Objective:Osteoporosis is a common metabolic disease that predominantly affects postmenopausal women.It is characterized by reduced bone density,destruction of the bone microstructure and increased bone fragility,which greatly increases the risk of fractures and seriously threatens the normal life of the patients.Estrogen deficiency in postmenopausal women causes the bone resorption of osteoclasts to exceed the bone formation of osteoblasts,but the specific pathogenesis is still unclear.At present,the treatment of osteoporosis is mainly based on drugs that inhibit osteoclasts.The mechanism is just to prevent further bone loss and the therapeutic effect is limited.Therefore,it is necessary to clarify the pathogenesis of postmenopausal osteoporosis and design drugs aimed at improving osteogenic ability.Some studies have found that oxidative stress damage is closely related to the development of PMOP.Oxidative stress is a pathological condition caused by excessive accumulation of reactive oxygen species in the body.Clinical studies demonstrated that variation of the serum oxidation-related biomarkers in postmenopausal women with osteoporosis,including antioxidant enzymes and advanced oxidation products,indicated that the patients were in a hyperoxidized state.Meanwhile,estrogen is a type of reducing hormone,and the lack of estrogen will inevitably lead to the decline of the body’s antioxidant capacity.At the cellular level,mitochondria are the key organelles in cells that eliminate free radicals and avoid oxidative stress damage.The decrease of its internal antioxidant enzyme activity leads to cell apoptosis through the mitochondrial apoptotic pathway,which is an important reason for the weakened function of osteoblasts in patients with osteoporosis.Recently,“conventional drug in new use”has increasingly become a hot topic for researchers.In particular,metformin has been shown to have therapeutic effects in many diseases.One of the most noteworthy is that metformin can delay the body’s aging,and the therapeutic mechanism is to eliminate excessive free radicals produced by the body,which suggests that metformin has a potential therapeutic effect in PMOP.Therefore,we aimed to explore the protective mechanism of metformin on oxidative stress damage of osteoblasts and its therapeutic effect on PMOP.Methods:1.CCK-8 reagent was used to detect the effect of metformin and H2O2 on osteoblast activity;2.Alkaline phosphatase kit was used to detect the effect of metformin on osteoblast differentiation;3.Alizarin red S staining method was used to detect the effect of metformin on osteoblast mineralization;4.Using transcriptome sequencing technology to detect the differentially expressed genes of metformin-induced osteoblast differentiation;5.Using Annexin V FITC kit to detect the protective effect of metformin on oxidative damage mediated apoptosis of osteoblasts.6.Using JC-1 kit to detect the change of mitochondrial membrane potential;7.Using RT-PCR to detect the m RNA level of target gene expression;8.Using Western blotting to detect the expression of target protein;9.Si RNA cell transfection technology was used to modulate the protein expression of target genes;10.Using micro-CT to detect the relevant parameters of mouse femoral cortex and cancellous bone;11.Using total SOD kit and T-AOC kit to detect mouse serum anti-oxidation Marker level;12.Statistical analysis:The results involved in the article have been carried out 3 repeated independent experiments,and analyzed using Graph Pad software.Two-sided t-test or one-way analysis of variance was used to determine statistical significance,p<0.05 has statistical significance.Results:1.With the concentration of H2O2 increasing,the inhibitory effect on the activity of osteoblasts was more significant.The osteoblasts were treated with 0m M;0.1m M;0.2m M;0.3m M H2O2 for 6 hours.The CCK8 measurement results showed that as the concentration of H2O2 increased,the osteoblast activity inhibition effect was lower.And the osteoblast activity was closer