Muscarinic acetylcholine M1 receptor regulates pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway

Jinying CHEN; Mi ZHANG; Yan ZENG; Yanping HUANG; Fangfang CHEN; Qianhui WANG; Chen XU

doi:10.16016/j.1000-5404.202105010

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Muscarinic acetylcholine M1 receptor regulates pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway

Basic Medicine | 更新时间：2026-01-09

- Muscarinic acetylcholine M1 receptor regulates pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway
- Journal of Third Military Medical University Vol. 43, Issue 18, Pages: 1796-1805(2021)
- 作者机构：
  
  400016 重庆，重庆医科大学生命科学研究院
- 作者简介：
- 基金信息：
  
  the Natural Science Foundation of Chongqing(CSTC2009BA5081)
- DOI：10.16016/j.1000-5404.202105010
  CLC： R394.2;R730.23;R737.25
- Received：06 May 2021，
  
  Revised：2021-07-16，
  
  Published：30 September 2021
- 稿件说明：
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Jinying CHEN, Mi ZHANG, Yan ZENG, et al. Muscarinic acetylcholine M1 receptor regulates pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway[J]. Journal of Third Military Medical University, 2021, 43(18): 1796-1805.
DOI：

Jinying CHEN, Mi ZHANG, Yan ZENG, et al. Muscarinic acetylcholine M1 receptor regulates pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway[J]. Journal of Third Military Medical University, 2021, 43(18): 1796-1805. DOI： 10.16016/j.1000-5404.202105010.

摘要

目的

探讨副交感神经M1受体(muscarinic acetylcholine M1 receptor，CHRM1)对前列腺癌细胞焦亡的影响及其可能的分子机制。

方法

在PC-3细胞中分别于12、24、48 h加入不同浓度CHRM1特异性抑制剂哌仑西平(pirenzepine，PIN)和激动剂氯贝胆碱(bethanechol，BECH)，后用慢病毒感染PC-3细胞构建CHRM1敲低的稳转株，再用质粒转染构建成孔蛋白gasdermin D(GSDMD)的敲低细胞模型，CCK-8实验检测细胞增殖能力，乳酸脱氢酶(lactate dehydrogenase，LDH)细胞毒性检测试剂检测LDH释放水平，流式细胞术及荧光倒置显微镜下观察Hoechst-PI和Annexin V-PI双染后的PI阳性细胞率，通过透射电镜观察细胞焦亡现象，Western blot检测Caspase-1/GSDMD通路相关分子的表达水平。

结果

CHRM1特异性抑制剂哌仑西平及慢病毒敲低CHRM1可使前列腺癌细胞PC-3细胞活力下降(

0.05)，PI阳性细胞数显著增加(

0.01)，同时在透射电镜下可观察到明显细胞焦亡现象，而相对的加入激动剂氯贝胆碱可使其细胞活力在一定浓度范围内上升(

0.05)，PI阳性细胞率减少(

0.01)；通过药物和慢病毒下调CHRM1之后，Caspase-1/GSDMD通路中的天冬氨酸蛋白水解酶-1 (Caspase-1)、天冬氨酸蛋白水解酶剪切体-1(cleaved Caspase-1)、凋亡相关斑点样蛋白(ASC)、炎症复合体(NLRP3)及GSDMD水平明显上升(

0.05)；敲低GSDMD之后，PI阳性细胞率降低(

0.05)，LDH释放率降低(

0.001)，各相关蛋白水平显著下降(

0.01)，且抑制或敲低CHRM1后PI阳性细胞率、LDH释放率及各相关蛋白水平无明显回复。

结论

下调CHRM1可通过Caspase-1/GSDMD通路诱导前列腺癌细胞焦亡。

Abstract

Objective

To determine the effect of muscarinic acetylcholine M1 receptor (CHRM1) on pyroptosis of prostate cancer cells and investigate its possible molecular mechanism.

Methods

Pirenzepine (PIN)

a specific CHRM1 antagonist

and bethanechol (BETH)

its agonist

were used to treat PC-3 cells at different doses for different time periods (12

24 and 48 h). Then a stable PC-3 cell strain cells with CHRM1 knockdown was established by lentivirus infection. A knockdown cell model of gasdermin D (GSDMD) was constructed by plasmid transfection. Cell proliferation ability was detected by CCK8 assay

LDH release level was measured by lactate dehydrogenase (LDH) cytotoxicity assay

and the rate of PI positive cells after staining with Hoechst-PI and Annexin V-PI was observed by flow cytometry and fluorescence inversion microscopy. Cell pyroptosis was observed by transmission electron microscopy and protein expression levels related to pyroptosis pathway were detected by Western blotting.

Results

The specific inhibitor of CHRM1

pirenzepine

and lentivirus knockdown of CHRM1 reduc

ed the cell viability of PC-3 cells (

0.05)

and increased the rate of PI positive cells significantly (

0.01). At the same time

the phenomenon of cell pyroptosis was observed under electron microscope

and the treatment of the BETH enhanced the cell viability (

0.05) and reduced the rate of PI positive cells in a certain concentration range (

0.01). After the down-regulation of CHRM1 by drugs and lentiviruses

the proteins levels of Caspase-1/GSDMD pathway

Caspase-1

cleaved Caspase-1

apoptosis associated speck-like protein (ASC)

NOD-

LRR- and pyrin domain-containing protein 3 (NLRP3) and GSDMD were significantly increased (

0.05). After the GSDMD was knocked down

the rate of PI positive cells was decreased (

0.05)

LDH release was reduced (

0.001)

and the levels of related proteins were reduced significantly (

0.01). Treatment of CHRM1 inhibitor or knockdown of CHRM1 had no effects on the rate of PI positive cells

LDH release

and the levels of related proteins.

Conclusion

Down-regulation of CHRM1 induces pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway.

关键词

Keywords

references

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