Laboratory of Physiology and Toxicology of Reproduction, Institute of Zoology and Biomedical Research, Jagiellonian University, Cracow, Poland.
Doctoral School of Exact and Natural Sciences, Jagiellonian University, Cracow, Poland.
J Physiol Pharmacol. 2023 Feb;74(1). doi: 10.26402/jpp.2023.1.03. Epub 2023 May 23.
The formation and luteolysis of the corpus luteum (CL) is strictly controlled by many factors. Imbalance between proliferation and apoptosis processes leads to deficiency of the luteal phase and infertility. Our previous study showed resistin expression in porcine luteal cells and an inhibitory effect on progesterone synthesis. Thus, the aim of the present study was to examine the in vitro effect of resistin on the proliferation/viability, apoptosis and autophagy of porcine luteal cells as well as the involvement of mitogen-activated kinase (MAP3/1), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) in these processes. Porcine luteal cells were incubated with resistin (0.1-10 ng/mL) for 24-72 h and viability was assessed using the alamarBlue or 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the time-dependent effect of resistin on mRNA and protein expression of proliferating cell nuclear antigen (PCNA), caspase 3, BCL2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), beclin1, microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal-associated membrane protein 1 (LAMP1) was measured by real-time polymerase chain reaction (PCR) and immunoblotting, respectively. We found that resistin enhanced luteal cell viability with no effect on caspase 3 mRNA and protein, increased the BAX/BCL2 mRNA and protein ratio and significantly stimulated the initiation of autophagy, which promotes the maintenance of CL function rather than its regression. Additionally, using pharmacological inhibitors of MAP3/1 (PD98059), AKT (LY294002) and STAT3 (AG490), we observed that the effect of resistin was reversed to the control level in viability and, by influence, MAP3/1 and STAT3 in autophagy. Taken together, our results suggest that resistin, in addition to its well-known effect on granulosa cell function has direct influence on CL luteolysis and the formation and maintenance of luteal cell function.
黄体（CL）的形成和黄体溶解受到许多因素的严格控制。增殖和凋亡过程之间的失衡导致黄体期不足和不孕。我们之前的研究表明抵抗素在猪黄体细胞中的表达及其对孕激素合成的抑制作用。因此，本研究旨在研究抵抗素对猪黄体细胞增殖/活力、凋亡和自噬的体外影响，以及丝裂原活化蛋白激酶（MAP3/1）、蛋白激酶 B（AKT）和信号转导和转录激活因子 3（STAT3）在这些过程中的参与情况。将猪黄体细胞与抵抗素（0.1-10ng/ml）孵育 24-72 小时，并用 alamarBlue 或 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐（MTT）测定法评估活力。然后，通过实时聚合酶链反应（PCR）和免疫印迹法分别测量抵抗素对增殖细胞核抗原（PCNA）、半胱天冬酶 3、B 细胞淋巴瘤 2 样蛋白 4（BAX）、B 细胞淋巴瘤 2（BCL2）、beclin1、微管相关蛋白 1A/1B-轻链 3（LC3）和溶酶体相关膜蛋白 1（LAMP1）mRNA 和蛋白表达的时间依赖性影响。我们发现，抵抗素增强黄体细胞活力，对半胱天冬酶 3 mRNA 和蛋白无影响，增加 BAX/BCL2 mRNA 和蛋白比值，并显著刺激自噬的启动，从而促进 CL 功能的维持而不是其退化。此外，使用丝裂原活化蛋白激酶 3/1（PD98059）、蛋白激酶 B（LY294002）和信号转导和转录激活因子 3（AG490）的药理学抑制剂，我们观察到抵抗素对活力的影响在自噬方面逆转至对照水平，并通过影响 MAP3/1 和 STAT3。综上所述，我们的结果表明，抵抗素除了对颗粒细胞功能的众所周知的影响外，还直接影响黄体溶解以及黄体细胞功能的形成和维持。
