Exploring the Effects of Electroacupuncture on Bone Metabolism in Ovariectomized Rats via the Gut Microbiota-Th17/Treg Axis

ObjectiveTo observe the effects of electroacupuncture at Pishu and Shenshu acupoints on gut microbiota, Th17/Treg cells, and bone metabolism in bilateral ovariectomized rats, and exploring the intrinsic relationship of electroacupuncture in regulating bone metabolism and the gut microbiota-Th17/Treg axis in postmenopausal osteoporosis (PMOP).MethodsTwenty SPF-grade 3-month-old female Sprague-Dawley rats were selected. They were subjected to bilateral ovariectomy (OVX) to establish a postmenopausal osteoporosis (PMOP) rat model. The rats were randomly divided into two groups using a random number table method: the model group and the electroacupuncture group, with 10 rats in each group. The electroacupuncture group began electroacupuncture treatment 3 days after modeling, while the model group underwent daily handling similar to the electroacupuncture group without any additional interventions. After 12 weeks of intervention, on the following morning, each rat had approximately 1 g of fecal sample collected into sterile EP tubes and stored at -80°C. The rats were then anesthetized with 2% pentobarbital sodium (0.2 mL/100 g) via intraperitoneal injection, euthanized, and tissue collection was performed for subsequent experiments. ELISA was used to detect the level of E2. The gut microbiota of fecal samples from both groups was analyzed using 16S rDNA technology, followed by microbial profiling analysis, intergroup difference analysis, and LEfSe Multi-Level Species Difference Discriminant Analysis. Flow cytometry was used to determine the levels of Th17 and Treg cells in the colonic mesenteric lymph nodes. ELISA was also used to measure bone metabolism-related biochemical markers, including I-type procollagen N-terminal propeptide (PINP), β-collagen degradation product (β-CTx), interleukin-17 (IL-17), interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), hydroxyvitamin D3 (25-OH-D3), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β). Bone density was measured using a dual-energy X-ray absorptiometry (DXA) scanner.ResultsCompared with the model group, the estradiol (E2) content in peripheral blood increased in the electroacupuncture group(<italic>P</italic>&lt;0.05). Compared with the model group, the gut microbiota health index (GMHI) was significantly increased in the electroacupuncture group (<italic>P</italic>&lt;0.05); compared with the model group, the microbiota dysbiosis index (MDI) value was significantly decreased in the electroacupuncture group (<italic>P</italic>&lt;0.05); compared with the model group, the abundance of Lactobacillaceae and Bifidobacteriaceae in the gut microbiota were both increased in the electroacupuncture group(<italic>P</italic>&lt;0.05); compared with the model group, beneficial bacteria such as Bifidobacterium and Lactobacillus were significantly enriched in the gut of the electroacupuncture group (<italic>P</italic>&lt;0.05). Compared with the model group, the content of Th17 cells in the colonic lymph nodes was significantly reduced in the electroacupuncture group (<italic>P</italic>&lt;0.05); compared with the model group, the content of Treg cells in the colonic lymph nodes was significantly increased in the electroacupuncture group(<italic>P</italic>&lt;0.05). Compared with the model group, the expression levels of PINP, β-CTx, IL-17, IL-6, and TNF-αwere all decreased in the electroacupuncture group(<italic>P</italic>&lt;0.05); the expression levels of 25(OH)D3, IL-10, and TGF-β were all increased (<italic>P</italic>&lt;0.05). Compared with the model group, bone mineral density was significantly increased in the electroacupuncture group(<italic>P</italic>&lt;0.05).ConclusionElectroacupuncture at Pishu and Shenshu can increase E2 levels, upregulate the abundance of Bifidobacteriaceae and Lactobacillaceae, regulate the gut microbiota-Th17/Treg axis, effectively improve bone metabolic imbalance caused by estrogen deficiency, then prevent and treat PMOP.