Effect Of Quercetin On Bone Metabolism In Ovarian Ovariectomized Rats And Its Mechanism

Effect of Quercetin on Bone Metabolism in Ovarian Ovariectomized Rats and Its MechanismPart one:Effect of quercetin on osteoporosis in ovariectomized ratsBackground:Osteoporosis is a clinically common metabolic disease,mainly characterized by loss of bone mass and destruction of trabecular bone microstructure.After menopause,due to the lack of estrogen in the body,the immune system is activated.The inflammatory factor is chronically increased,the osteogenesis and osteoclast balance are broken,and the osteoclast activity is higher than that of osteogenesis,which eventually leads to further aggravation of osteoporosis.A large number of studies have confirmed that Eucommia ulmoides leaves have anti-osteoporosis effects.Compared with other ingredients,Eucommia ulmoides has the strongest anti-menopausal osteoporosis ability.It has been confirmed that quercetin has an anti-osteoporosis effect as an active ingredient of eucommia flavonoids.In this study,we investigated the effect of quercetin on osteoporosis in ovariectomized rats,and provided an effective clinical basis for the development of traditional Chinese medicine for postmenopausal osteoporosis.Objective:The aim of this study was to comparative and analysis of quercetin curative effect in the treatment of osteoporosis in ovariectomized rats.Method:30 female Sprague-Dawley rats were randomly divided into sham operation group,model group,quercetin group.The sham operation group and the model group were given sodium carboxymethylcellulose 3ml/d.The quercetin group was given quercetin 50mg/kg/d.One time a day,8W.(1)After 8 weeks,the rats were anesthetized by intraperitoneal injection of 1%sodium pentobarbital solution,blood was collected from the femoral artery,and serum levels of calcium(S-Ca)and phosphorus(S-P)were measured.(2)Urine was extracted from the bladder of rats and the levels of urinary calcium(U-Ca)and phosphorus(U-P)were analyzed.(3)Micro-CT was used to detect the changes of trabecular bone microstructure in femoral metaphysis.(4)Statistical analysis:All experimental data were analyzed by SPSS22.0.All data were compared by analysis of variance and expressed as x±s.Results:(1)Quercetin can reduce the loss of calcium and phosphorus in urine.(2)The effect of quercetin on S-Ca,S-P in rats was not yet indicated.(3)Quercetin can improve postmenopausal osteoporosis,bone microstructure,increase bone density,but it can not make the trabecular structure fully restored.Conclusion:(1)Quercetin as a eucommia flavonoid has anti-osteoporosis effect on ovariectomized rats.(2)Quercetin achieves anti-osteoporosis by improving bone metabolism,increasing BMD,and improving bone morphological parameters.Part two:Effect of quercetin on bone formation induced by BMSCsBackground:BMSCs are an important source of osteogenic precursor cells.Recent studies have found that increased apoptosis of BMSCs is a key factor leading to the reduction of osteogenic precursor cells and an important cause of postmenopausal osteoporosis.Studies have shown that quercetin can not only induce osteoclast apoptosis,inhibit bone marrow absorption by osteoclasts,but also enhance the differentiation activity of osteoblasts,and fundamentally alleviate the symptoms of osteoporosis.The focus of this section is to investigate whether quercetin can increase the osteogenic differentiation of BMSCs and increase the number of osteoblasts in vitro,thereby increasing postmenopausal osteoporotic bone density.This part of the study provides a theoretical basis for the study of the molecular mechanism of quercetin in promoting osteogenic differentiation.Objective:The focus of this section is to investigate whether quercetin can increase the osteogenic differentiation of BMSCs and increase the number of osteoblasts in vitro,thereby increasing postmenopausal osteoporotic bone density.Method:(1)40 SD rats were randomly divided into Sham group and OVX group.After 8 weeks,BMSCs from each group were isolated and cultured in vitro.(2)BMSCs from sham group and OVX group were extracted,subcultured and BMSCs cell surface antigen were identified.(3)The BMSCs in the OVX group were treated with different concentrations of quercetin(5μM、10μM、20μM).The effects of quercetin on the proliferation of BMSCs in OVX rats were observed by MTT assay.(4)Effects of different concentrations of quercetin on the induction of post-osteogenesis calcification nodules in BMSCs cells by alizarin red staining(5)PNPP method was used to detect the effect of quercetin on the activity of alkaline phosphatase ALP in BMSCs cells induced by osteogenic cells.(6)Statistical analysis:All experimental data were analyzed by SPSS22.0.All data were compared by analysis of variance and expressed as x±s.Results:(1)The cell surface antigens of BMSCs in each group were identified by flow cytometry,and CD29.