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

<bold>Objective</bold>To observe the effects of electroacupuncture at Pishu and Shenshu acupoints on gut microbiota, Th17/Treg cells, and bone metabolism in bilateral ovariectomized rats, and to explore the intrinsic relationship between electroacupuncture in regulating bone metabolism and the gut microbiota-Th17/Treg axis in postmenopausal osteoporosis (PMOP).<bold>Methods</bold>A total of twenty SPF-grade 3-month-old female Sprague-Dawley rats were selected. They were subjected to bilateral ovariectomy (OVX) to establish PMOP rat models. The rats were randomly divided into two groups using a random number table method: model group and 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 ℃ refrigerator. The rats were then anesthetized with 2% pentobarbital sodium (0.2 mL/100 g) via intraperitoneal injection, and euthanized for tissue collection to conduct 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, inter-group 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 procollagen type Ⅰ 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 mineral density was measured using dual-energy X-ray absorptiometry (DXA).<bold>Results</bold>Compared 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) significantly increased in the electroacupuncture group (<italic>P</italic>&lt;0.05); compared with the model group, the microbiota dysbiosis index (MDI) value 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 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 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 all increased (<italic>P</italic>&lt;0.05). Compared with the model group, bone mineral density significantly increased in the electroacupuncture group (<italic>P</italic>&lt;0.05).<bold>Conclusion</bold>Electroacupuncture at Pishu and Shenshu can increase E₂ levels, upregulate the abundance of Bifidobacteriaceae and Lactobacillaceae, and regulate the gut microbiota-Th17/Treg axis. These effects can effectively improve bone metabolic imbalance caused by estrogen deficiency, and then prevent and treat PMOP.