Metformin Reverses Purine Metabolism Disorder-induced Oxidative Damage Of Osteoblasts For The Treatment Of Postmenopausal Osteoporosis

Objective:Osteoporosis is a systemic metabolic disease that occurs in bone tissue induced by the imbalance between bone formation of osteoblasts and osteoclast bone resorption,which is common as the primary osteoporosis in menopausal women.The onset of osteoporosis is relatively insidious and often asymptomatic.The occurrence of thoracic and low back pain,spinal deformation and other symptoms indicate that bone mass has decreased significantly.Imaging examination may indicate obvious collapse of the vertebral body with double concave shape or wedge shape.At the same time,bone destruction and loss of bone mass greatly increase the risk of fractures,resulting in serious health and economic costs for patients.At present,estrogen receptor modulators and estrogen drugs are the main treatments for postmenopausal osteoporosis.Hormone replacement therapy is supposed to fundamentally relieve the bone imbalance caused by estrogen deficiency,but it is associated with the risk of reproductive tumors,endometriosis,and endocrine disruption in women.Therefore,identifying the pathophysiological states of bone tissue microenvironment changes caused by estrogen deficiency after menopause and targeting therapy will improve the effectiveness and safety of treatment.Oxidative stress,a negative effect of free radicals in the body,is believed to be the chief of accelerated aging in postmenopausal women.Estrogen is an antioxidant hormone,and the increase of oxidation-related biomarkers and the decrease of antioxidant capacity in postmenopausal women indicate that postmenopausal women with osteoporosis are in a state of oxidative stress.In bone metabolism,oxidative stress caused by estrogen deficiency has been shown to significantly promote osteoblast apoptosis.Oxygen-derived free radicals also known as reactive oxygen species are agents of cellular oxidative stress.The purine metabolism catalyzed by xanthine oxidase is the main way to produce reactive oxygen species.After improving oxidative damage of osteoblasts induced by abnormal purine metabolism with estrogen deficiency may be an effective means to treat postmenopausal osteoporosis.Recently,metformin has been shown to delay the aging process by reducing the production of reactive oxygen species.Our previous studies have also shown that metformin can alleviate hydrogen peroxide-induced osteoblast apoptosis,which greatly increases the feasibility of metformin in the treatment of postmenopausal osteoporosis.Therefore,we aimed to continue to explore the upstream mechanism of metformin reversing hydrogen peroxide-induced apoptosis of osteoblasts,and to clarify the therapeutic effect of metformin on the oxidative damage of osteoblasts induced by purine metabolic disorders.Methods:1.GSEA enrichment analysis was performed using R language for differential genes expressed in osteoblasts with and without metformin treatment（gene function enrichment analysis:cell composition,molecular function,biological processes;KEGG pathway analysis）;2.CCK8 was used to detect the effect of xanthine/xanthine oxidase on the activity of osteoblasts and the therapeutic effect of metformin.3.JC-1 kit was used to detect the changes of mitochondrial membrane potential induced by H₂O₂with metformin treatment;4.DCFH-DA kit was used to detect the level of intracellular reactive oxygen species induced by H₂O₂with metformin treatment;5.Fluo-4AM fluorescent probe was used to detect the intracellular calcium ion concentration of osteoblasts induced by H₂O₂with metformin treatment.6.Flow cytometry was used to detect the protective effect of metformin on osteoblasts apoptosis after adding calcium channel agonist Bay K8644 with Annexin V FITC staining.7.Protein expression of mitochondrial apoptosis genes was detected by Western blotting.8.The expression level of GSK-3βin osteoblasts was detected by immunofluorescence.9.GSK-3β-si RNA was transfected into osteoblasts to inhibit the expression of GSK-3β.10.Mito SOX kit was used to detect the level of superoxide in mitochondria.11.Network pharmacology and metabonomics were combined to analyze the mechanism of metformin in the treatment of postmenopausal osteoporosis.12.Statistical analysis:The experimental data in this article were obtained from three repeated and independent experiments.Bilateral t test or one-way variance analysis were used for statistical analysis,and the standard of statistical significance was p<0.05.Results:1.Gene functional enrichment（GO）analysis showed that differentially expressed genes were mainly enriched in mitochondrial components（mitochondrial inner membrane and mitochondrial protein-containing complex）for cell composition.For molecular function,differentially expressed genes are closely related to transcriptional regulation（transcriptional coregulatory activity and catalytic activity acting on RNA）.For biological process,differentially expressed genes are mainly enriched in protein modification（negative regulation of protein modification process and regulation of protein stability）.2.Metformin could improve mitochondrial function of osteoblasts induced by H₂O₂.The changes of mitochondrial membrane potential and reactive oxygen species in mitochondria were detected by JC-1 and DCFH-DA fluorescence staining.The results showed that the mitochondrial membrane potential of osteoblasts was significantly decreased and the level of reactive oxygen species in mitochondria was significantly increased with H₂O₂induction.The mitochondrial membrane potential was improved and the level of reactive oxygen species