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RIPK1/RIPK3-Mediated Necroptosis is Involved in Sevoflurane-Induced Neonatal Neurotoxicity in the Rat Hippocampus

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Abstract

Recent studies have shown that exposure to sevoflurane in developing brains causes neuronal apoptosis and cognitive dysfunction. “Necroptosis” is a novel pathway of necrosis. We introduced the caspase-specific inhibitor Z-VAD in addition to the receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-1, to ascertain the existence and importance of necroptosis. Sprague–Dawley rat pups postnatal day 7 were randomly assigned into one of five groups: control, sevoflurane + Z-VAD, sevoflurane + Nec-1, sevoflurane + Z-VAD + Nec-1 and 3% sevoflurane group. Neuronal apoptosis was evaluated by hematoxylin and eosin staining. The MTT assay was performed to evaluate cell viability. Immunofluorescence was employed to measure expression of RIPK1 and RIPK3. Western blots showing expression of RIPK1, RIPK3 and phosphorylation of mixed lineage kinase domain-like (p-MLKL) were used to explore the role of necroptosis. Binding of RIPK1/RIPK3 was detected via co-immunoprecipitation. Finally, the Morris water maze test was used to determine cognitive function. Exposure to 3% sevoflurane for 6 h induced neurotoxicity and inhibited cell viability. Neuron viability was low in the SEV, SEV + Z-VAD and SEV + Nec-1 groups. The study revealed that RIPK1 and RIPK3 protein expression increased significantly, but there was no significant differences between the SEV and SEV + Z-VAD groups. The expression of p-MLKL significantly increased in the SEV and SEV + Z-VAD groups, but not in the SEV + Nec-1 group or SEV + Z-VAD + Nec-1 group compared to the control group. Co-immunoprecipitation results showed that sevoflurane exposure enhanced binding of RIPK1/RIPK3 protein significantly. Blockade of apoptosis and necroptosis alleviated sevoflurane-induced cognitive impairment. Sevoflurane exposure elicited neurotoxicity within neonatal hippocampal neurons and tissues. Blockade of apoptosis or necroptosis alone did not attenuate sevoflurane-induced neurotoxicity (SIN). RIPK1/RIPK3-mediated necroptosis was involved in SIN in hippocampal neurons. SIN could be attenuated only by inhibiting both apoptosis and necroptosis.

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Abbreviations

Z-VAD:

ZVAD-fmk

Nec-1:

Necrostatin-1

RIPK1:

Receptor-interacting protein kinase 1

RIPK3:

Receptor-interacting protein kinase 3

FDA:

Food and Drug Administration

GABA:

Gamma-aminobutyric acid

NMDA:

N-methyl-d-aspartate

MLKL:

Mixed lineage kinase domain-like

FBS:

Fetal bovine serum

DMSO:

Dimethyl sulfoxide

TBST:

Tris buffer solution Tween

HE:

Hematoxylin staining

MTT:

Methyl thiazolyl tetrazolium

NSE:

Neuron-specific enolase

DAPI:

4′,6-Diamidino-2-phenylindole

SEV:

Sevoflurane

Co-IP:

Co-immunoprecipitation

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Funding

This research was funded by Grant of National Natural Science Foundation 81901093 (to Y.L.) and National Natural Science Foundation 81671059 (to Y.L.).

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Author notes

  1. Rui Xu and Yun Zhu contributed equally to this study and share first authorship.

Authors and Affiliations

  1. Department of Anesthesiology, The Eye, Ear, Nose and Throat Hospital, Fudan University, Fenyang Road #83, Shanghai, 200031, People’s Republic of China

    Rui Xu, Jie Jia, Wen Xian Li & Yi Lu

  2. Department of Oro-Maxillofacial Head and Neck Oncology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China

    Yun Zhu

Authors
  1. Rui Xu
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  2. Yun Zhu
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  3. Jie Jia
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  4. Wen Xian Li
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  5. Yi Lu
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Contributions

Conceptualization, YL and WL; methodology, RX and YZ; validation, JJ; formal analysis, WL; data curation, RX; writing—original draft preparation, RX and YZ; writing—review and editing, YL and WL; funding YL.

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Correspondence to Wen Xian Li or Yi Lu.

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Xu, R., Zhu, Y., Jia, J. et al. RIPK1/RIPK3-Mediated Necroptosis is Involved in Sevoflurane-Induced Neonatal Neurotoxicity in the Rat Hippocampus. Cell Mol Neurobiol 42, 2235–2244 (2022). https://doi.org/10.1007/s10571-021-01098-z

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