressions of IL-1β, TNF-α, IL-18, IL-6, and MDA but increased SOD and GSH expressions in CLP-induced septic mice. The expressions of NLRP3, GSDMD-N, ASC, and caspase 1 were decreased by PHLDA1 silencing.


Conclusion: Knockdown of PHLDA1 inhibited lung inflammation and pyroptosis in mice with sepsis-induced ALI by down-regulating NLRP3.

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References

1. Napolitano LM. Sepsis 2018: Definitions and guideline changes. Surg Inf. 2018;19(2):117–25. 10.1089/sur.2017.278

2. Li W, Li D, Chen Y, Abudou H, Wang H, Cai J, et al. Classic signaling pathways in alveolar injury and repair involved in sepsis-induced ALI/ARDS: New research progress and prospect. Dis Markers. 2022;2022:6362344. 10.1155/2022/6362344

3. Wang L, Hauenstein AV. The NLRP3 inflammasome: Mechanism of action, role in disease and therapies. Mol Aspects Med. 2020;76:100889. 10.1016/j.mam.2020.100889

4. Coll RC, Schroder K, Pelegrín P. NLRP3 and pyroptosis blockers for treating inflammatory diseases. Trends Pharmacol Sci. 2022;43(8):653–68. 10.1016/j.tips.2022.04.003

5. Shi X, Li T, Liu Y, Yin L, Xiao L, Fu L, et al. HSF1 protects sepsis-induced acute lung injury by inhibiting NLRP3-inflammasome activation. Front Immunol. 2022;13:781003. 10.3389/fimmu.2022.781003

6. Yu C, Chen P, Miao L, Di G. The role of the NLRP3 inflammasome and programmed cell death in acute liver injury. Int J Mol Sci. 2023;24(4):3067. 10.3390/ijms24043067

7. Li N, Xiong R, He R, Liu B, Wang B, Geng Q. Mangiferin mitigates lipopolysaccharide-induced lung injury by inhibiting NLRP3 inflammasome activation. J Inflam Res. 2021;14:2289–300. 10.2147/JIR.S304492

8. Chen Y, Takikawa M, Tsutsumi S, Yamaguchi Y, Okabe A, Shimada M, et al. PHLDA1, another PHLDA family protein that inhibits Akt. Cancer Sci. 2018;109(11):3532–42. 10.1111/cas.13796

9. Peng H, Wang J, Song X, Huang J, Hua H, Wang F, et al. PHLDA1 suppresses TLR4-triggered proinflammatory cytokine production by interaction with tollip. Front Immunol. 2022;13:731500. 10.3389/fimmu.2022.731500

10. Wang J, Yao N, Hu Y, Lei M, Wang M, Yang L, et al. PHLDA1 promotes glioblastoma cell growth via sustaining the activation state of Ras. Cell Mol Life Sci (CMLS). 2022;79(10):520. 10.1007/s00018-022-04538-1

11. Jiao HW, Jia XX, Zhao TJ, Rong H, Zhang JN, Cheng Y, et al. Up-regulation of TDAG51 is a dependent factor of LPS-induced RAW264.7 macrophages proliferation and cell cycle progression. Immunopharmacol Immunotoxicol. 2016;38(2):124–30. 10.3109/08923973.2016.1138968

12. Wang S, Zhang H, Wang A, Huang D, Fan J, Lu L, et al. PHLDA1 promotes lung contusion by regulating the toll-like receptor 2 signaling pathway. Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol. 2016;40(5):1198–206. 10.1159/000453173

13. Zhao H, Liu Y, Chen N, Yu H, Liu S, Qian M, et al. PHLDA1 blockade alleviates cerebral ischemia/reperfusion injury by affecting microglial M1/M2 polarization and NLRP3 inflammasome activation. Neuroscience. 2022;487:66–77. 10.1016/j.neuroscience.2022.01.018

14. Drechsler S, Osuchowski M. Cecal ligation and puncture. Methods Mol Biol (Clifton, NJ). 2021;2321:1–8. 10.1007/978-1-0716-1488-4_1

15. Silva IAN, Gvazava N, Bölükbas DA, Stenlo M, Dong J, Hyllen S, et al. A semi-quantitative scoring system for green histopathological evaluation of large animal models of acute lung injury. Bio-protocol. 2022;12(16):e4493. 10.21769/BioProtoc.4493

16. Rahmati M, Shariatzadeh Joneydi M, Koyanagi A, Yang G, Ji B, Won Lee S, et al. Resistance training restores skeletal muscle atrophy and satellite cell content in an animal model of Alzheimer’s disease. Sci Rep. 2023;13(1):2535. 10.1038/s41598-023-29406-1

17. Panahzadeh F, Mirnasuri R, Rahmati M. Exercise and syzygium aromaticum reverse memory deficits, apoptosis and mitochondrial dysfunction of the hippocampus in Alzheimer’s disease. J Ethnopharmacol. 2022;286:114871. 10.1016/j.jep.2021.114871

