Study on Moxibustion Improving Reproductive Function of Rats in Cold Stress Model by Activating Hypothalamic Arcuate Nucleus Kisspeptin Neurons to Regulate the Kisspeptin/GPR54 Pathway

Abstract

Objective: This study examined how moxibustion affects reproductive function, the Kisspeptin/G protein-coupled receptor 54 (GPR54) pathway, and arcuate nucleus (ARC) Kisspeptin neurons in cold-stressed rats. This study explored the mechanisms by which moxibustion enhances reproductive function in cold-stressed rats, through the Kisspeptin/GPR54 pathway, and clarified the role of Kisspeptin neuron activation in the improvement of reproductive function by moxibustion. Methods: A total of 72 female Sprague Dawley rats (SPF, 6–8 weeks, regular estrous cycles) were divided into six groups: blank, cold, moxibustion, TAK488, chemogenetics, and chemogenetics + moxibustion. Cold-stress model was induced through ice-water baths and epinephrine injections. Moxibustion and chemogenetics + moxibustion groups received daily 20 min treatments. The TAK488 group was administered the KISS1R agonist TAK488 subcutaneously every other day for 14 days. Except for the blank and TAK488 groups, the remaining four groups received AAV chemogenetics virus injections into the ARC. Three weeks post-injection, the chemogenetics and chemogenetics + moxibustion groups received intraperitoneal CNO for 14 days. Assessments included cold symptom scores, ovarian histology, ELISA for serum and hypothalamic tissue, immunohistochemistry (IHC), real-time quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence (IF) of the hypothalamus or ovary. Results: Compared with the blank group, cold rats showed higher cold symptom scores, decreased serum 6-keto-prostaglandin F_1α (6-keto-PGF_1α), increased thromboxane B2(TXB₂), reduced reproductive hormones [estradiol 2 (E₂), follicle-stimulating hormone (FSH), luteinizing hormone (LH), gonadotropin-releasing hormone (GnRH)], and downregulated Kisspeptin/GPR54 pathway factors. Moxibustion lowered cold scores, enhanced blood circulation, elevated reproductive hormones, and activated the Kisspeptin/GPR54 pathway. Kisspeptin neuron activation similarly improved rat condition; elevated serum levels of E₂, FSH, and LH reduced levels of dopamine (DA) and neuropeptide Y (NPY); and activated the pathway. The combination of chemogenetics activation and moxibustion yielded superior results. Conclusion: Moxibustion enhances reproductive function by activating ARC Kisspeptin neurons and regulating the Kisspeptin/GPR54 pathway.

Graphical abstract

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