OBJECTIVE: To observe the effects of Mongolian medical warm acupuncture on hypothalamic biological clock genes such as Clock, Bmal1, Per1, and Per2 in insomnia rats, and to explore the mechanism by which Mongolian medical warm acupuncture regulates biological rhythms in insomniac rats.
METHODS: SPF-grade SD rats were divided into groups, and a PCPA-induced insomnia rat model was established. Mongolian medical warm acupuncture was applied to acupoints such as "Dinghui", "Heyi", and "Heart". General behavior, spontaneous activity, and pentobarbital-induced sleep tests were observed before and after treatment; Western blot and IHC were used to analyze the expression of Clock, Bmal1, Per1, and Per2 proteins in the hypothalamus; Realtime Quantitative PCR was used to analyze mRNA expression of Clock, Bmal1, Per1, and Per2 in the hypothalamus; HE staining was used to observe morphological changes in hypothalamic neurons.
RESULTS: After intervention with Mongolian medical warm acupuncture, insomniac rats showed quiet behavior and reduced activity, with drinking, eating, and mental state gradually returning to normal; Western blot results showed that, compared to the blank group, Clock and Bmal1 protein expression in the hypothalamus of the model group rats was significantly decreased (p<0.05), while Per1 and Per2 expression was significantly increased (p<0.05). Compared to the model group, the warm acupuncture group showed significantly increased Clock and Bmal1 protein expression (p<0.05), and significantly decreased Per1 and Per2 protein expression (p<0.05). IHC results showed that Clock and Bmal1 positive expression in the model group was significantly reduced, while expression in the warm acupuncture group and drug group was significantly increased; PCR results showed that, compared to the blank group, Clock and Bmal1 expression in the hypothalamus of the model group rats was significantly decreased (p<0.05), while Per1 and Per2 expression was significantly increased (p<0.05). Compared to the model group, the warm acupuncture group showed significantly increased Clock and Bmal1 expression (p<0.05), and significantly decreased Per1 and Per2 expression (p<0.05). HE staining results showed that in the blank group, cell membranes were intact, nuclei were located centrally, and nucleoli were clearly visible; in the model group, cell numbers were significantly reduced, boundaries were unclear, cells were swollen, and many nuclei were displaced; in the warm acupuncture group and drug group, hypothalamic cells were densely and orderly arranged, structures were clear, and cell damage was significantly restored.
DISCUSSION: The findings suggest that Mongolian medical warm acupuncture exerts a regulatory effect on hypothalamic biological clock gene expression, which may contribute to the restoration of circadian rhythm balance and reduction of hyperarousal in insomnia rats.
CONCLUSION: Mongolian medical warm acupuncture significantly reduces excitability in insomniac rats. The sleep-promoting mechanism of Mongolian medical warm acupuncture is closely related to its regulation of hypothalamic expression of biological clock genes Clock, Bmal1, Per1, and Per2 in insomniac rats.
