Zuogui Pills Alleviate Iron Overload-Induced Osteoporosis by Attenuating ROS-Mediated Osteoblast Apoptosis via the PI3K-AKT Pathway and Mitigating Mitochondrial Damage
Ethnopharmacological relevance Zuogui Pill (ZGP) is a classic herbal formula in Traditional Chinese Medicine, primarily used to tonify the kidney and replenish essence, and is widely applied in treating various kidney deficiency-related conditions. Over time, ZGP has demonstrated significant efficacy in addressing symptoms such as fatigue, weakness, and soreness of the lower back and knees, which are often caused by kidney deficiency. According to Traditional Chinese Medicine theory, the kidneys govern the bones, meaning that sufficient kidney essence is closely related to bone strength. By nourishing the kidneys and replenishing essence, ZGP helps to increase bone density and improve bone microstructure, making it an important therapeutic option for osteoporosis. Aim of the study To investigate the protective effects of ZGP in iron overload-induced osteoporosis and elucidate its molecular mechanisms through the activation of the Phosphoinositide 3-Kinase (PI3K)/Protein Kinase B (AKT) and Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) pathways, which reduce oxidative stress, inhibit osteoblast apoptosis, and promote osteoblast differentiation and mineralization. Materials and methods An in vivo mouse model of iron overload-induced osteoporosis and an in vitro MC3T3-E1 osteoblast model were used. In vitro experiments involved the use of ZGP containing-serum, along with transcriptomic analysis, Western blot, flow cytometry, TUNEL staining, and immunofluorescence, to assess the effects on oxidative stress, mitochondrial function, and apoptosis. In vivo experiments evaluated the effects of ZGP on bone mass, oxidative stress, and apoptosis using Micro-computed tomography (micro-CT), Hematoxylin and eosin staining (H&E), TUNEL staining, and immunohistochemistry. Results The study found that ZGP containing-serum significantly enhanced the viability of osteoblasts induced by iron overload and reduced apoptosis through the reactive oxygen species (ROS)-mediated Phosphoinositide 3-Kinase (PI3K) / Protein Kinase B (AKT) pathway while mitigating mitochondrial damage. In vivo , micro-computed tomography results showed that ZGP improved bone mass, and decreased ROS and apoptosis, consistent with the in vitro findings. Conclusion ZGP demonstrates significant antioxidant and anti-apoptotic effects in iron overload-induced osteoporosis, primarily through the ROS-mediated PI3K/AKT pathway and by reducing mitochondrial damage. These findings suggest that ZGP may be a promising therapeutic agent for treating osteoporosis associated with iron overload.
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