Proteomic analysis reveals that cigarette smoke exposure diminishes ovarian reserve in mice by disrupting the CREB1-mediated ovarian granulosa cell proliferation-apoptosis balance

Exposure to cigarette smoke (CS) adversely affects ovarian health and it is currently unknown how CS exposure causes ovarian injury. This study compared the differences in proteomics between CS exposure and healthy control groups using liquid chromatography-tandem mass spectrometry quantitative proteomics to further understand the molecular mechanism of ovarian cell injury in mice exposed to CS. Furthermore, western blotting and qPCR were carried out to validate the proteomic analysis outcomes. CREB1 was selected from the differentially expressed proteins, and then the down-regulation of CREB1 and phosphorylated CREB1(Ser133) expressions were confirmed in mice ovarian tissue and human ovarian granulosa cells (KGN cells) after CS exposure. In addition, the expressions of apoptosis-related proteins Bcl-2 and Bcl-xL were downregulated, and Bax expression was up-regulated. Moreover, the results of cellular immunofluorescence, flow cytometry, and transmission electron microscopy (TEM) showed that cigarette smoke extract (CSE) efficiently stimulated the production of Reactive Oxygen Species, Apoptosis, G1 phase arrest, mitochondrial membrane potential decreases, and ultrastructural changes in KGN cells. KG-501 (CREB inhibitor) aggravated CSE-induced mitochondrial dysfunction and apoptosis-proliferation imbalance in KGN cells mediated by down-regulated CREB1/Bcl-2 axis. In addition, CREB1 over-expression partially restores mitochondrial dysfunction and apoptosis-proliferation imbalance of KGN cells induced by CSE. The results suggested that CSE diminished ovarian reserve in mice by disrupting the CREB1-mediated ovarian granulosa cell (GCs) proliferation-apoptosis balance and provided possible therapeutic targets for the clinical intervention of premature ovarian failure (POI) caused by CS exposure.

CREB1; Cigarette smoke exposure; Granulosa cell; Premature ovarian insufficiency; Proliferation-apoptosis balance; Proteomics.