Mechanism of acupuncture regulating cAMP signaling pathway to improve ovarian dysfunction in sleep-deprived mice

To explore the potential mechanism by which acupuncture alleviates sleep deprivation (SD)-induced ovarian dysfunction in mice via regulating the cyclic adenosine monophosphate (cAMP) signaling pathway based on transcriptomics.

Female C57BL/6 mice with regular estrous cycles were randomly divided into blank group, SD group and acupuncture group, with 15 mice in each group. Mice in the blank group maintained a normal light-dark schedule for 6 weeks; mice in the SD group and acupuncture group were subjected to 20-hour SD daily from 16: 00 to 12: 00 the next day for 6 consecutive weeks. Starting from the 4^th week, mice in the acupuncture group received acupuncture treatment for 20 min per session at non-SD time points, 6 d a week for 3 consecutive weeks. The estrous cycle changes of mice were observed via vaginal exfoliative cell smear staining. Body weight, ovarian wet weight were measured and ovarian index was calculated. Hematoxylin-eosin (HE) staining was adopted to detect ovarian tissue morphology. ELISA was used to detect serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH), estradiol (E2), as well as ovarian tissue cAMP content. Transcriptomic technology was applied to screen differentially expressed genes (DEGs) in ovarian tissues of mice in each group followed by functional enrichment analysis. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expressions of follicle-stimulating hormone receptor (FSHR), cAMP response element-binding protein 1 (CREB1), protein kinase A catalytic subunit α (PRKACA) and adenylate cyclase 5 (ADCY5), respectively.

After treatment, compared with the blank group, the rate of estrous cycle disorder was significantly elevated in the SD group (P<0.01); body weight, ovarian wet weight and ovarian index were markedly decreased (P<0.01); the morphological structure of ovarian tissue was damaged with increased atretic follicles; serum FSH and LH levels were up-regulated (P<0.01), while AMH and E2 levels were down-regulated (P<0.01). Compared with the SD group, the acupuncture group exhibited a reduced rate of estrous cycle disorder (P<0.01); increased body weight, ovarian wet weight and ovarian index (P<0.01); ameliorated pathological ovarian structure and fewer atretic follicles; decreased serum FSH and LH levels (P<0.01), and elevated serum AMH and E2 levels (P<0.01). A total of 173 intersecting DEGs with a callback trend were identified via transcriptomic sequencing. Gene ontology analysis revealed that these intersecting DEGs were mainly involved in biological processes such as response to other organisms, localized in cellular components including extracellular region, and associated with molecular functions like 2’-5’-oligoadenylate synthetase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis demonstrated that the intersecting DEGs were primarily enriched in the cAMP signaling pathway. Gene set enrichment analysis indicated that the cAMP signaling pathway was suppressed in the SD group but activated after acupuncture intervention. Compared with the blank group, the SD group showed significantly reduced ovarian cAMP content, mRNA expressions of FSHR, CREB1, ADCY5 and PRKACA, protein levels of FSHR, ADCY5, PRKACA, as well as p-CREB1/CREB1 ratio (P<0.01). Relative to the SD group, the acupuncture group presented increased ovarian cAMP content, upregulated mRNA expressions of FSHR, CREB1, ADCY5 and PRKACA, elevated protein levels of FSHR, ADCY5, PRKACA, and an increased p-CREB1/CREB1 ratio (P<0.01, P<0.05).

Acupuncture can ameliorate SD-induced ovarian dysfunction in mice, and its therapeutic mechanism may be related to regulating the expressions of key molecules including CREB1, FSHR, ADCY5 and PRKACA in the cAMP signaling pathway.