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1,2,4-Trimethoxybenzene selectively inhibits NLRP3 inflammasome activation and attenuates experimental autoimmune encephalomyelitis

A Correction to this article was published on 13 April 2021

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Abstract

NOD-like receptor (NLR) family pyrin domain-containing-3 (NLRP3) inflammasome is implicated in inflammation-associated diseases such as multiple sclerosis, Parkinson’s disease, and stroke. Targeting the NLRP3 inflammasome is beneficial to these diseases, but few NLRP3 inflammasome-selective inhibitors are identified to date. Essential oils (EOs) are liquid mixtures of volatile and low molecular-weight organic compounds extracted from aromatic plants, which show various pharmacological activities, including antibacterial, antifungal, antiviral, antioxidant, and anti-inflammatory properties. In this study we screened active ingredients from essential oils, and identified 1,2,4-trimethoxybenzene (1,2,4-TTB) as a selective NLRP3 inflammasome inhibitor. We showed that 1,2,4-TTB (1 mM) markedly suppressed nigericin- or ATP-induced NLRP3 inflammasome activation, thus decreased caspase-1 activation and IL-1β secretion in immortalized murine bone marrow-derived macrophages (iBMDMs) and in primary mouse microglia. Moreover, 1,2,4-TTB specifically inhibited the activation of NLRP3 inflammasome without affecting absent in melanoma 2 (AIM2) inflammasome activation. We further demonstrated that 1,2,4-TTB inhibited oligomerization of the apoptosis-associated speck-like protein containing a CARD (ASC) and protein–protein interaction between NLRP3 and ASC, thus blocking NLRP3 inflammasome assembly in iBMDMs and in primary mouse macrophages. In mice with experimental autoimmune encephalomyelitis (EAE), administration of 1,2,4-TTB (200 mg · kg−1 · d−1, i.g. for 17 days) significantly ameliorated EAE progression and demyelination. In conclusion, our results demonstrate that 1,2,4-TTB is an NLRP3 inflammasome inhibitor and attenuates the clinical symptom and inflammation of EAE, suggesting that 1,2,4-TTB is a potential candidate compound for treating NLRP3 inflammasome-driven diseases, such as multiple sclerosis.

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Fig. 1: Screening of NLRP3 inflammasome-targeting natural compounds derived from essential oils.
Fig. 2: 1,2,4-TTB inhibits the activation of the NLRP3 inflammasome in vitro.
Fig. 3: The effect of TTB on NLRP3 inflammasome activation is structure dependent.
Fig. 4: 1,2,4-TTB had no significant effect on the activation of the AIM2 inflammasome.
Fig. 5: Effect of 1,2,4-TTB on the formation of ASC specks.
Fig. 6: 1,2,4-TTB inhibits the protein interactions of NLRP3.
Fig. 7: 1,2,4-TTB ameliorates the clinical symptoms of EAE model mice.
Fig. 8: 1,2,4-TTB inhibits activation of the NLRP3 inflammasome in vivo.
Fig. 9: Schematic diagram of our study.

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Acknowledgements

We sincerely thank Prof. Feng Shao (National Institute of Biological Sciences, Beijing, China) for providing the ASC knockout mice. This work was supported by the National Natural Science Foundation of China (81701187 to YJL), the Open Project of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine (TCM-201914 to YJL), and the Open Project of NHC Key Laboratory of Birth Defects Research, Prevention and Treatment (KF2019001 to YJL).

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YJL conceived, designed, and performed the experiments with the assistance of RYP and XXK; HYZ, ZCW, and LD performed the virtual screening and established a library of the bioactive compounds of EOs; CY provided technical support on confocal imaging. YJL, RYP, XXK, ZQY, JBC, and YC analyzed the data; and RYP and YJL wrote the paper.

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Correspondence to Hai-yan Zhang or Ya-jin Liao.

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Pan, Ry., Kong, Xx., Cheng, Y. et al. 1,2,4-Trimethoxybenzene selectively inhibits NLRP3 inflammasome activation and attenuates experimental autoimmune encephalomyelitis. Acta Pharmacol Sin (2021). https://doi.org/10.1038/s41401-021-00613-8

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Keywords

  • essential oils
  • 1,2,4-Trimethoxybenzene
  • NLRP3 inflammasome
  • ASC
  • experimental autoimmune encephalomyelitis

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