Wheat-grain moxibustion improves sleep in perimenopausal insomnia rats by regulating the SIRT1/Nrf2 signaling pathway

To observe the effect of wheat-grain moxibustion (WGM) at “Guanyuan” (CV4), “Mingmen” (GV4), and bilateral “Xuanzhong”(GB39) on the oxidative stress and pyroptosis, and the activity of the Sirtuin 1 (SIRT1)/nuclear factor erythroid-2-related factor 2(Nrf2) signaling pathway in the hypothalamus of perimenopausal insomnia (PI) rats, so as to explore its underlying mechanism in improving PI.

Thirty-two female SD rats were randomly divided into four groups: sham, model, WGM, and WGM+EX527 (n=8 per group). The PI model was established by bilateral ovariectomy combined with an intraperitoneal injection of 4-Chloro-DL-phenylalanine suspension (350 mg/kg) for 2 consecutive days. Rats in the WGM and WGM+EX527 groups received WGM at CV4, GV4 and GB39, with 6 cones for each acupoint. The rats in the WGM+EX527 group received additional intraperitoneal injection of the SIRT1 inhibitor EX527 (10 mg/kg) 30 min before each moxibustion session. The interventions were conducted once daily for 14 consecutive days. The sleep latency and sleep duration were assessed using the pentobarbital sodium-induced sleep test (righting reflex), and the spontaneous activities (total distance moved, center zone dwelling time, and rearing times in 5 min) were measured using the open field test (OFT). The hematoxylin-eosin (H.E.) staining was used to observe morphological changes in the hypothalamic tissue. Western blot was used to detect protein expression levels of SIRT1, Nrf2, Kelch-like ECH-associated protein1 (Keap1), heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteingl aspartate specific protease 1 (Caspase-1), apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin-D (GSDMD) in the hypothalamus. The qPCR was used to measure mRNA expression levels of SIRT1, Nrf2, Keap1, and HO-1 in the hypothalamus. The reactive oxygen species (ROS) assay kit was used to detect ROS level of hypothalamic tissue. The ELISA was used to measure the content of interleukin (IL)-1β and IL-18 in the hypothalamus.

Compared to the sham group, the model group showed a striking decrease in the total distance traveled, central zone dwelling time and rearing times in the OFT (P<0.01), and significantly shortened sleep duration and prolonged sleep latency (P<0.01). In comparison with the model group and the WGM+EX527 group, the WGM group showed an obvious increase in the total distance traveled, central zone dwelling time and rearing times (P<0.01), and shortened sleep latency, prolonged sleep duration (P<0.01). Additionally, in comparison with the sham group, the model group had a significant down-regulation in the expression levels of hypothalamic SIRT1, Nrf2, Keap1, and HO-1 proteins and mRNAs, and SOD and CAT proteins (P<0.01), and an obvious up-regulation in the expression levels of NLRP3, Caspase-1, ASC, GSDMD proteins, ROS level, and contents of IL-1β and IL-18 (P<0.01) in the hypothalamus. In comparison with the model group and the WGM+EX527 group, both the down-regulation and up-regulation of expression levels of the proteins and mRNAs mentioned above, and ROS level and the contents of IL-1β and IL-18 were reversed in the WGM group (P<0.01,P<0.05). H.E. staining showed that in comparison with the sham group, more hypothalamic neurons were wrinkled with unclear cell structures, varying sizes and uneven distribution in the model and WGM+EX527 groups, which was alleviated in the degree of cell injury in the WGM group, for instance clearer cell structure and uniform distribution.

WGM can improve the sleep and spontaneous activity of PI rats, which may be related to its functions in alleviation of hypothalamic oxidative stress and pyroptosis by regulating the SIRT1/Nrf2 signaling pathway.