Exogenous hydrogen sulfide mitigates LPS + ATP-induced inflammation by inhibiting NLRP3 inflammasome activation and promoting autophagy in L02 cells

  • Dongdong Wu
  • Peiyu Zhong
  • Jun Wang
  • Honggang WangEmail author


The aim of this study is to investigate whether exogenous hydrogen sulfide (H2S) could mitigate lipopolysaccharide (LPS) + Adenosine Triphosphate (ATP)-induced inflammation by inhibiting nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome activation and promoting autophagy in L02 cells. We stimulated L02 cells with different concentrations of LPS, then the cell viability, cell apoptosis, and the protein level of NLRP3 inflammasome were detected by MTT and western blot to determine the appropriate LPS concentration used in this study. The cells were divided into 4 group: the cells in control group were cultured with RPMI-1640 for 23.5 h; the cells in LPS + ATP group were cultured with RPMI-1640 for 0.5 h, then were stimulated with 100 ng/ml LPS for 18 h followed by stimulation with 5 mM ATP for 5 h; the cells in Sodium hydrosulfide (NaHS) + LPS + ATP group were pretreated with NaHS for 0.5 h before exposure to LPS for 18 h and ATP for 5 h; the cells in NaHS group were treated with NaHS for 0.5 h, then were cultured with RPMI-1640 for 23 h. Subsequently, the cells in each group were collected, the protein levels of NLRP3, pro-caspase-1, cleaved caspase-1, P62, toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), LC3, Beclin-1, and interleukin (IL)-1 beta (β) were detected by western blot and enzyme-linked immunosorbent assay. Our results showed that exogenous H2S reduced the protein levels of NLRP3, cleaved caspase-1, TLR4, NF-κB, P62, and IL-1β induced by LPS + ATP and increased the ratio of LC3-II/I and the protein levels of Beclin 1 suppressed by LPS + ATP. This study demonstrated that H2S might suppress LPS + ATP-induced inflammation by inhibiting NLRP3 inflammasome and promoting autophagy. In conclusion, H2S might have potential applications in the treatment of aseptic hepatitis.


Hydrogen sulfide LPS ATP NLRP3 inflammasome Autophagy 



This work was supported by grants from the National Natural Science Foundation of China (No. 31300884), and the key scientific research project of colleges and universities in Henan, China (No. 16A310001).

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.


