Exploring the Mechanism of Acupuncture in Improving Ovarian Function in Rats with Poor Ovarian Response Using High-Throughput Sequencing

Objective: This study aimed to investigate the possible mechanism through which acupuncture protects ovaries with Poor Ovarian Response (POR) in rats based on microRNA (miRNA).

Methods: Thirty-six SPF SD female non-pregnant rats aged 8 weeks were randomly divided into the blank group, model group, and acupuncture group, with 12 rats in each group. According to the group, the rats were given gavage of Tripterygium wilfordii polyglycosides suspension for 14 days to establish the model of POR, and then the rats were treated with acupuncture for 2 weeks, once a day, for 20 minutes. Afterward, their hormone levels were measured, and HE staining was performed on the ovaries after the intervention. Three rats from each group were randomly selected for ovarian tissue miRNA sequencing, and differential miRNAs were subjected to Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, and Quantitative Polymerase Chain Reaction (QPCR) verification. By using TargetScan to predict the target genes of differential miRNAs, we validated the results with a dual luciferase reporter gene assay.

Results: Compared with the blank group, the estrus cycle of the model group was significantly prolonged (P<0.01). Anti-Müllerian Hormone (AMH) (P<0.01) and Estrogen (E2) were significantly decreased (P<0.05). Follicle-Stimulating Hormone (FSH)(P<0.05) and Luteinizing Hormone (LH) increased sharply (P<0.01). Compared with the model group, the estrus cycle was significantly shortened in the acupuncture group (P<0.01). AMH and E2 were markedly raised (P<0.05). FSH (P<0.05) and LH (P<0.01) were significantly declined. miRNA sequencing showed that there were 23 miRNAs significantly different between the model group and the blank group (P<0.05), and 30 miRNAs significantly different between the acupuncture group and the model group (P<0.05). GO demonstrated that the network was mainly involved in cellular components, cells, cellular metabolic processes, binding, and single biological processes; KEGG signaling pathway enrichment showed that it was mainly related to MAPK, adhesion junction, calcium signaling pathways, etc. Q-PCR results showed that after modeling, the expression rose, and after acupuncture, the expression of the following miRNA decreased: miR-154-5p (P<0.01), miR-300-5p (P < 0.05), miR-376c-5p (P<0.05). The dual luciferase reporter assay showed that the relative luciferase activity of the miR-300-5p mimics+MAP3K1-WT group was significantly lower than that of the NC+MAP3K1-WT group (P<0.01). HE results demonstrated that the number of primordial follicles and primary follicles in the model group was significantly lower than that in the blank group (P<0.05). Moreover, the morphology was poorer, and the granulosa cell layer was thinner. Compared with the model group, the number of primary follicles in the acupuncture group increased (P<0.05); the morphology and granulosa cell structure of the ovary were improved to different degrees, and mature follicles could be seen.

Conclusion: Acupuncture may improve the ovarian responsiveness of POR rats by regulating miR-154- 5p,miR-300-5p, and miR-376c-5p. Furthermore, miR-300-5p can specifically bind to the 3'-UTR of MAP3K1, and MAP3K1 may be the target of miR-300-5p.