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Modulatory effects of bufalin, an active ingredient from toad venom on voltage-gated sodium channels

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Abstract

Chan-su (toad venom) has been used as an analgesic agent in China from ancient to modern times. Bufalin, a non-peptide toxin extracted from toad venom, is considered as one of the analgesic components. The molecular mechanism underlying the anti-nociceptive effects of bufalin remains unclear so far. In this study, we investigated the pharmacological effects of bufalin on pain-related ion channels as well as animal models through patch clamping, calcium imaging and animal behavior observation. Using the whole-cell recording, bufalin caused remarkable suppressive effect on the peak currents of Nav channels (voltage gated sodium channels, VGSCs) of dorsal root ganglion neuroblastomas (ND7-23 cell) in a dose-dependent manner. Bufalin facilitated the voltage-dependent activation and induced a negative shift on the fast inactivation of VGSCs. The recovery kinetics of VGSCs were significantly slowed and the recovery proportion were reduced after administering bufalin. However, bufalin prompted no significant effect not only on Kv4.2, Kv4.3 and BK channels heterologously expressed in HEK293T cells, but also on the capsaicin and allyl isothiocyanate induced Ca2+ influx. What’s more, bufalin could observably relieve formalin-induced spontaneous flinching and licking response as well as carrageenan-induced thermal and mechanical hyperalgesia in dose-dependent manner in agreement with the results of in vitro experiments. The present results imply that the remarkable anti-nociceptive effects produced by bufalin are probably ascribed to its specific regulation on Nav channels. Bufalin inhibits the Nav channels in a dose-dependent manner, which will provide references for the optimal dose selection of analgesia drugs.

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Abbreviations

VGSCs:

Voltage-gated sodium channels

BK:

Large-conductance calcium-activated potassium channels

Kv :

Voltage-gated K+ channels

DRG:

Dorsal root ganglion

TRPV1:

Transient receptor potential vanilloid 1

TRPA1:

Transient receptor potential ankyrin 1

INaT :

Transient sodium currents

INaSS :

Steady-state sodium current

NPPB:

5-Nitro-2-(3-phenylpropylamino)benzoic acid

TTX:

Tetrodotoxin

AITC:

Allyl isothiocyanate

[Ca2+]i :

Intracellular calcium concentration

PWT:

Paw withdrawal threshold

PWL:

Paw withdrawal latency

HBSS:

Hank’s balanced salt solution

PARP1:

Ploy (ADP-ribose) polymerase 1

iNOS:

Nitric oxide synthase

TNF-α:

Tumor necrosis factor-α

NF-κB:

Nuclear factor kappa B

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Acknowledgements

We are grateful to Prof. Tong Liu (Soochow University), Prof. Ren Lai (Kunming Institute of Zoology, Chinese Academy of Sciences), Prof. Kaoru Yamaoka (Tokyo University of Agriculture and Technology), Prof. Noel Davies (University of Leicester), Prof. Jiuping Ding (Huazhong University of Science and Technology) and Prof. Ping Song (Yale University) for providing the plasmids comprising genes of hSloα, hKv4.2, hKv4.3, rNav 1.5, mNav 1.7, TRPA1 and TRPV1, respectively.

Funding

This work was supported by National Science Foundation of China (No. 81603410, 81473482 and 81402903), Innovation Fund of Putuo District Health System (No. 17-PT-10), the “Twelfth Five Year” Key Subject (Integrated Chinese and Western Medicine) of State Administration of Traditional Medicine of China, Shanghai Municipal Commission of Health and Family Planning Fund (No. 20134Y022) and Research Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine (No. 2016208A).

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Author notes

  1. Jie Tao, Feng Jiang and Cheng Liu have contributed equally to this work.

Authors and Affiliations

  1. Department of Central Laboratory and Neurosurgery, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China

    Jie Tao, Cheng Liu, Yudan Zhu, Jian Xu, Yiqin Ge, Kan Xu & Peihao Yin

  2. Xinhua Hospital (Chongming) Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai Chongming Xinhua Translational Medical Institute for Cancer Pain, Shanghai, China

    Feng Jiang

  3. Department of Pharmacology, Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zhirui Liu

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  1. Jie Tao
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Contributions

Conceived and designed the experiments: JT, PY, KX. Performed the experiments: JT, FJ, CL, YZ, JX. Analyzed the data: JT, JX, ZL. Contributed reagents/Materials/Analysis tools: FJ, ZL, YG. Wrote the paper: JT, CL.

Corresponding authors

Correspondence to Jie Tao, Kan Xu or Peihao Yin.

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Conflict of interest

All authors have reviewed the final version of the manuscript and approve it for publication. This manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. The authors have no conflicts of interest to declare.

Ethical approval

The protocols of animal experiments complied with the current ethical considerations of Shanghai University of Traditional Chinese Medicine’s Animal Ethic Committee, which is in accordance with the National Research Council criteria. All animal experiments and procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai University of Traditional Chinese Medicine and were performed in accordance with the relevant guidelines and regulations as well as approved by the guidelines on ethical standards for investigation of experimental pain in conscious animals.

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Tao, J., Jiang, F., Liu, C. et al. Modulatory effects of bufalin, an active ingredient from toad venom on voltage-gated sodium channels. Mol Biol Rep 45, 721–740 (2018). https://doi.org/10.1007/s11033-018-4213-9

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