  1. 1.
    Matzinger P (2002) The danger model: a renewed sense of self. Science 296:301–305. CrossRefPubMedGoogle Scholar
  2. 2.
    Elliott EI, Sutterwala FS (2015) Initiation and perpetuation of NLRP3 inflammasome activation and assembly. Immunol Rev 265:35–52. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Jo EK, Kim JK, Shin DM, Sasakawa C (2016) Molecular mechanisms regulating NLRP3 inflammasome activation. Cell Mol Immunol 13:148–159. CrossRefPubMedGoogle Scholar
  4. 4.
    Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 10:417–426CrossRefPubMedGoogle Scholar
  5. 5.
    Sir D, Tian Y, Chen WL, Ann DK, Yen TS, Ou JH (2010) The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication. Proc Natl Acad Sci USA 107:4383–4388. CrossRefPubMedGoogle Scholar
  6. 6.
    Qiu DM, Wang GL, Chen L, Xu YY, He S, Cao XL, Qin J, Zhou JM, Zhang YX, E Q (2014) The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance. BMC Cancer 14:327. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Murrow L, Debnath J (2013) Autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease. Annu Rev Pathol 8:105–137. CrossRefPubMedGoogle Scholar
  8. 8.
    Kimura H (2014) The physiological role of hydrogen sulfide and beyond. Nitric Oxide 41:4–10. CrossRefPubMedGoogle Scholar
  9. 9.
    Gomes LR, Menck CFM, Cuervo AM (2017) Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation. Autophagy 13:928–940. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Parzych KR, Klionsky DJ (2014) An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signa 20:460–473. CrossRefGoogle Scholar
  11. 11.
    Yin F, Lu LM (2017) Advances in mechanism of autophagy in renal tubular injury. Acta Physiol Sin 69:723–729Google Scholar
  12. 12.
    Jiang C, Jiang L, Li Q, Liu X, Zhang T, Dong L, Liu T, Liu L, Hu G, Sun X, Jiang L (2018) Acrolein induces NLRP3 inflammasome-mediated pyroptosis and suppresses migration via ROS-dependent autophagy in vascular endothelial cells. Toxicology 410:26–40. CrossRefPubMedGoogle Scholar
  13. 13.
    Wang Z, Li Z, Feng D, Zu G, Li Y, Zhao Y, Wang G, Ning S, Zhu J, Zhang F, Yao J, Tian X (2018) Autophagy induction ameliorates inflammatory responses in intestinal ischemia-reperfusion through inhibiting NLRP3 inflammasome activation. Shock. Google Scholar
  14. 14.
    Li L, Rose P, Moore PK (2011) Hydrogen sulfide and cell signaling. Annu Rev Pharmacol Toxico 51:169–187. CrossRefGoogle Scholar
  15. 15.
    Olson KR (2012) A practical look at the chemistry and biology of hydrogen sulfide. Antioxid Redox Signal 17:32–44. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wang R (2012) Physiological implications of hydrogen sulfide: a Whiff exploration that blossomed. Physiol Rev 9:791–896. CrossRefGoogle Scholar
  17. 17.
    Kolluru GK, Shen X, Bir SC, Kevil CG (2013) Hydrogen sulfide chemical biology: pathophysiological roles and detection. Nitric Oxide 35:5–20. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Paul BD, Snyder SH (2012) H2S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol 13:499–507. CrossRefPubMedGoogle Scholar
  19. 19.
    Szabo C (2016) Gasotransmitters in cancer: from pathophysiology to experimental therapy. Nat Rev Drug Discov 15:185–203. CrossRefPubMedGoogle Scholar
  20. 20.
    Cao X, Bian JS (2016) The role of hydrogen sulfide in renal system. Front Pharmacol 7:385. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Xu D, Jin H, Wen J, Chen J, Chen D, Cai N, Wang Y, Wang J, Chen Y, Zhang X, Wang X (2017) Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration. Pharmacol Res 117:357–369. CrossRefPubMedGoogle Scholar
  22. 22.
    Feliers D, Lee HJ, Kasinath BS (2016) Hydrogen sulfide in renal physiology and disease. Antioxid Redox Signal 25:720–731. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ali FF, Abdel-Hamid HA, Toni ND (2018) H2S attenuates acute lung inflammation induced by administration of lipopolysaccharide in adult male rats. Gen Physiol Biophys.
  24. 24.
    Zhu Q, Lin F (2016) Molecular markers of autophagy. Acta Pharmaceutica Sin 51:33–38Google Scholar
  25. 25.
    Cullen SP, Kearney CJ, Clancy DM, Martin SJ (2015) Diverse activators of the NLRP3 inflammasome promote IL-1β secretion by triggering necrosis. Cell Rep 11:1535–1548. CrossRefPubMedGoogle Scholar
  26. 26.
    Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, Zhuang Y, Cai T, Wang F, Shao F (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526:660–665. CrossRefPubMedGoogle Scholar
  27. 27.
    Dunton CL, Purves JT, Hughes FM Jr, Jin H, Nagatomi J (2018) Elevated hydrostatic pressure stimulates ATP release which mediates activation of the NLRP3 inflammasome via P2 × 4 in rat urothelial cells. Int Urol Nephrol 50:1607–1617. CrossRefPubMedGoogle Scholar
  28. 28.
    Wang Y, Jia J, Ao G, Hu L, Liu H, Xiao Y, Du H, Alkayed NJ, Liu CF, Cheng J (2014) Hydrogen sulfide protects blood-brain barrier integrity following cerebral ischemia. J Neurochem 129:827–838. CrossRefPubMedGoogle Scholar
  29. 29.
    Yin J, Tu C, Zhao J, Ou D, Chen G, Liu Y, Xiao X (2013) Exogenous hydrogen sulfide protects against global cerebral ischemia/reperfusion injury via its anti-oxidative, anti-inflammatory and anti-apoptotic effects in rats. Brain Res 1491:188–196. CrossRefPubMedGoogle Scholar
  30. 30.
    Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G, Wallace JL (2006) Hydrogen sulfide is an endogenous modulator of leukocyte-mediated inflammation. FASEB J 20: 2118–2120. CrossRefPubMedGoogle Scholar