、CD90 were positive,but CD45 was negative,which was consistent with the surface markers of BMSCs(2)Quercetin can increase the proliferation of BMSCs in ovariectomized rats,and the optimal concentration is 10μM(3)Quercetin can induce osteogenic differentiation of BMSCs in ovarian ovariectomized rats and the optimal concentration is 10μM.Conclusion:Quercetin can promote the proliferation and osteogenic differentiation of BMSCs The optimal concentration of quercetin is 10μM.Part three:FoxO1 mediates quercetin anti-TNF-α-induced apoptosis of BMSCsBackground:The post-menopausal women’s estrogen expression levels are reduced,and the chronic sustained activation of the immune system produces a large number of inflammatory factors.TNF-a is one of the most important inflammatory factors,which plays an important role in inducing apoptosis of osteogenic precursor cells.TNF-a can not only activate NF-κB signaling,but also increase the apoptosis of BMSCs induced by reactive oxygen species(reactive oxygen species,ROS).In addition,the expression of FoxO1 gene(Forkhead box transcription factor O1,FoxO1)was increased,and the increased of FoxO1 gene expression could increase the anti-apoptotic ability of BMSCsStudies have shown that PI3K(phosphoinositide3-kinase)signaling pathway is a key factor regulating apoptosis and oxidative balance,and oxidative stress can also activate PI3K,transforming different extracellular stimuli(environmental stress and pro-apoptotic factors nuclear).In addition,there is evidence that the FoxO protein family is an important downstream target of the PI3K/AKT pathway,involved in apoptosis,promotes the inactivation of apoptotic proteins,and attenuates the induction of reactive oxygen species.However,it is still unclear how this protective mechanism of antioxidant stress works.Objective:To observe the effect of quercetin on the apoptosis of BMSCs induced by TNF-a,and further explore its mechanism of action.Method:(1)The fourth generation BMSCs were divided into normal control group,TNF-αgroup,quercetin + TNF-α group,quercetin + TNF-α + PI3K inhibitor group,quercetin+ TNF-α group + FoxO1 inhibitor group,using MTT method.The cell viability of each group of BMSCs was observed.(2)The intracellular apoptosis levels of Sham control group,TNF-group,quercetin +TNF-group,quercetin +TNF-group,PI3K inhibitor group and quercetin+TNF-group +FoxO1 inhibitor group were detected by flow method.(3)Intracellular ROS levels were detected in Sham control group,TNF-group,quercetin +TNF-group,quercetin +TNF-group,PI3K inhibitor group,quercetin+TNF-group and FoxO1 inhibitor group.(4)The antioxidant levels of the normal control group,TNF-α group,quercetin +TNF-α group,quercetin + TNF-α + PI3K inhibitor group,quercetin + TNF-α group +FoxO1 inhibitor group were detected by SOD and MDA methods.(5)The mRNAtranscription level and protein expression level of PI3K and FoxO1 in each group were detected by qPCR and Western blot.Results:(1)Quercetin can significantly increase the proliferation rate of rat BMSCs,and the proliferation rate is the highest at a concentration of 10 μM.(2)Compared with Sham group,the intracellular apoptosis rate of TNF-group was significantly increased,while the cell apoptosis rate was decreased after quercetin intervention.After adding PI3K and FoxO1 inhibitors to quercetin +TNF-group,their apoptosis levels were significantly increased.(3)Compared with Sham control group,the intracellular ROS content in TNF-group was significantly increased,indicating that the intracellular TNF-level increased and the intracellular ROS content increased.After adding quercetin,the ROS content was significantly reduced,indicating that quercetin can promote the decrease of intracellular ROS content.The ROS levels of quercetin +TNF-group increased significantly after adding PI3K and FoxO1 inhibitors respectively.(4)Compared with the Sham group,the SOD content in the TNF-a group was significantly decreased,while the SOD content was significantly increased after the intervention with quercetin.After adding PI3K and FoxO1 inhibitors to the quercetin TNF-a group,the SOD content was significantly reduced.(5)Compared with Sham group,MDA content in TNF-group was significantly increased,while that in quercetin group was significantly decreased.The content of MDA in quercetin +TNF-group increased significantly after adding PI3K and FoxO1 inhibitors.