to the cells in the true state of oxidative damage when treated with 0.2m M H2O2.The appropriate concentration of metformin promoted the increase of osteoblast activity.Osteoblasts was treated with 0m M;0.1m M;0.2m M;0.3m M;0.4m M metformin for 24 hours,48 hours and 72 hours respectively.The results showed that the activity of osteoblasts was higher with time,and the cell activity was highest when treated with 0.2m M metformin,but 0.4m M metformin showed an inhibitory effect on osteoblast activity compared to untreated.2.Metformin could promote osteoblast differentiation and reverse apoptosis caused by oxidative damage.The osteoblasts were treated with 0m M;0.1m M;0.2m M;0.3m M metformin and osteogenic medium for 7 days respectively.The ALP level of the cells was detected by alkaline phosphatase kit,and The m RNA levels of Collagen I,OCN,Runx2 were detected by RT-PCR.Western blotting was used to detect the protein levels of Collagen I,OCN,and Runx2 of osteoblasts.The results showed that the osteoblast differentiation indexes were the highest when treated with 0.2m M metformin.In addition,the osteoblasts were treated with 0m M;0.1m M;0.2m M;0.3m M metformin and 0.2m M H2O2 respectively for 6h.The Annexin V FITC kit was used to detect the apoptosis rate of osteoblasts.The results showed that metformin treatment can reduce the apoptosis rate of osteoblasts when treated with H2O2alone,and the apoptosis rate was the lowest when0.2m M metformin and 0.2m M H2O2 were mixed for treatment.3.Metformin enhanced the mineralization of osteoblasts.0m M;0.1m M;0.2m M;0.3m M metformin and osteogenic medium were mixed respectively to treat osteoblasts for 28 days.Then these cells were stained with Alizarin Red S.The approximate changes of cells were observed by eyes and the mineralized nodules were observed by using a microscope.The results indicated that metformin could enhance the mineralization of osteoblasts and the effect was most significant when treated with 0.2m M metformin.4.Metformin attenuated the mitochondrial apoptosis of osteoblasts induced by H2O2.The osteoblasts were cultured in the blank group,0.2m M metformin group,0.2m M H2O2 group and 0.2m M metformin mixed with 0.2m M H2O2 group.Western blotting was used to detect the protein expression of mitochondrial apoptosis indicators Bax,Bcl-2,Caspase3,Cleaved-caspase3,Mitochondrial Cytochrome C and Cytosolic Cytochrome C.The results showed that Bax,Cleaved-caspase3,Cytosolic Cytochrome C protein expression decreased,and Bcl-2,Caspase3,Mitochondrial Cytochrome C protein expression increased when adding metformin compared with the blank group and H2O2 group.5.Transcriptome sequencing data indicated that the PI3K/AKT pathway and oxidation-reducation reaction mediated metformin to promote osteoblast differentiation.The osteoblasts were cultured in osteogenic medium and a mixed medium of osteogenic medium and 0.2m M metformin for 3 days.Transcriptome sequencing was used to analyze the differential expression genes of these two groups.The results showed that there were 1946 up-regulated genes and 1544 down-regulated genes after metformin treatment compared with osteogenic medium alone.The GO gene function and KEGG pathway analysis of the up-regulated genes showed that these up-regulated genes were enriched in the oxidation-reduction reaction and PI3K/AKT pathway.6.Metformin could reverse the inhibitory effect of H2O2 on osteoblast differentiation.The osteoblasts were divided into osteogenic induction group,0.2m M metformin plus osteogenic induction group,0.2m M H2O2 plus osteogenic induction group,and 0.2m M metformin mixed with0.2m M H2O2plus osteogenic induction group.Western blotting was used to detect the protein levels of Collagen I,OCN,and Runx2 in osteoblasts.The results showed that the protein levels of Collagen I,OCN,and Runx2 were increased after metformin treatment compared with the osteogenic induction group and the H2O2plus osteogenic induction group.7.PI3K/AKT pathway inhibitor inhibited the effect of metformin on the