OBJECTIVE: To observe the effect of Huayu Tongluo moxibustion on the NOD-like receptor protein 3 (NLRP3)/cysteine-aspartic acid protease-1 (Caspase-1)/gasdermin D (GSDMD) signaling pathway in rats with vascular dementia (VD), and to explore its mechanism in improving learning and memory ability and alleviating neuronal injury in the hippocampal CA1 region. METHODS: A total of 80 SPF-grade male Wistar rats were included. Three rats were excluded based on the Morris water maze test. From the remaining rats, 12 were randomly selected as the sham operation group. The rest were used to establish VD models via modified bilateral common carotid artery ligation. Thirty-six successfully modeled rats were randomly divided into a model group, a medication group, and a moxibustion group, with 12 rats in each group. The medication group was treated with nimodipine solution (12 mg/kg) via gavage. The moxibustion group was treated with Huayu Tongluo moxibustion. The suspended moxibustion was applied at Shenting (GV24) and Dazhui (GV14), and aconite cake-separated moxibustion was applied at Baihui (GV20), with each acupoint treated for 20 min. All treatments were administered once daily for 21 consecutive days. Before and after modeling, and after intervention, the Morris water maze test was used to assess cognitive function. After intervention, the activation and morphology of microglia in the hippocampal CA1 region were observed by immunofluorescence. Ultrastructure of hippocampal CA1 neurons was examined by transmission electron microscopy. Western blot was used to detect protein expression of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, GSDMD, and interleukin-1β (IL-1β) in the hippocampal CA1 region. ELISA was used to detect the content of IL-6, IL-8, and tumor necrosis factor-α (TNF-α) in the hippocampal CA1 region. RESULTS: Compared with the sham operation group, the model group showed longer mean escape latency (P<0.01) and fewer platform crossings (P<0.01); the microglial processes in the hippocampal CA1 region were thickened, cytoplasm was hypertrophic, and relative fluorescence intensity of ionized calcium-binding adapter molecule 1 (IBA-1) was increased (P<0.05); the neuronal ultrastructure in the CA1 region was severely damaged, rough endoplasmic reticulum was swollen, mitochondria were deformed and swollen, some cristae were ruptured or dissolved, showing vacuolar changes; the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β, as well as levels of IL-6, IL-8, and TNF-α were significantly elevated (P<0.001). Compared with the model group, both the medication group and the moxibustion group showed shortened mean escape latency (P<0.01) and increased platform crossings (P<0.01); the microglial processes were thinner, and IBA-1 fluorescence intensity was decreased (P<0.05); the neuronal ultrastructure in the CA1 region was partially improved; the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β, and levels of IL-6, IL-8, and TNF-α were significantly reduced (P<0.001). Compared with the medication group, the moxibustion group showed shortened mean escape latency (P<0.05) and more platform crossings (P<0.05); the IBA-1 fluorescence intensity was decreased (P<0.05); the neuronal ultrastructure in the CA1 region was improved; the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β, as well as levels of IL-6, IL-8, and TNF-α, were significantly lower (P<0.001). CONCLUSION The Huayu Tongluo moxibustion could enhance learning and memory abilities in VD rats, inhibit excessive activation of microglia, and alleviate neuronal injury in the hippocampal CA1 region. Its mechanism may involve modulation of the NLRP3/Caspase-1/GSDMD signaling pathway, reduction of inflammatory responses.
Animals ; Male ; Dementia, Vascular/physiopathology* ; Rats ; Signal Transduction ; Moxibustion ; Rats, Wistar ; CA1 Region, Hippocampal/injuries* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Caspase 1/genetics* ; Memory ; Humans ; Neurons/metabolism* ; Learning

The aim of the present study was to investigate the effects of different courses of electroacupuncture on synaptic structure and synaptic function-related proteins expression in the hippocampal CA1 region of radiation-induced brain injury mice. Sixty C57BL/6J male mice were randomly divided into control group, radiation-induced brain injury model group, 1-week electroacupuncture group (EA1), 2-week electroacupuncture group (EA2), 3-week electroacupuncture group (EA3), and electroacupuncture-control (EA-Ctrl) group. The mice in model group were exposed to X-ray irradiation (8 Gy, 10 min) to establish radiation-induced brain injury model. The mice in EA groups were acupunctured at electroacupuncture points (Baihui, Fengfu and bilateral Shenshu) for 1 week, 2 weeks and 3 weeks respectively after radiation. Immunohistochemistry was used to observe synaptic structure in hippocampal CA1 region. The expressions of brain-derived neurotrophic factor (BDNF), synapsin-1 and postsynaptic density 95 (PSD95) in the hippocampal CA1 region of each group were detected by RT-PCR and Western blotting. The results showed that the nuclear gap in model and EA-Ctrl groups was significantly decreased compared to control group, however nucleus to cytoplasm ratio was significantly increased. The synaptic cleft, postsynaptic density (PSD) thickness, the mitochondrial surface density, volume density and specific surface area were significantly reduced. Compared with model group, the nucleus to cytoplasm ratio of EA2 group was significantly decreased, the PSD thickness and mitochondrial volume density were significantly increased; the nuclear gap of EA3 group was significantly increased, nucleus to cytoplasm ratio was significantly decreased, synaptic cleft and PSD thickness were significantly increased, and the mitochondrial surface density and specific surface area were all increased significantly. In addition, compared with the control group, the gene and protein expressions of BDNF, synapsin-1 and PSD95 in the hippocampal CA1 region of the model group and EA-Ctrl group were significantly decreased. However, compared with the model group, the gene expression of synapsin-1 in EA groups was significantly up-regulated, the gene expression of BDNF in EA1 and EA2 groups was significantly up-regulated, and the gene expression of PSD95 in EA2 group was significantly up-regulated. Moreover, the protein expressions of BDNF, synapsin-1 and PSD95 of EA groups were significantly up-regulated compared with the model group. These results indicate that the synaptic structure and the expression of synaptic function-related proteins in hippocampal CA1 region were injured by radiation exposure, whereas electroacupuncture intervention can significantly improve the synaptic structure and function damage caused by radiation.
Animals ; Brain Injuries ; CA1 Region, Hippocampal ; Electroacupuncture ; Hippocampus ; Male ; Mice ; Mice, Inbred C57BL