Study On Aucubin Promotes Osteoblast Differentiation And Inhibits Osteoporosis Through Nrf2/Keap1 Signaling Pathway

With the continuous improvement of people's pursuit of quality of life,more and more adult orthodontic patients have entered our field of vision.It is not uncommon for these adult orthodontic patients to be accompanied by osteoporosis.At the same time,there are many elderly patients who need to use orthodontics to lower the teeth of the jaws.These patients are high-risk people with osteoporosis,and we must pay attention to it.Osteoporosis?OP?is a systemic bone caused by a decrease in bone tissue content and degradation of bone microstructure,resulting in increased bone fragility and reduced strength resulting in fractures.disease.Osteoporosis is more than just a disease and has become a serious social problem.Aucubin?AU?,also known as coral glucoside,is a cyclic olefinic mushroom compound extracted from plants.It has anti-oxidation,anti-photoaging and collagen synthesis effects.It is a potential Pure natural anti-aging protective agent.Eucommia,one of the sources of aucubin,has been confirmed by many scholars to promote osteogenesis and inhibit bone breakage.However,whether or not the extract of Eucommia ulmoides has a role in promoting osteogenesis,there are few reports.Oxidative stress damage is now recognized as one of the important pathogenesis of osteoporosis,Nrf2/Keap1/ARE signaling pathway is a classic pathway of oxidative stress.The pathway is inside the body resistance to external stimulation such as oxidation and chemical defensive transduction pathways.Oxidative stress was confirmed by Nrf2/Keap1/ARE signaling pathways mediating osteoporosis.BMP2 is a kind of bone morphogenetic proteins,bone formation and bone reconstruction is an important factor,it can induce mesenchymal cells into the bone formation.The increase of the level of BMP2 can directly stimulate the ALP,OCN,Collagen I and Integrin beta 1 the osteogenetic differentiation related protein expression.BMPs and two main types of transmembrane Ser/Thr kinase receptor,to activate the Smads and non Smads signal pathway.Smads can directly activate Osterix within hours,Smad1/5/8 can improve the ALP,OCN and Collagen I level.BMP2 induction of Smad pathway with a family and Akt MAPKs signaling pathways,both can induce osteoblast differentiation.BMP2 can through different BMP type I receptor activation of MAPKs family,occurs after the phosphorylation of MAPKs?Erk,JNK and P38lightning?combined with target protein,further regulate OPN,ALP and Collagen I.Akt signaling pathway can enhance the stability of Osterix transcription activity in order to regulate osteoblast differentiation.Therefore,this study to osteoblast and osteoporosis mice as the research object,peach leaf coral glycosides as the experimental tools,through the research of peach leaf coral glycosides and the function of osteoblast proliferation and differentiation of mice role in contributing to bone osteoporosis conditions,coral glycosides of peach leaf through the research of the new Chinese medicine monomer for the prevention and treatment of osteoporosis to provide new experimental basis,to provide a new direction of the orthodontic treatment of osteoporosis.This study first studied the effect of AU on osteoblast MG63 differentiation and its preliminary mechanism in vitro.The results showed that AU had no significant effect on the viability of MG63 cells;AU can induce differentiation of MG63 cells and increase osteoblasts.Differentiation factor Alkaline phosphatase?ALP?activity promotes mineralization in MG63 cells;osteoblast differentiation marker protein assay results show that AU can promote type I collagen in osteoblasts?Collagen I,the expression of Integrin?1,recombinant protein?Osterix?,osteocalcin?OCN?and Osteopontin?OPN?protein,further confirm that AU can promote osteoblast differentiation;AU can Promotes the expression of Bone morphogenetic protein 2?BMP2?