Neferine inhibits LPS-ATP-induced endothelial cell pyroptosis via regulation of ROS/NLRP3/Caspase-1 signaling pathway

  • Yang-Shuo Tang
  • Yan-Hua Zhao
  • Yong Zhong
  • Xiao-Zhao Li
  • Jia-Xi Pu
  • Yan-Cheng Luo
  • Qiao-Ling ZhouEmail author
Original Research Paper



Oxidative stress-induced endothelial dysfunction and pyroptosis play an important role during chronic kidney disease (CKD) progression. Neferine, which is an alkaloid ingredient from the lotus seed embryo, has many biological actions such as anti-inflammatory, anticancer and antioxidant. However, the role of neferine in endothelial cell pyroptosis and the involved mechanism remain obscure. The aim is to probe the protective effects of neferine on cell pyroptosis and the involved underlying mechanism.


After the HUVECs were primed with neferine treatment for 2 h prior to LPS and ATP exposure for 24 h, the cell proliferation was determined by BrdU; the cell LDH release was detected by LDH kits; the levels of intracellular ROS, MDA and SOD were tested by detection kits; Caspase-1 activity kit was used to determine caspase-1 activity; the contents of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD were tested by RT-PCR and western blot.


We found that neferine could inhibit LPS-ATP-induced oxidative stress and the activation of NLRP3 inflammasome signaling, and increased the endothelial cell viability and SOD production. siRNA which mediated the knockdown of NLRP3 promoted the neferine-induced inhibition effects of cell pyroptosis. Furthermore, these neferine-induced effects were reversed by the over-expression of NLRP3.


Our findings indicated neferine may reduce ROS by anti-oxidation and inhibit LPS-ATP-induced endothelial cell pyroptosis via blocking ROS/NLRP3/Caspase-1 signaling pathway, which provides the evidence for therapeutic effect in CKD.


Neferine Cell pyroptosis NLRP3 inflammasome Chronic kidney disease (CKD) ROS/NLRP3/Caspase-1 signaling pathway 



We would like to give our sincere gratitude to the reviewers for their constructive comments.


This work was supported by grants from the National Natural Science Foundation of China [no. 81470933].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All the procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study. We would like to give our sincere gratitude to the reviewers for their constructive comments.

Supplementary material

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Supplementary material 1 (TIFF 1526 kb)
11_2019_1256_MOESM2_ESM.docx (16 kb)
Supplementary material 2 (DOCX 15 kb)


