Hojo Takuo, Piotrowska-Tomala Katarzyna K, Jonczyk Agnieszka W, Lukasik Karolina, Jankowska Katarzyna, Okuda Kiyoshi, Witek Krzysztof J, Skarzynski Dariusz J
Division of Biology of Reprodcution, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland.
Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan; Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
Theriogenology. 2019 Apr 1;128:193-200. doi: 10.1016/j.theriogenology.2019.01.035. Epub 2019 Feb 4.
Necroptosis is an alternative form of programmed cell death regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent. In the present study, to clarify if necroptosis in luteal endothelial cells (LECs) participates and contributes for bovine luteolysis, we investigated RIPK1 and RIPK3 localization in luteal tissue and their expression in cultured LECs after treatment with selected immune factors - mediators of luteolytic action of prostaglandin F2α (PGF). In addition, effects of tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM), and/or nitric oxide donor - NONOate (100 μM) on viability and CASP3 activity in the cultured LECs were investigated. Furthermore, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on RIPKs and CASPs expression, were evaluated. Localization of RIPK1 and RIPK3 protein in the cultured LECs were determined. In cultured LECs, expression of RIPKs mRNA were up-regulated by TNF + IFNG at 12 h, and by PGF (1 μM) or NONOate at 24 h, respectively (P < 0.05). Although NONOate decreased cell viability, it prevented TNF + IFNG-stimulated CASP3 activity in cultured LECs. Nec-1 prevented TNF + IFNG-induced RIPK1 and CASP3 mRNA expression at 12 h and prevented RIPK3 mRNA expression. These findings suggest that RIPKs-dependent necroptosis which are induced by TNF + IFNG, PGF or NO could be potent mechanism responsible for LECs cell death and disappearance of luteal capillaries in regressing bovine CL.
坏死性凋亡是一种由受体相互作用蛋白激酶（RIPK）1和3依赖性调节的程序性细胞死亡的替代形式。在本研究中，为了阐明黄体内皮细胞（LECs）中的坏死性凋亡是否参与并促成牛黄体溶解，我们研究了RIPK1和RIPK3在黄体组织中的定位以及在用选定的免疫因子——前列腺素F2α（PGF）黄体溶解作用的介质处理后它们在培养的LECs中的表达。此外，还研究了肿瘤坏死因子α（TNF；2.3 nM）与干扰素γ（IFNG；2.5 nM）和/或一氧化氮供体——NONOate（100 μM）联合使用对培养的LECs活力和CASP3活性的影响。此外，还评估了RIPK1抑制剂（坏死素-1，Nec-1；50 μM）对RIPKs和CASPs表达的影响。确定了RIPK1和RIPK3蛋白在培养的LECs中的定位。在培养的LECs中，RIPKs mRNA的表达在12小时时被TNF + IFNG上调，在24小时时分别被PGF（1 μM）或NONOate上调（P < 0.05）。虽然NONOate降低了细胞活力，但它阻止了TNF + IFNG刺激的培养LECs中的CASP3活性。Nec-1在12小时时阻止了TNF + IFNG诱导的RIPK1和CASP3 mRNA表达，并阻止了RIPK3 mRNA表达。这些发现表明，由TNF + IFNG、PGF或NO诱导的RIPKs依赖性坏死性凋亡可能是导致退化的牛黄体中LECs细胞死亡和黄体毛细血管消失的潜在机制。