  31. 31.
    Zhang M, Wu X, Xu Y, He M, Yang J, Li J, Li Y, Ao G, Cheng J, Jia J (2017) The cystathionine β-synthase/hydrogen sulfide pathway contributes to microglia-mediated neuroinflammation following cerebral ischemia. Brain Behav Immun 66:332–346. CrossRefPubMedGoogle Scholar
  32. 32.
    Chen Y, Jin S, Teng X, Hu Z, Zhang Z, Qiu X, Tian D, Wu Y (2018) Hydrogen sulfide attenuates LPS-induced acute kidney injury by inhibiting inflammation and oxidative stress. Oxid Med Cell Longev 2018:6717212. PubMedPubMedCentralGoogle Scholar
  33. 33.
    Castelblanco M, Lugrin J, Ehirchiou D, Nasi S, Ishii I, So A, Martinon F, Busso N (2017) Hydrogen sulfide inhibits the NLRP3 inflammasome and reduces cytokine production both in vitro and in a mouse model of inflammation. J Biol Chem 293:2546–2557. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Lin S, Lian D, Liu W, Haig A, Lobb I, Hijazi A, Razvi H, Burton J, Whiteman M, Sener A (2018) Daily therapy with a slow-releasing H2S donor GYY4137 enables early functional recovery and ameliorates renal injury associated with urinary obstruction. Nitric Oxide 76:16–28. CrossRefPubMedGoogle Scholar
  35. 35.
    Wei X, Zhang B, Zhang Y, Li H, Cheng L, Zhao X, Yin J, Wang G (2015) Hydrogen sulfide inhalation improves neurological outcome via NF-κB-mediated inflammatory pathway in a rat model of cardiac arrest and resuscitation. Cell Physiol Biochem 36:1527–1538. CrossRefPubMedGoogle Scholar
  36. 36.
    Huang Z, Zhuang X, Xie C, Hu X, Dong X, Guo Y, Li S, Liao X (2016) Exogenous hydrogen sulfide attenuates high glucose-induced cardiotoxicity by inhibiting NLRP3 inflammasome activation by suppressing TLR4/NF-κB pathway in H9c2 cells.Cell Physiol Biochem 40:1578–1590. CrossRefPubMedGoogle Scholar
  37. 37.
    Luo ZL, Ren JD2, Huang Z, Wang T, Xiang K, Cheng L, Tang LJ (2017) TThe role of exogenous hydrogen sulfide in free fatty acids induced inflammationin macrophages. Cell Physiol Biochem 42:1635–1644. CrossRefPubMedGoogle Scholar
  38. 38.
    Wu D, Luo N, Wang L, Zhao Z, Bu H, Xu G, Yan Y, Che X, Jiao Z, Zhao T, Chen J, Ji A, Li Y, Lee GD (2017) Hydrogen sulfide ameliorates chronic renal failure in rats by inhibiting apoptosis and inflammation through ROS/MAPK and NF-κB signaling pathways. Sci Rep 7:455. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Lin Z, Altaf N, Li C, Chen M, Pan L, Wang D, Xie L, Zheng Y, Fu H, Han Y, Ji Y (2018) Hydrogen sulfide attenuates oxidative stress-induced NLRP3 inflammasome activation via S-sulfhydrating c-Jun at Cys269 in macrophages. Biochim Biophys Acta Mol Basis Dis 1864:2890–2900. CrossRefPubMedGoogle Scholar
  40. 40.
    Yang B, Bai Y, Yin C, Qian H, Xing G, Wang S, Li F, Bian J, Aschner M, Lu R (2018) Activation of autophagic flux and the Nrf2/ARE signaling pathway by hydrogen sulfide protects against acrylonitrile-induced neurotoxicity in primary rat astrocytes. Arch Toxicol 92:2093–2108. CrossRefPubMedGoogle Scholar
  41. 41.
    Wang SS, Chen YH, Chen N, Wang LJ, Chen DX, Weng HL, Dooley S, Ding HG (2017) Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/Akt/mTOR signaling pathway. Cell Death Dis 8:e2688. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Arroyo DS, Gaviglio EA, Peralta Ramos JM, Bussi C, Rodriguez-Galan MC, Iribarren P (2014) Autophagy in inflammation, infection, neurodegeneration and cancer. Int Immunopharmacol 18:55–65. CrossRefPubMedGoogle Scholar
  43. 43.
    Lai M, Yao H, Shah SZA, Wu W, Wang D, Zhao Y, Wang L, Zhou X, Zhao D, Yang L (2018) The NLRP3-caspase 1 inflammasome negatively regulates autophagy via TLR4-TRIF in prion peptide-infected microglia. Front Aging Neurosci 10:116. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Xue Z, Zhang Z, Liu H, Li W, Guo X, Zhang Z, Liu Y, Jia L, Li Y, Ren Y, Yang H, Zhang L, Zhang Q, Da Y, Hao J, Yao Z, Zhang R (2019) lincRNA-Cox2 regulates NLRP3 inflammasome and autophagy mediated neuroinflammation. Cell Death Differ 26:130–145. CrossRefPubMedGoogle Scholar
  45. 45.
    Meng Y, Pan M, Zheng B, Chen Y, Li W, Yang Q, Zheng Z, Sun N, Zhang Y, Li X (2019) Autophagy attenuates angiotensin II-induced pulmonary fibrosis by inhibiting redox imbalance-mediated NLRP3 inflammasome activation. Antioxid Redox Signal 30:520–554. CrossRefPubMedGoogle Scholar
  46. 46.
    Huang S, Huang P, Lin Z, Liu X, Xu X, Guo L, Shen X, Li C, Zhong Y (2018) Hydrogen sulfide supplement attenuates the apoptosis of retinal ganglion cells in experimental glaucoma. Exp Eye Res 168:33–48. CrossRefPubMedGoogle Scholar
  47. 47.
    Xu K, Wu F, Xu K, Li Z, Wei X, Lu Q, Jiang T, Wu F, Xu X, Xiao J, Chen D, Zhang H (2018) NaHS restores mitochondrial function and inhibits autophagy by activating the PI3K/Akt/mTOR signalling pathway to improve functional recovery after traumatic brain injury. Chem Biol Interac 286:96–105. CrossRefGoogle Scholar
  48. 48.
    Jiang WW, Huang BS, Han Y, Deng LH, Wu LX (2017) Sodium hydrosulfide attenuates cerebral ischemia/reperfusion injury by suppressing overactivated autophagy in rats. FEBS Open Bio 7:1686–1695. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Wei K, Wang P, Miao CY (2012) A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury. CNS Neurosci Ther 18:879–886. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Basic Medical ScienceHenan UniversityKaifengChina

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