BACKGROUND Pyroptosis belongs to a novel inflammatory programmed cell death pathway, with the possible prognosis of endometrial cancer related to the terminal protein GSDMD. Hydrogen exerts a biphasic effect on cancer by promoting tumor cell death and protecting normal cells, which might initiate GSDMD pathway-mediated pyroptosis. METHODS We performed immunohistochemical staining and western immunoblotting analysis to observe expression of NLRP3, caspase-1, and GSDMD in human and xenograft mice endometrial cancer tissue and cell lines. We investigated treatment with hydrogen could boost ROS accumulation in endometrial cancer cells by intracellular and mitochondrial sources. GSDMD shRNA lentivirus was used to transfect endometrial cancer cells to investigate the function of GSDMD protein in pyroptosis. Propidium iodide (PI) staining, TUNEL assay, measurement of lactate dehydrogenase (LDH) release and IL-1β ELISA were used to analysis pyroptosis between hydrogen-supplemented or normal culture medium. We conducted in vivo human endometrial tumor xenograft mice model to observe anti-tumor effect in hydrogen supplementation. RESULTS We observed overexpression of NLRP3, caspase-1, and GSDMD in human endometrial cancer and cell lines by IHC and western immunoblotting. Hydrogen pretreatment upregulated ROS and the expression of pyroptosis-related proteins, and increased the number of PI- and TUNEL-positive cells, as well as the release of LDH and IL-1β, however, GSDMD depletion reduced their release. We further demonstrated that hydrogen supplementation in mice was sufficient for the anti-tumor effect to inhibit xenograft volume and weight of endometrial tumors, as mice subjected to hydrogen-rich water displayed decreased radiance. Tumor tissue sections in the HRW groups presented moderate-to-strong positive expression of NLRP3, caspase-1 and GSDMD. Hydrogen attenuated tumor volume and weight in a xenograft mouse model though the pyroptotic pathway. CONCLUSIONS This study extended our original analysis of the ability of hydrogen to stimulate NLRP3 inflammasome/GSDMD activation in pyroptosis and revealed possible mechanism (s) for improvement of anti-tumor effects in the clinical management of endometrial cancer.

中文翻译：

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氢通过ROS / NLRP3 / caspase-1 / GSDMD介导的焦磷酸途径抑制子宫内膜癌的生长。

背景技术细胞凋亡属于一种新型的炎症程序性细胞死亡途径，其可能与子宫内膜癌GSDMD有关。氢通过促进肿瘤细胞死亡和保护正常细胞而对癌症产生双相作用，这可能引发GSDMD途径介导的细胞凋亡。方法我们进行了免疫组织化学染色和免疫印迹分析，以观察NLRP3，caspase-1和GSDMD在人和异种移植小鼠子宫内膜癌组织和细胞系中的表达。我们调查了氢处理可以通过细胞内和线粒体来源促进子宫内膜癌细胞中ROS的积累。GSDMD shRNA慢病毒被用于转染子宫内膜癌细胞，以研究GSDMD蛋白在凋亡中的功能。碘化丙啶（PI）染色，TUNEL分析，乳酸脱氢酶（LDH）释放的测定和IL-1βELISA用于分析补充氢的培养基或正常培养基之间的热解。我们进行了体内人子宫内膜肿瘤异种移植小鼠模型，以观察补充氢气中的抗肿瘤作用。结果我们观察了通过IHC和Western免疫印迹在人子宫内膜癌和细胞系中NLRP3，caspase-1和GSDMD的过度表达。氢预处理上调了ROS和焦磷酸相关蛋白的表达，并增加了PI和TUNEL阳性细胞的数量，以及LDH和IL-1β的释放，但是GSDMD耗竭降低了它们的释放。我们进一步证明，在小鼠体内补充氢足以抑制异种移植物体积和子宫内膜肿瘤重量的抗肿瘤作用，因为接受富含氢的水的小鼠显示出降低的辐射。HRW组的肿瘤组织切片呈现NLRP3，caspase-1和GSDMD的中等至强阳性表达。氢通过焦磷酸途径减弱了异种移植小鼠模型中的肿瘤体积和重量。结论这项研究扩展了我们对氢刺激焦磷酸化中的NLRP3炎性体/ GSDMD活化能力的最初分析，并揭示了改善子宫内膜癌临床治疗中抗肿瘤作用的可能机制。