副交感神经M1受体通过Caspase-1/GSDMD通路调控前列腺癌细胞焦亡  

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

作　　者：陈津滢 张咪 曾燕 黄燕萍 陈芳芳 王千慧 徐晨[1] CHEN Jinying;ZHANG Mi;ZENG Yan;HUANG Yanping;CHEN Fangfang;WANG Qianhui;XU Chen(Institution of Life Science,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学生命科学研究院,重庆400016

出　　处：《第三军医大学学报》2021年第18期1796-1805,共10页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(cstc2009BA5081)。

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(P<0.05),PI阳性细胞数显著增加(P<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(P<0.05),PI阳性细胞率减少(P<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(P<0.05);敲低GSDMD之后,PI阳性细胞率降低(P<0.05),LDH释放率降低(P<0.001),各相关蛋白水平显著下降(P<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。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 reduced the cell viability of PC-3 cells(P<0.05), and increased the rate of PI positive cells significantly(P<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(P<0.05) and reduced the rate of PI positive cells in a certain concentration range(P<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(P<0.05). After the GSDMD was knocked down, the rate of PI positive cells was decreased(P<0.05), LDH release was reduced(P<0.001), and the levels of related proteins were reduced significantly(P<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 r

关 键 词：细胞焦亡 副交感神经M1受体 前列腺癌 CASPASE-1 GSDMD 

分 类 号：R394.2[医药卫生—医学遗传学] R730.23[医药卫生—基础医学]