Zongyuan Tian, Xueran Wang, Yingyi Luan, Ruixia Liu
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引用次数: 0
Abstract
Objective
The principal objective of this project is to elucidate the ameliorating function of telomerase reverse transcriptase (TERT) on placental trophoblast dysfunction and its associated mitochondrial dysfunction.
Methods
We utilised 50 μM hydrogen peroxide (H2O2) to induce an oxidative stress model in HTR-8/SVneo cells (NC group and OS group), subsequently establishing three experimental conditions via lentiviral transfection: untreated controls (Con group), TERT-overexpressing cells (OE group), and empty vector-transfected negative cells (Neg group). In order to explore the role of mitochondrial TERT, we employed the Src kinase familyinhibitor PP1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a cell-permeable small molecule specifically blocking the translocation of TERT in HTR-8/SVneo cells with overexpressed TERT. We established four experimental conditions: untreated overexpression group (OE group), H2O2-stimulated group (OS group), co-treatment group of H2O2 and PP1 (PP1 OS group), and PP1 treatment group alone (PP1 group). The expression and localization of TERT were analysed via qRT-PCR, immunofluorescence, and Western blotting. In addition, the cellular functions (viability, migration, invasion) were assessed using CCK-8, Calcein AM/PI staining, wound healing assay and Transwell assay. Mitochondrial integrity was thoroughly assessed using a mitochondrial membrane potential assay, an ATP content assay, a mtDNA copy number quantification and a mitochondrial morphology assay.
Results
The OE group demonstrated significantly reduced reactive oxygen species (ROS) levels compared to Con and Neg groups, accompanied by enhanced cellular viability and improved migratory and invasive capacities. Mitochondrial functional analyses revealed superior outcomes in the OE group, including stabilized mitochondrial membrane potential and ATP production, maintained mtDNA copy numbers, and preserved mitochondrial ultrastructure. Conversely, pharmacological inhibition with PP1 - a Src kinase family inhibitor that blocks TERT mitochondrial translocation - effectively abolished these protective effects. PP1-treated cells exhibited exacerbated oxidative stress, impaired cell viability, and diminished motility, confirming the essential role of mitochondrial-localized TERT in cellular homeostasis.
Conclusions
Elevated mitochondrial TERT levels were able to effectively mitigate the cellular functional damage induced by elevated ROS levels by maintaining mitochondrial stability. The above findings provide a theoretical basis for the development of intervention strategies for pregnancy-related disorders based on the regulation of mitochondrial TERT function.
期刊介绍:
Drawing from a large number of disciplines, Reproductive Toxicology publishes timely, original research on the influence of chemical and physical agents on reproduction. Written by and for obstetricians, pediatricians, embryologists, teratologists, geneticists, toxicologists, andrologists, and others interested in detecting potential reproductive hazards, the journal is a forum for communication among researchers and practitioners. Articles focus on the application of in vitro, animal and clinical research to the practice of clinical medicine.
All aspects of reproduction are within the scope of Reproductive Toxicology, including the formation and maturation of male and female gametes, sexual function, the events surrounding the fusion of gametes and the development of the fertilized ovum, nourishment and transport of the conceptus within the genital tract, implantation, embryogenesis, intrauterine growth, placentation and placental function, parturition, lactation and neonatal survival. Adverse reproductive effects in males will be considered as significant as adverse effects occurring in females. To provide a balanced presentation of approaches, equal emphasis will be given to clinical and animal or in vitro work. Typical end points that will be studied by contributors include infertility, sexual dysfunction, spontaneous abortion, malformations, abnormal histogenesis, stillbirth, intrauterine growth retardation, prematurity, behavioral abnormalities, and perinatal mortality.