-Smads,Mitogen-activated protein kinase?MAPKs?and Akt/mTOR/p70s6k proteins in pathways related to osteoblast differentiation;Preliminary indicates that AU can promote the differentiation of osteoblasts.In vitro,this study used dexamethasone?Dex?to establish a model of osteoblast MG63injury,first using AU pretreatment for 2 h,and then with glucocorticoid Dex for 24 h,by detecting osteoblasts The expression of death,related proteins,expression of differentiation-related proteins,oxidative stress and changes of antioxidant proteins were studied to study the protective effect of AU on osteoblasts.The results showed that AU can inhibit the apoptosis rate of osteoblasts induced by Dex.Reduce the accumulation of intracellular ROS and reverse the decrease of mitochondrial transmembrane potential?MMP?;AU can inhibit the expression of differentiation-related proteins OCN,OPN,Collagen I and Osterix in MG63 cells,and promote the key protein of osteoblast differentiation.Expression of BMP2 and its downstream Smads protein;AU can up-regulate the expression of Nrf2/Keap1 anti-oxidation pathway protein and increase the antioxidant enzymes Heme oxygenase-1?HO-1?and heme oxygenase-2?Heme oxygenase 2,HO-2?,Superoxide Dismutase-1?SOD-1?,Superoxide Dismutase-2?Superoxide Dismutase 2?The activity of SOD-2?indicates that AU has a protective effect on Dex-damaged osteoblasts,which is related to the Nrf2-induced oxidative stress pathway.Based on the above results,the oxidative damage model of osteoblast MG63 was established by using oxidant hydrogen peroxide?H₂O₂?.Firstly,AU pretreatment was used for 2 h,and then treated with H₂O₂ for 24 h to detect apoptosis of osteoblasts.And the expression of related proteins,the expression of differentiation-related proteins,oxidative stress and changes of antioxidant proteins,etc.,studied the protective effect of AU on oxidative damage of osteoblasts,and the results showed that AU can inhibit the apoptosis rate of osteoblasts induced by H₂O₂.Reduces the accumulation of intracellular ROS and reverses the decrease of MMP in cells;AU can inhibit the expression of differentiation-related proteins OCN,OPN,Collagen I and Osterix in MG63 cells,and promote the expression of BMP2 and its downstream Smads proteins.AU can up-regulate the expression of Nrf2/Keap1 anti-oxidation pathway protein and increase the activities of antioxidant enzymes HO-1,HO-2,SOD-1 and SOD-2,indicating that AU has protective effect on oxidatively injured osteoblasts.Protection is related to the Nrf2 antioxidant stress pathway.In an in vivo experiment,a mouse model of osteoporosis was established using dexamethasone sodium phosphate injection,and a simultaneous method of modeling and administration was used.After 8 weeks of AU treatment,the tissue morphology and tissue structure of the femur and tibia of the mice were observed,the mineral content in the peripheral blood of the mice was detected,and the expression levels of osteoblast differentiation-related factors in the peripheral blood and bone tissues of the mice were detected.The expression levels of oxidative stress-related factors in peripheral blood and bone tissues of mice were measured.The therapeutic effect of AU on osteoporosis mice induced by Dex was studied.The results showed that the femurs of mice in the AU group were observed by femoral section staining.The cortical bone showed continuous continuity,the number of osteoclasts decreased,the number of trabecular bone cells increased,and the trabecular bone density increased.Micro-CT of mouse bone tissue observed in the AU-treated group compared with the model group.The rat cortical bone is thickened and continuous,the cortical bone mass is increased,the bone density is increased,the bone cancellous structure is dense,and the bone mineral density?BMD?and Bone volume fraction?BV/TV?of the mouse are obtained.Trabecular thickness?Tb.Th?and Trabecular number?Tb.N?have significant increases,trabecular spacing?Trabecular spacing?Tb.Sp)showed a decreasing trend;in addition,the effect of AU can increase the levels of Ca and Pi in peripheral blood of mice;the effect of AU can increase the osteoblast differentiation factors ALP,Collagen I,OCN,OPN in peripheral blood of mice.,BMP-2 and Bone morphogenetic protein receptor type II?BMPR-2?expression levels,promote Collagen I,Osterix,OCN in mouse bone tissue,OPN,BMP-2 protein expression levels and phosphorylation levels of Smads and Akt proteins;AU can inhibit the level of ROS in peripheral blood of mice and promote the antioxidant enzyme catalase in peripheral blood of mice?Catalase,The expression levels of CAT?and SOD promote the expression levels of oxidative stress Nrf2 signaling pathway-related proteins Nrf2,Keap1,CAT,HO-1,HO-2,SOD-1,SOD-2 in mouse bone tissue,thereby promoting Oxidative stress inhibits the expression of signaling pathways;this result further indicates that AU can promote osteoblast differentiation and has a therapeutic effect on osteoporotic mice,which is related to the Nrf2 antioxidant pathway.In summary,AU has the effects on promoting differentiation and injury protection of osteoblast MG63,and has the therapeutic effect on Dex-induced osteoporosis mice,which is related to Nrf2-mediated oxidative stress pathway.This study provides a potential therapeutic agent on the treatment of osteoporosis.