in mitochondria was also significantly decreased after adding metformin.3.Metformin could reduce the intracellular calcium concentration of osteoblasts induced by H₂O₂.Intracellular calcium concentration was detected by Fluo-4AM fluorescence probe.The results showed that the intracellular calcium concentration of osteoblasts increased significantly with H₂O₂induction,which was improved by metformin,but the effect of metformin on calcium ions was reversed after the addition of calcium channel agonist Bay K8644.4.Metformin inhibits H₂O₂-induced osteoblast apoptosis by reducing intracellular calcium ion concentration.The apoptosis rate of osteoblasts was detected by flow cytometry with Annexin V FITC staining,and the protein expressions of mitochondrial apoptosis genes Bax,Bcl-2,Caspase3 and Cleaved caspase3 were detected by Western blotting.The results showed that metformin reduced the apoptosis rate,inhibited the expression of Bax,Cleaved caspase3 and promoted the expression of Bcl-2 and Caspase3 of osteoblasts induced by H₂O₂.However,the protective effect of metformin on osteoblast apoptosis was reversed after the addition of Bay K8644.5.GSK-3β-si RNA transfection could reverse the protective effect of metformin on H₂O₂-induced osteoblast apoptosis.The apoptosis rate of osteoblasts was detected by flow cytometry with Annexin V FITC staining,and the protein expressions of mitochondrial apoptosis genes Bax,Bcl-2,Caspase3 and Cleaved caspase3 were detected by Western blotting.The results showed that after transfection of GSK-3β-si RNA,the apoptosis rate of osteoblasts was increased again after metformin treatment,and the expression levels of Bax,Cleaved caspase3 were increased,and the expression levels of Bcl-2,Caspase3 were decreased.6.GSK-3β-si RNA transfection could reverse the protective effect of metformin on mitochondrial damage of osteoblasts induced by H₂O₂.The changes of mitochondrial membrane potential,reactive oxygen species（ROS）and superoxide levels in mitochondria were detected by JC-1,DCFH-DA and Mito SOX fluorescence staining.The results showed that after transfection of GSK-3β-si RNA,the mitochondrial membrane potential decreased,and the levels of reactive oxygen species and superoxide in mitochondria increased of osteoblasts with metformin treatment.7.GSK-3β-si RNA transfection could reverse the inhibitory effect of metformin on calcium concentration in osteoblasts induced by H₂O₂.Intracellular calcium concentration was detected by Fluo-4AM fluorescence probe.The results showed that the calcium concentration in osteoblasts increased again with metformin treatment after GSK-3β-si RNA transfection.8.Metformin reversed the oxidative damage of osteoblasts by promoting the phosphorylation of GSK-3β.The phosphorylation level of GSK-3βwas detected by Western blotting.The results showed that H₂O₂significantly inhibited the phosphorylation of GSK-3β,which was reversed by metformin.9.KEGG pathway enrichment analysis suggested that EGFR tyrosine kinase inhibitor resistance pathway mediated the antioxidant damage effect of metformin on osteoblasts.10.Metformin reversed oxidative damage in osteoblasts by promoting EGFR phosphorylation.The phosphorylation level of EGFR was detected by Western blotting.The results showed that metformin reversed H₂O₂-inhibited EGFR phosphorylation.11.Metformin could prevent bone loss in mice after ovariectomy.The menopausal model was constructed by bilateral ovariectomy of C57/B6L mice.The postmenopausal mice were given metformin saline solution（100mg/kg/day）by intestinal gavage starting from the third day after modeling,and the menopausal model group were given the same amount of saline gavage for two months.micro-CT was used to detect the changes of bone mass in mice.The results showed that the menopausal model group had significant bone destruction and bone mass decrease.Intragastric metformin improved bone quality and mass.12.Metformin improved bone mass in postmenopausal mice by reversing purine metabolism disorders.Combined analysis of metabomics and network pharmacology was used to reveal the association of serum metabolites with metformin in postmenopausal mice and potential target of metformin in the treatment of osteoporosis.The results show that CANT1 and HPRT1 were two potential targets,and improving purine metabolism abnormalities was a potential pathway of metformin in the treatment of postmenopausal osteoporosis.13.Xanthine/xanthine oxidase had an obvious inhibitory effect on the activity of osteoblasts.Osteoblasts were treated with 100μm xanthine mixed with 0-25munit/m L xanthine oxidase for 24 h,and the activity of osteoblasts was detected by CCK8.The results showed that the activity of osteoblasts decreased with the increase of xanthine concentration.14.Metformin could reverse xanthine/xanthine oxidase-induced osteoblast apoptosis.The activity,apoptosis rate and protein expression of mitochondrial apoptosis genes Bax,Bcl-2,Caspase3,Cleaved caspase3 of osteoblasts were detected by CCK8,flow cytometry with Annexin V FITC staining and Western blotting,respectively.The results showed that metformin increased the activity reduced the apoptosis rate,inhibited the expression of Bax,Cleaved caspase3,and promoted the expression of Bcl-2 and Caspase3 of osteoblasts under the condition of xanthine/xanthine oxidase.Conclusion:1.Metformin improves H₂O₂-induced osteoblast apoptosis by reducing intracellular calcium ion concentration.2.Metformin improves H₂O₂-induced mitochondrial damage in osteoblasts by promoting EGFR/GSK-3βphosphorylation.3.Metformin improves bone mass in postmenopausal mice by reversing oxidative stress damage induced by purine metabolic disorders.