  1. 1.
    Levey AS, Coresh J. Chronic kidney disease. Lancet. 2012;379:165–80.CrossRefGoogle Scholar
  2. 2.
    Madan P, Kalra OP, Agarwal S, Tandon OP. Cognitive impairment in chronic kidney disease. J Am Geriatr Soc. 2014;36:116–7.Google Scholar
  3. 3.
    Perlman RL, Finkelstein FO, Liu L, Roys E, Kiser M, Eisele G, et al. Quality of life in chronic kidney disease (CKD): a cross-sectional analysis in the Renal Research Institute-CKD study. Am J Kidney Dis. 2005;45:658–66.CrossRefGoogle Scholar
  4. 4.
    Popolo A, Autore G, Pinto A, Marzocco S. Oxidative stress in patients with cardiovascular disease and chronic renal failure. Free Radical Res. 2013;47:346–56.CrossRefGoogle Scholar
  5. 5.
    Agarwal R, Vasavada N, Sachs NG, Chase S. Oxidative stress and renal injury with intravenous iron in patients with chronic kidney disease. Kidney Int. 2004;65:2279–89.CrossRefGoogle Scholar
  6. 6.
    Basile DP. The endothelial cell in ischemic acute kidney injury: implications for acute and chronic function. Kidney Int. 2007;72:151–6.CrossRefGoogle Scholar
  7. 7.
    Olgaard K, Lewin E. Use (or misuse) of vitamin D treatment in CKD and dialysis patients: a recent meta-analysis on vitamin D compounds in chronic kidney disease [1] and an editorial comment [2] accompanying this meta-analysis have already been published. We believe that these. Nephrol Dial Transpl. 2008;23:1789–93.CrossRefGoogle Scholar
  8. 8.
    Vecchio LD, Longhi S, Locatelli F. Safety concerns about intravenous iron therapy in patients with chronic kidney disease. Clin Kidney J. 2016;9:260–7.CrossRefGoogle Scholar
  9. 9.
    Grima DT, Dunn ES, Bernard LM, Mendelssohn DC. Impact of sevelamer versus calcium-based binders on hospitalizations and missed in-center dialysis treatments among CKD patients on dialysis: a modeled analysis. Curr Med Res Opin. 2013;29:109–15.CrossRefGoogle Scholar
  10. 10.
    Impellizzeri D, Esposito E, Attley J, Cuzzocrea S. Targeting inflammation: new therapeutic approaches in chronic kidney disease (CKD). Pharmacol Res. 2014;81:91–102.CrossRefGoogle Scholar
  11. 11.
    Guo H, Callaway JB, Ting JP. Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat Med. 2015;21:677–87.CrossRefGoogle Scholar
  12. 12.
    Chow MTC. Danger signal receptors and cancer: NLRP3 inflammasome and Toll-like receptor 3. 2013.Google Scholar
  13. 13.
    Granata S, Masola V, Zoratti E, Scupoli MT, Baruzzi A, Messa M, et al. NLRP3 inflammasome activation in dialyzed chronic kidney disease patients. PLoS One. 2015;10:e0122272.CrossRefGoogle Scholar
  14. 14.
    Wang S, Li Y, Fan J, Zhang X, Luan J, Bian Q, et al. Interleukin-22 ameliorated renal injury and fibrosis in diabetic nephropathy through inhibition of NLRP3 inflammasome activation. Cell Death Dis. 2017;8:e2937.CrossRefGoogle Scholar
  15. 15.
    Hutton HL, Ooi JD, Holdsworth SR, Kitching AR. The NLRP3 inflammasome in kidney disease and autoimmunity. Nephrology. 2016;21:736–44.CrossRefGoogle Scholar
  16. 16.
    Su WJ, Zhang Y, Chen Y, Gong H, Lian YJ, Peng W, et al. NLRP3 gene knockout blocks NF-κB and MAPK signaling pathway in CUMS-induced depression mouse model. Behav Brain Res. 2017;322:1–8.CrossRefGoogle Scholar
  17. 17.
    Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A, et al. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat Immunol. 2010;11:1136–42.CrossRefGoogle Scholar
  18. 18.
    Miao EA, Rajan JV, Aderem A. Caspase-1-induced pyroptotic cell death. Immunol Rev. 2011;243:206–14.CrossRefGoogle Scholar
  19. 19.
    Zhang Z, Shao X, Jiang N, Mou S, Gu L, Li S, et al. Caspase-11-mediated tubular epithelial pyroptosis underlies contrast-induced acute kidney injury. Cell Death Dis. 2018;9:983.CrossRefGoogle Scholar
  20. 20.
    Garg JP, Vucic D. Targeting cell death pathways for therapeutic intervention in kidney diseases. Semin Nephrol. 2016;36:153–61.CrossRefGoogle Scholar
  21. 21.
    Darisipudi MN, Knauf F. An update on the role of the inflammasomes in the pathogenesis of kidney diseases. Pediatr Nephrol. 2016;31:1–10.CrossRefGoogle Scholar
  22. 22.
    Forestoneto O, Ávila VF, Arias S, Zambom F, Rempel L, Faustino VD, et al. NLRP3 inflammasome inhibition ameliorates tubulointerstitial injury in the remnant kidney model. Lab Invest. 2018;98:1.CrossRefGoogle Scholar
  23. 23.
    Chen H, Lu Y, Cao Z, Ma Q, Pi H, Fang Y, et al. Cadmium induces NLRP3 inflammasome-dependent pyroptosis in vascular endothelial cells. Toxicol Lett. 2016;246:7–16.CrossRefGoogle Scholar
  24. 24.
    Kadioglu O, Law B, Mok S, Xu SW, Efferth T, Wong V. Mode of action analyses of neferine, a bisbenzylisoquinoline alkaloid of lotus (Nelumbo nucifera) against multidrug-resistant tumor cells. Front Pharmacol. 2017;8:238.CrossRefGoogle Scholar
  25. 25.
    Marthandam AS, Mariappan R, Muthusamy S, Velmurugan BK. Pharmacological benefits of neferine—a comprehensive review. Life Sci. 2018;199:60–70.CrossRefGoogle Scholar
  26. 26.
    Zhao L, Wang X, Chang Q, Xu J, Ying H, Guo Q, et al. neferine, a bisbenzylisoquinline alkaloid attenuates bleomycin-induced pulmonary fibrosis. Eur J Pharmacol. 2010;627:304–12.CrossRefGoogle Scholar
  27. 27.
    Abais JM, Xia M, Zhang Y, Boini KM, Li PL. Redox regulation of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid Redox Signal. 2015;22:1111.CrossRefGoogle Scholar
  28. 28.
    Zhang X, Liu Z, Xu B, Sun Z, Gong Y, Shao C. Neferine, an alkaloid ingredient in lotus seed embryo, inhibits proliferation of human osteosarcoma cells by promoting p38 MAPK-mediated p21 stabilization. Eur J Pharmacol. 2012;677:47–54.CrossRefGoogle Scholar
  29. 29.
    Pan Y, Cai B, Wang K, Wang S, Zhou S, Yu X, et al. Neferine enhances insulin sensitivity in insulin resistant rats. J Ethnopharmacol. 2009;124:98–102.CrossRefGoogle Scholar
  30. 30.
    Liu XD, Li H, Wang CZ, Zhao HD, Xiao P, Nephrology DO, et al. Mechanism of neferine in antioxidant stress. China J Mod Med; 2018.Google Scholar
  31. 31.
    Yoon JS, Kim HM, Yadunandam AK, Kim NH, Jung HA, Choi JS, et al. Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: molecular mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response. Phytomedicine. 2013;20:1013–22.CrossRefGoogle Scholar
  32. 32.
    Lalitha G, Poornima P, Archanah A, et al. Protective effect of neferine against isoproterenol-induced cardiac; toxicity. Cardiovasc Toxicol. 2013;13:168–79.CrossRefGoogle Scholar
  33. 33.
    Li G, Gao Y, Xu H, et al. Neferine inhibits the upregulation of CCL5 and CCR1 in vascular; endothelial cells during chronic high glucose treatment. Inflammation. 2013;36:300–8.CrossRefGoogle Scholar
  34. 34.
    De ND, Latz E. NLRP3 inflammasomes link inflammation and metabolic disease. Trends Immunol. 2011;32:373–9.CrossRefGoogle Scholar
  35. 35.
    Takahashi M. NLRP3 inflammasome as a novel player in myocardial infarction. Int Heart J. 2014;55:101–5.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of NephrologyXiangya Hospital, Central South UniversityChangshaPeople’s Republic of China
  2. 2.Department of ObstetricsXiangya Hospital, Central South UniversityChangshaPeople’s Republic of China

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