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.

Down-regulated Smyd1 participated in the inhibition of myoblast differentiation induced by cigarette smoke extract.

The histone methyltransferase Smyd1 is essential for muscle development; however, its role in smoking-induced skeletal muscle atrophy and dysfunction has not been investigated thus far. In this study, Smyd1 was overexpressed or knocked down in C2C12 myoblasts by an adenovirus vector and cultured in differentiation medium containing 5% cigarette smoke extract (CSE) for 4 days. CSE exposure resulted in inhibition of C2C12 cell differentiation and downregulation of Smyd1 expression, whereas Smyd1 overexpression reduced the degree of inhibition of myotube differentiation caused by CSE exposure. CSE exposure activated P2RX7-mediated apoptosis and pyroptosis, caused increased intracellular reactive oxygen species (ROS) levels, and impaired mitochondrial biogenesis and increased protein degradation by downregulating PGC1α, whereas Smyd1 overexpression partially restored the altered protein levels caused by CSE exposure. Smyd1 knockdown alone produced a phenotype similar to CSE exposure, and Smyd1 knockdown during CSE exposure aggravated the degree of inhibition of myotube differentiation and the degree of activation of P2RX7. CSE exposure suppressed H3K4me2 expression, and chromatin immunoprecipitation confirmed the transcriptional regulation of P2rx7 by H3K4me2 modification. Our findings suggest that CSE exposure mediates C2C12 cell apoptosis and pyroptosis through the Smyd1-H3K4me2-P2RX7 axis, and inhibits PGC1α expression to impair mitochondrial biosynthesis and increase protein degradation by inhibiting Smyd1 expression, ultimately leading to abnormal C2C12 myoblasts differentiation and impaired myotube formation.

Single-nucleus RNA Sequencing reveals the mechanism of cigarette smoke exposure on diminished ovarian reserve in mice.

The systematic toxicological mechanism of cigarette smoke (CS) on ovarian reserve has not been extensively investigated. Female 8-week-old C57BL/6 mice at peak fertility were exposed to CS or indoor air only for 30 days (100 mice per group) and the effects of CS on ovarian reserve were assessed using Single-Nucleus RNA Sequencing (snRNA-seq). In addition, further biochemical experiments, including immunohistochemical staining, ELISA, immunofluorescence staining, transmission electron microscopy, cell counting kit-8 assay, flow cytometry analysis, senescence-associated ß-galactosidase staining, and western blotting, were accomplished to confirm the snRNA-seq results. We identified nine main cell types in adult ovaries and the cell-type-specific differentially expressed genes (DEGs) induced by CS exposure. Western blot results verified that down-regulation of antioxidant genes (Gpx1 and Wnt10b) and the steroid biosynthesis gene (Fdx1) occurred in both ovarian tissue and human granulosa cell-like tumor cell line (KGN cells) after CS exposure. Five percent cigarette smoke extract (CSE) effectively stimulated the production of reactive oxygen species (ROS), DNA damage, cellular senescence and markedly inhibited KGN cell proliferation by inducing G1-phase cell cycle arrest. Moreover, down-regulation of Gja1, Lama1 and the Ferroptosis indicator (Gpx4) in granulosa cells plays a significant role in ultrastructural changes in the ovary induced by CS exposure. These observations suggest that CS exposure impaired ovarian follicle reserve might be caused by REDOX imbalance in granulosa cells. The current study systematically determined the damage caused by CS in mouse ovaries and provides a theoretical basis for early clinical prediction, diagnosis and intervention of CS exposure-associated primary ovarian insufficiency (POI), and is of great significance in improving female reproductive health.