(6)Compared with Sham group,the transcriptional levels of Bax and P65 in TNF-group were significantly increased,and the transcriptional levels of bcl-2 and FOXO1 were significantly decreased.After the intervention with quercetin,the transcriptional levels of FOXO1 and bcl-2 in TNF-group were significantly increased,and the transcriptional levels of Bax and P65 were significantly decreased,indicating that quercetin can increase the transcriptional levels of FOXO1 and bcl-2 in cells,and significantly reduce the transcriptional levels of Bax and P65.After the addition of PI3K and FoxO1 inhibitors to the quercetin +TNF-group,the transcription levels of FoxO1 and bcl-2 in the cells were significantly decreased,and the transcription levels of Bax and P65 were significantly increased.In other words,PI3K and FoxO1 could promote the transcription levels of FoxO1 and bcl-2 and reduce the transcription levels of Bax and P65.Conclusion:Quercetin can inhibit the apoptosis of BMSCs induced by TNF-α.The anti-apoptotic ability of quercetin to enhance BMSCs may be achieved by regulating PI3K/Akt/Fox01/NF-kB signaling pathway and inhibiting free oxygen release.Part four:Quercetin promotes bone formation through the FoxO1 signaling pathwayBackground:Postmenopausal osteoporosis is a systemic aging of the bone or a disease associated with the degradation of waste.Its main features are reduced bone mass,degeneration of bone tissue,and increased bone fragility.Bone marrow mesenchymal stem cells(bone marrow mesenchymal stem cells,BMSCs)are important sources of osteogenic precursor cells,and BMSCs have good proliferative capacity and multi-tissue differentiation potential.BNSCs can differentiate into different cell lines such as osteoblasts,chondroblasts,and adipocytes under the induction of specific microenvironments.Quercetin belongs to eucommia flavonoids and is an effective phenolic antioxidant.It can protect the body by reducing the level of lipid peroxide and increasing the antioxidant system.Our previous research in this topic also confirmed that quercetin is the main component of eucommia flavonoids against osteoporosis.Osteoporosis model rats received quercetin treatment,bone mineral density and trabecular bone number increased significantly.Quercetin can increase the anti-apoptotic ability of BMSCs and the ability to differentiate into osteoblasts,it can achieve the effect of treating osteoporosis.But the exact mechanism needs further research.Objective:To explore the relationship between quercetin induced osteogenic and the mechanism of FoxO1 signaling pathways.Method:(1)The fourth generation BMSCs were divided into Sham group,OVX group,OVX+quercetin group,OVX+quercetin+PI3K inhibitor group,OVX+quercetin+FoxO1 inhibitor group.(2)The osteogenic differentiation of Sham group,OVX group,OVX+quercetin group,OVX+quercetin+PI3K inhibitor group,OVX+quercetin+Foxo1 inhibitor group were analyzed by alizarin red staining.(3)The osteogenic differentiation of Sham group,OVX group,OVX+quercetin group,OVX+quercetin+PI3K inhibitor group,OVX+quercetin+FoxO1 inhibitor group were analyzed by PNPP method.(4)The transcription levels of FoxO1,P65,Runx2,and Osterix in the Sham group,the OVX group,the OVX+quercetin group,the OVX+quercetin+PI3K inhibitor group,and the OVX+quercetin+FoxO1 inhibitor group were detected by qPCR.(5)The expressions of FoxO1,P65,Runx2,and Osterix in the Sham group,the OVX group,the OVX+quercetin group,the OVX+quercetin+PI3K inhibitor group,and the OVX+quercetin+FoxO1 inhibitor group were detected by the WB method.Results:(1)Compared with the Sham group,the number of intracellular mineralized nodules in the OVX group was significantly reduced;compared with the OVX group,the number of mineralized nodules in the quercetin+OVX group was significantly increased;compared with the quercetin+OVX group,PI3K inhibitor and FoxO1 inhibitor were added respectively,and the number of mineralized nodules were significantly reduced.(2)Compared with the Sham group,the intracellular ALP level of BMSCs in the OVX group was significantly decreased.Compared with the OVX group,the ALP level in the quercetin+OVX group was significantly increased;compared with the quercetin+ OVX group,PI3K inhibitor and FoxO1 inhibitor were added respectively,and there ALP level were significantly reduced.(3)Compared with OVX group,the transcriptional and translation levels of FOXOl、Osterix and Runx2 in cells of Sham group were significantly increased,and the transcriptional and translation levels of P65 were significantly decreased,indicating that the rat model of osteoporosis was successfully established.After quercetin intervention,the transcription and translation levels of P65 in OVX group were significantly decreased,and the transcription and translation levels of FOXO1、Osterix and Runx2 were significantly increased,indicating that quercetin could increased the transcription and translation levels of FOXO1、Osterix and Runx2 in OVX group.Compared with the ossification group induced by quercetin +OVX,the transcriptional and translation levels of FoxO1,Osterix and Runx2 in cells were significantly decreased after the addition of PI3K and FoxO1 inhibitors respectively,and the transcriptional and translation levels of P65 were significantly increased,that is PI3K and FoxO1 could promote the increase of the transcriptional and translation levels of FoxO1、Osterix and Runx2,and reduce the transcriptional and translation levels of P65.Conclusion:The induction of osteogenesis by quercetin may be through the PI3K/Akt/Foxo1/NF-KB signaling pathway,which regulates the expression of Runx2/Osterix.