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Intrauterine exposure to oxidative stress induces caspase-1-dependent enteric nerve cell pyroptosis

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Abstract

Purpose

This study determined whether oxidative stress causes the developmental abnormalities of the enteric nervous system during the embryonic period.

Methods

Using the test results of tissue specimens of children with Hirschsprung disease (HSCR), we established a pregnant rat model of oxidative stress and a cellular oxidative stress model to conduct related molecular, cellular, and histopathological experiments for exploration and validation.

Results

The results of the quantitative real-time polymerase chain reaction assay indicated overexpression of pyroptosis markers (NLRP3, ASC, and caspase-1) in HSCR lesions and newborn pups in the oxidative stress group (treated with d-galactose). The expression of cathepsin D was significantly decreased in intestinal tissues of newborn pups in the oxidative stress group compared to the control group. Reactive oxygen species scavengers (N-acetyl-cysteine, NAC), the caspase-1 inhibitor (VX-765), and the NLRP3 siRNA could reverse the release of LDH, decrease the number of propidium iodide stained cells, and reduce the percentage of TUNEL/caspase-3 double-positive cells in the H2O2-treated group.

Conclusion

Oxidative stress can induce the death of enteric nerve cells by activating caspase-1-dependent pyroptosis through NLRP3 inflammasomes, which may contribute to abnormal enteric nervous system development.

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Funding

This work was supported by the National Natural Science Foundation of China (81801496, 81900460, and 81700449).

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Author notes

  1. Lingling Zhou and Bingyu Wang have contributed equally to this work and share first authorship.

Authors and Affiliations

  1. Department of Neonatal Surgery, Children’s Hospital of Nanjing Medical University, Nanjing, People’s Republic of China

    Lingling Zhou, Bingyu Wang, Hua Xie, Chunxia Du, Jie Tang & Weibing Tang

  2. Department of General Surgery, Children’s Hospital of Wujiang District, Suzhou, People’s Republic of China

    Lingling Zhou

  3. Department of Pediatric Surgery, Huai’an First People’s Hospital, Nanjing Medical University, Nanjing, People’s Republic of China

    Bingyu Wang

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  1. Lingling Zhou
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  3. Hua Xie
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  4. Chunxia Du
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  5. Jie Tang
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Contributions

WT and JT conceived the project and designed the experiments. LZ and BW performed experiments. LZ, HX, and CD analyzed the data. LZ draught the manuscript under the guidance of JT and WT. All authors reviewed the manuscript.

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Correspondence to Jie Tang or Weibing Tang.

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Supplementary Information

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Supplementary file1: Supplementary Table 1: Primers used for qRT-PCR.(PDF 32 KB)

383_2022_5199_MOESM2_ESM.tif

Supplementary file2: Supplementary Figure 1. (A) The relative ROS was analyzed by Image J. Treatment with H2O2 fostered intracellular ROS production in SH-SY5Y cells. (B) The percentage of tunel/caspase-1 double-positive cells was increased in the H2O2-treated groups. (C) NAC (ROS inhibitor) alleviated the ROS production in SH-SY5Y cells with the treatment of H2O2. (D) NAC reversed the change of the percentage of tunel/caspase-1 double-positive cells caused by H2O2 treatment. (E) Caspase-1 selective inhibitor (VX-765) decreased H2O2-induced pyroptotic cell death, as demonstrated by reducing the percentage of Tunel/Caspase-1 double-positive cells. (F) The expression of NLRP3 mRNA was significantly decreased by siRNA. (G) NLRP3 siRNA decreased the percentage of tunel/caspase-1 double-positive cells induced by H2O2. *P < 0.05; **P < 0.01; ***P < 0.001 (TIF 1672 KB)

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Zhou, L., Wang, B., Xie, H. et al. Intrauterine exposure to oxidative stress induces caspase-1-dependent enteric nerve cell pyroptosis. Pediatr Surg Int 38, 1555–1567 (2022). https://doi.org/10.1007/s00383-022-05199-8

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