differentiation and mineralization of osteoblasts.The PI3K/AKT pathway inhibitor LY294002 was added to the mixed medium of osteogenic medium and metformin.Western blotting was used to detect the protein levels of Collagen I,OCN,and Runx2 in osteoblasts.The results showed that after adding LY294002,the protein levels of Collagen I,OCN,and Runx2 decreased.Using Alizarin Red S staining,it was found that after adding LY294002,the mineralization ability of osteoblasts was weakened,and the number of mineralized nodules was reduced.8.Metformin protected H2O2-induced mitochondrial damage and decreasing antioxidant capacity.JC-I detection kit was used to detect the changes of osteoblast mitochondrial membrane potential and Western blotting was used to detect the protein levels of SOD 1,CAT in osteoblasts.The results showed that the mitochondrial membrane potential decreased,and the protein expression level of antioxidant enzyme SOD 1,CAT also decreased with H2O2 treatment.But after adding metformin on the basis of H2O2,the mitochondrial membrane potential level and the antioxidant enzyme SOD 1,CAT protein expression increased.9.SIRT3-si RNA downregulated the expression of SIRT3 gene and reversed the protective effect of metformin on H2O2-induced osteoblast apoptosis.The Annexin V FITC kit was used to detect the apoptosis rate of osteoblasts and Western blotting was used to detect the protein expression of mitochondrial apoptosis indicators Bax,Bcl-2,Caspase3,Cleaved-caspase3,Mitochondrial Cytochrome C and Cytosolic Cytochrome C.The results showed that after SIRT3-si RNA transfection,the apoptotic rate increased,Bax,Cleaved-caspase3,Cytosolic Cytochrome C protein expression increased,and Bcl-2,Caspase3,Mitochondrial Cytochrome C protein expression decreased.10.SIRT3-si RNA down-regulated the expression of SIRT3 gene and reversed the protective effect of metformin on H2O2-induced mitochondrial damage.JC-I detection kit was used to detect the changes of osteoblast mitochondrial membrane potential and Western blotting was used to detect the protein levels of osteoblast SOD 1,CAT.The results showed that after SIRT3-si RNA transfection,the level of mitochondrial membrane potential and the protein expression of antioxidant enzymes SOD 1,CAT decreased.11.Metformin reversed the oxidative stress damage of osteoblasts induced by H2O2 through PI3K/AKT/Nrf2/HO-1 pathway.Western blotting was used to detect the protein expression levels of p-PI3K,PI3K,p-AKT,AKT,nuclear Nrf2,HO-1.The results showed that compared with H2O2 treatment,the protein expression levels of p-PI3K,p-AKT,nuclear Nrf2,and HO-1 increased after adding metformin.AKT inhibitor MK2206 was added with metformin,and Western blotting was used to detect the protein expression level of nuclear Nrf2 and HO-1 in osteoblasts.The results showed that the protein expression levels of nuclear Nrf2 and HO-1 decreased.12.Metformin improved bone mass and serum antioxidant capacity in postmenopausal mice.Bilateral ovariectomy was taken to mimic the menopausal model of mice and injected with100mg/kg/day metformin intragastrically for 8 weeks.Micro-CT was used to detect mouse femoral cortex and cancellous bone parameters,western blotting was used to detect the protein levels of Collagen I,OCN,Runx2,SOD 1 and CAT in mouse femur,and Elisa was used to detect serum SOD and T-AOC levels in mice.The results showed that bone mass lost,bone quality decreased,femoral Collagen I,OCN,Runx2,SOD 1,CAT protein levels decreased,serum SOD and T-AOC levels decreased in postmenopausal mice,but after the above indicators were all improved with intragastrical metformin.Conclusion:1.Metformin promotes the proliferation and differentiation of osteoblasts.2.Metformin promotes osteoblast differentiation through PI3K/AKT pathway.3.Metformin protects mitochondrial function by up-regulating SIRT3 expression and attenuates the oxidative stress damage of osteoblasts induced by H2O2 via the PI3K/AKT/Nrf2/HO-1pathway.4.Metformin improves bone mass and serum antioxidation levels in OVX mice. |