Abstract
Postmenopausal osteoporosis poses a significant clinical challenge, as conventional therapies are often ineffective or poorly tolerated owing to adverse effects or underlying health conditions, underscoring the need for alternative treatments. This study investigated the anti-osteoporotic effects of a novel probiotic mixture combining Limosilactobacillus reuteri MGE 3301 (LR) and Weissella cibaria MGE 3110 (WC), which were selected for their anti-inflammatory properties and ability to modulate bone metabolism, in an ovariectomized rat model. Thirty-five female Wistar rats were randomly assigned to five groups: Sham, Ovariectomy (OVX), OVX with LR supplementation (OVX/LR), OVX with WC (OVX/WC), and OVX with a combination of LR and WC (OVX/LR/WC), under ARRIVE guidelines and ethical approval. Each probiotic group received 1 × 10⁹ CFU/mL/day for 16 weeks starting at 5 weeks post-OVX. Micro-computed tomography and histopathological analyses revealed that the OVX/LR/WC group had superior trabecular bone preservation compared with that in the OVX control group, with significant improvements in bone mineral density (+ 54.2%), bone volume fraction (+ 24.8%), trabecular thickness (+ 13.6%), and trabecular number (+ 20%), along with decreased trabecular separation (- 8.1%; p < 0.05). RT-qPCR analysis of bone marrow demonstrated that LR/WC suppressed osteoclastogenic mediators (RANKL: -1.35-fold; TNF-α: -2.5-fold; IL-6: -1.9-fold) while elevating osteoprotective osteoprotegerin expression (+ 3.14-fold; p < 0.05). Serum analysis showed reduced CTX-I (- 38.9%) and elevated calcium (+ 30.8%) levels in OVX/LR/WC versus OVX rats (p < 0.05), indicating suppressed bone resorption and enhanced mineral homeostasis. These findings indicate that LR/WC probiotic supplementation attenuates OVX-induced bone loss by modulating bone turnover markers and inflammatory cytokines. To our knowledge, this is the first study to assess the combined effects of LR and WC in an osteoporosis animal model, highlighting its potential as an adjunctive therapeutic candidate for osteoporosis. However, few notable imitations include undefined human dosing and the unassessed long-term safety of probiotics. Future clinical trials must validate the efficacy, elucidate mechanisms (e.g., gut-bone axis interactions), and assess safety in postmenopausal women to advance therapeutic applicability.