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ObjectiveTo investigate the effect of Jingui Shenqiwan on diabetic osteoporosis （DOP） in mice by regulating the advanced glycation end products （AGEs）/receptor activator of nuclear factor-κB ligand （RANKL）/nuclear factor-κB （NF-κB） signaling pathway based on the theory of "kidneys governing bones". MethodForty 6-week-old male and female skeletal-muscle-specific， dominant negative insulin-like growth factor-1 receptor （MKR） mice were selected and fed on a high-fat diet for eight weeks to establish the DOP model. The model mice were randomly divided into a model group， low- and high-dose Jingui Shenqiwan group （1.3， 2.6 g·kg-1）， and an alendronate sodium group （0.01 g·kg-1）， with 10 mice in each group. Additionally， 10 FVB/N mice of the same age were assigned to the normal group. The corresponding drugs were administered orally to each group once a day for four weeks. After the administration period， fasting blood glucose （FBG） measurement and oral glucose tolerance test （OGTT） were conducted. Kidney function and kidney index were measured. Renal tissue pathological changes were observed through hematoxylin-eosin （HE） and Masson staining. Immunohistochemistry was performed to assess the protein expression levels of AGEs， phosphorylated NF-κB （p-NF-κB）， and RANKL in renal tissues. Western blot analysis was conducted to measure the expression of proteins related to the AGEs/RANKL/NF-κB signaling pathway， osteoprotegerin （OPG）， and Runt-related transcription factor 2 （RUNX2） proteins in femoral bone tissues. ResultCompared with the normal group， mice in the model group exhibited significantly increased FBG （P<0.01）， trabecular bone degeneration， abnormal bone morphological parameters， significantly increased area under the curve （AUC） of OGTT （P<0.01）， enlarged kidney volume， significantly increased kidney function indicators and kidney index （P<0.01）， disrupted renal glomeruli and renal tubule structures， significantly increased expression of AGEs， RANKL， and p-NF-κB/NF-κB in renal tissues （P<0.05）， and significantly decreased expression of OPG and RUNX2 in femoral bone tissues （P<0.01）. Compared with the model group， mice in the Jingui Shenqiwan groups showed a significant decrease in OGTT AUC （P<0.01）. Histopathological analysis revealed alleviated structural lesions in renal glomeruli and renal tubules. Furthermore， the expression of AGEs， RANKL， and p-NF-κB/NF-κB in renal tissues was significantly reduced （P<0.05， P<0.01）， and the expression of RUNX2 and OPG in femoral bone tissues was significantly increased （P<0.05， P<0.01）. ConclusionJingui Shenqiwan can improve kidney function and downregulate the AGEs/RANKL/NF-κB signaling pathway to inhibit inflammatory reactions， thereby alleviating the symptoms of DOP in mice， demonstrating a therapeutic effect on DOP from the perspective of the kidney.
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OBJECTIVE@#To explore the interaction between reactive oxygen species (ROS) and ferroptosis in methylglyoxalinduced injury of mouse embryonic osteoblasts (MC3T3-E1 cells).@*METHODS@#MC3T3-E1 cells were treated with methylglyoxal to establish a cell model of diabetic osteoporosis. CCK-8 assay was used to detect the viability of MC3T3-E1 cells. Rhodamine 123 staining followed by photofluorography was used to examine mitochondrial membrane potential (MMP). The intracellular ROS level was detected by 2', 7'-dichlorodihydrofluorescein diacetate staining with photofluorograph. Alkaline phosphatase (ALP) activity in the cells was detected using an ALP kit, the number of mineralized nodules was determined with alizarin red S staining, and the level of iron ions was detected using a detection kit. The expression level of glutathione peroxidase 4 (GPX4, a marker protein that inhibits ferroptosis) in the osteoblasts was determined using Western blotting.@*RESULTS@#Treatment of MC3T3-E1 cells with 0.6 mmol/L methylglyoxal for 24 h significantly inhibited the expression level of GPX4 (P < 0.001), increased intracellular iron ion concentration, decreased the cell viability, increased the loss of MMP and intracellular ROS level, decreased both ALP activity and the number of mineralized nodules in the cells (P < 0.001). Co-treatment of MC3T3-E1 cells with 2 mmol/L N-acetylcysteine (NAC, a ROS scavenger) and methylglyoxal significantly increased the expression level of GPX4 (P < 0.01); co-treatment with 4 mmo/L FER-1 (a ferroptosis inhibitor) and methylglyoxal obviously decreased the intracellular ROS level (P < 0.001). Co-treatment of the cells either with NAC and methylglyoxal or with FER-1 and methylglyoxal attenuated methylglyoxal-induced injuries in the osteoblasts (P < 0.001).@*CONCLUSION@#The interaction between ROS and ferroptosis pathway plays an important role in methylglyoxal-induced injury of mouse embryonic osteoblasts.