18. Wang S, Chen Y. Deoxyelephantopin alleviates lipopolysaccharide-induced septic lung injury through inhibiting NF-ĸB/STAT3 axis. Allergol Immunopathol. 2022;50(5):39–46. 10.15586/aei.v50i5.626

19. Nagai MA. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and cancer. Biomed Rep. 2016;4(3):275–81. 10.3892/br.2016.580

20. Nagai MA, Fregnani JH, Netto MM, Brentani MM, Soares FA. Down-regulation of PHLDA1 gene expression is associated with breast cancer progression. Breast Cancer Res Treat. 2007;106(1):49–56. 10.1007/s10549-006-9475-6

21. Zhao P, Lu Y, Liu L. Correlation of decreased expression of PHLDA1 protein with malignant phenotype of gastric adenocarcinoma. Int J Clin Exp Pathol. 2015;8(5):5230–5.

22. Sakthianandeswaren A, Christie M, D’Andreti C, Tsui C, Jorissen RN, Li S, et al. PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis. Cancer Res. 2011;71(10):3709–19. 10.1158/0008-5472.CAN-10-2342

23. Xu J, Bi G, Luo Q, Liu Y, Liu T, Li L, et al. PHLDA1 modulates the endoplasmic reticulum stress response and is required for resistance to oxidative stress-induced cell death in human ovarian cancer cells. J Cancer. 2021;12(18):5486–93. 10.7150/jca.45262

24. Baldavira CM, Machado-Rugolo J, Prieto TG, Bastos DR, Balancin M, Ab’Saber AM, et al. The expression patterns and prognostic significance of pleckstrin homology-like domain family A (PHLDA) in lung cancer and malignant mesothelioma. J Thorac Dis. 2021;13(2):689–707. 10.21037/jtd-20-2909

25. Gong M, Liang W, Lu Q, Zhang J. PHLDA1 knockdown inhibits inflammation and oxidative stress by regulating JNK/ERK pathway, and plays a protective role in sepsis-induced acute kidney injury. Allergol Immunopathol. 2022;50(6):1–9. 10.15586/aei.v50i6.671

26. Mangan MSJ, Olhava EJ, Roush WR, Seidel HM, Glick GD, Latz E. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov. 2018;17(8):588–606. 10.1038/nrd.2018.97

27. Blevins HM, Xu Y, Biby S, Zhang S. The NLRP3 inflammasome pathway: A review of mechanisms and inhibitors for the treatment of inflammatory diseases. Front Aging Neurosci. 2022;14:879021. 10.3389/fnagi.2022.879021

28. Li J, Xu Z, Ou Yang C, Wu X, Xie Y, Xie J. Protopine alleviates lipopolysaccharide-triggered intestinal epithelial cell injury through retarding the NLRP3 and NF-κB signaling pathways to reduce inflammation and oxidative stress. Allergol Immunopathol. 2022;50(6):84–92. 10.15586/aei.v50i6.669

29. Zhang S, Lai Q, Liu L, Yang Y, Wang J. Morroniside alleviates lipopolysaccharide-induced inflammatory and oxidative stress in inflammatory bowel disease by inhibiting NLRP3 and NF-κB signaling pathways. Allergol Immunopathol. 2022;50(6):93–9. 10.15586/aei.v50i6.674

30. Li Q, Feng H, Wang H, Wang Y, Mou W, Xu G, et al. Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interaction. EMBO Rep. 2022;23(2):e53499. 10.15252/embr.202153499

31. Zhong C, Wang R, Hua M, Zhang C, Han F, Xu M, et al. NLRP3 inflammasome promotes the progression of acute myeloid leukemia via IL-1β pathway. Front Immunol. 2021;12:661939. 10.3389/fimmu.2021.661939

32. Kong R, Sun L, Li H, Wang D. The role of NLRP3 inflammasome in the pathogenesis of rheumatic disease. Autoimmunity. 2022;55(1):1–7. 10.1080/08916934.2021.1995860

33. Shi X, Tan S, Tan S. NLRP3 inflammasome in sepsis (review). Mol Med Rep. 2021;24(1):514. 10.3892/mmr.2021.12153

34. Busch K, Kny M, Huang N, Klassert TE, Stock M, Hahn A, et al. Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy. J Cachexia Sarcopenia Muscle (JCSM). 2021;12(6):1653–68. 10.1002/jcsm.12763

35. Borges-Rodriguez M, Shields CA, Travis OK, Tramel RW, Baik CH, Giachelli CA, et al. Platelet inhibition prevents NLRP3 inflammasome activation and sepsis-induced kidney injury. Int J Mol Sci. 2021;22(19):10330. 10.3390/ijms221910330

36. Li T, Sun H, Li Y, Su L, Jiang J, Liu Y, et al. Downregulation of macrophage migration inhibitory factor attenuates NLRP3 inflammasome mediated pyroptosis in sepsis-induced AKI. Cell Death Discov. 2022;8(1):61. 10.1038/s41420-022-00859-z