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Animals , Mice , Cell Survival , Ferroptosis , Osteoblasts , Pyruvaldehyde/metabolism , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND: The method of promoting osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions to inhibit adipogenic differentiation can provide prevention and treatment ideas for the treatment of bone metabolic diseases such as diabetic osteoporosis. OBJECTIVE: To explore the effects of uncarboxylated osteocalcin on adipogenic and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions so as to reveal the action mechanism of uncarboxylated osteocalcin on the differentiation of bone marrow mesenchymal stem cells. METHODS: Mouse bone marrow mesenchymal stem cells were cultured by whole bone marrow culture and adherent purification. Cells were treated with uncarboxylated osteocalcin at different concentrations (0, 1, 3, 10, and 30 μg/L). Cell proliferation was detected by cell counting kit-8 to determine the best mass concentration. Passage 3 bone marrow mesenchymal stem cells were incubated with adipogenic (or osteogenic) differentiation medium, and assigned to four groups: control group, high glucose group, uncarboxylated osteocalci n group, and high glucose + uncarboxylated osteocalcin group. Corresponding groups received the addition of 25.5 mmol/L exogenous glucose and 3 μg/L uncarboxylated osteocalcin. Lipid droplets and calcium nodules were detected by oil red and alizarin red staining. Quantitati ve reverse transcription-polymerase chain reaction was used to detect the relative expression levels of adipogenic marker genes (Fabp4, PPARγ, Adipsin and FAS) and osteogenic differentiation marker genes (Runx2, Osx, alkaline phosphatase, and type I collagen). Kits were used to detect alkaline phosphatase activity and type I collagen levels. The relative expression levels of P-Erk and P-AMPKα were detected using signal pathway specific inhibitors (PD98059 and BML) and western blot assay. RESULTS AND CONCLUSION: (1) Uncarboxylated osteocalcin 3 μg/L promoted cell proliferation (P < 0.01). (2) Uncarboxylated osteocalcin promoted the formation of calcium nodules (P < 0.01) in bone marrow mesenchymal stem cells under high-glucose conditions but inhibited the formation of lipid droplets (P < 0.05), down-regulating the relative expression levels of adipogenic marker genes (PFabp4 < 0.01; PPPARγ < 0.05; PAdipsin < 0.01; PFAS < 0.01), but increasing the relative expression levels of osteogenic differentiation marker genes (PRunx2 < 0.05; POsx < 0.05; PALP < 0.01; PCOLI < 0.01). Uncarboxylated osteocalcin increased alkaline phosphatase activity (P < 0.01) and type I collagen level (P < 0.05). (3) Uncarboxylated osteocalcin up-regulated the expression levels of P-Erk (P < 0.01) and P-AMPKα (P < 0.01) under high-glucose conditions. (4) These results indicate that uncarboxylated osteocalcin promoted osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions through Erk/AMPKα signaling pathway and inhibited adipogenic differentiation.
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Insulin-like growth factor-1 receptor (IGF-1R) is involved in both glucose and bone metabolism. IGF-1R signaling regulates the canonical Wnt/β-catenin signaling pathway. In this study, we investigated whether the IGF-1R/ β-catenin signaling axis plays a role in the pathogenesis of diabetic osteoporosis (DOP). Serum from patients with or without DOP was collected to measure the IGF-1R level using enzyme-linked immunosorbent assay (ELISA). Rats were given streptozotocin following a four-week high-fat diet induction (DOP group), or received vehicle after the same period of a normal diet (control group). Dual energy X-ray absorption, a biomechanics test, and hematoxylin-eosin (HE) staining were performed to evaluate bone mass, bone strength, and histomorphology, respectively, in vertebrae. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to measure the total and phosphorylation levels of IGF-1R, glycogen synthase kinase-3β (GSK-3β), and β-catenin. The serum IGF-1R level was much higher in patients with DOP than in controls. DOP rats exhibited strikingly reduced bone mass and attenuated compression strength of the vertebrae compared with the control group. HE staining showed that the histo-morphology of DOP vertebrae was seriously impaired, which manifested as decreased and thinned trabeculae and increased lipid droplets within trabeculae. PCR analysis demonstrated that IGF-1R mRNA expression was significantly up-regulated, and western blotting detection showed that phosphorylation levels of IGF-1R, GSK-3β, and β-catenin were enhanced in DOP rat vertebrae. Our results suggest that the IGF-1R/β-catenin signaling axis plays a role in the pathogenesis of DOP. This may contribute to development of the underlying therapeutic target for DOP.
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Objective To investigate if the effects of integated traditional Chinese and westetrt medicine treat ing diabetic osteoporesis.Methods 120 patients aging from 50 to 70 years were randomly recruited into two groups according to theirgender,namely DN1 and DN2.DN1 groupwastreatedwitll vitamin D3 and DN2 groupwastreatedwitll vitaminD3 plus Tanggukang capsule.Results X-ray showed that the bone density in the DN2 group WaS higher than DNI group,and the difference was significant(P＜0.05).Conclusion The integrated traditional Chinese and western medicine has good effects in treating diabetic osteoporosis.