The Influence And Mechanism Of Fluorosis In Estrogen Deficiency Induced Osteoporosis

Part I:Establish and identify the estrogen deficiency-induced osteoporosis mice modelObjective Osteoporotic animal models are the essential tools to study of the pathogenesis of osteoporosis and the pharmacology of osteoporosis drugs.At present,there are 4 types of osteoporotic animal models:1)hormone intervention-induced osteoporosis animal models,2)immobilization-induced osteoporosis animal models,3)dietary intervention-induced osteoporosis models,and 4)other types of osteoporosis models.Among these models,the ovariectomized osteoporosis rat model is one of the most commonly used animal models.In this study,20 female Balb/c rats were used and divided into sham-and ovariectomized-group.And imaging,ELISA,and PCR assays were used to identify whether the model were successful,and to provide scientific evaluation methods for drug treatment of the disease.Methods 20 female 6-month-old Balb/c mice were randomly divided into two groups:sham operation group and castrated group after one week of adaptive feeding.Each group of 10.After 3 months,the animal body weight was weighed,and then the animals were sacrificed by cervical cutting.Remove the mouse femurs,HE staining,and to observe the bone tissue structure under light microscope;using Micro-CT detection of bone density,and calculate the mean trabecular width and spacing.Results Compared with the sham operation group,the weight of ovariectomized group did not changed significantly,and the weight of the uterine the Ovx group decreased by 76%.Morphological examination showed that compared with sham operation group,the thickness of cortical bone in the proximal tibia of the mice was significantly decreased,and the bone marrow was hollow.In addition,the trabecular bone volume of Ovx group third was decreased and thinned.In addition,the average width of trabecular bone was reduced and the spacing of trabecular bone increased.Bone mineral density(BMD)was significantly decreased in the ovariectomized group(Ovx).Conclusion The mouse model of osteoporosis,which is easy to use and easy to replicate,is a reliable method for the preparation of osteoporosis model.Part II:Study on the effect and molecular mechanism of fluorosis on estrogen-deficiency induced OP mice modelObjective Estrogen deficiency in postmenopausal women frequently activates osteoclasts(OC),accelerates bone resorption,and leads to osteoporosis(OP).Previous studies have demonstrated that interferon y(IFNy)could increase bone resorption and may be involved in postmenopausal OP.Fluorosis also increased the risk of fractures and dental fluorosis,and fluoride may enhance osteoclast formation and induce osteoclastic bone destruction in postmenopausal women,but the underlying mechanisms are as yet unknown.In this study,we investigated the effects of fluorine on ovx-induced bone loss,and tried to find out the probably mechanism.Methods To investigate whether NaF involved in PO,we tested the differences of bone morphometric parameters such as BV/TV,Ob.S/BS,Oc.S/BS,and Tb.N,Tb.Sp,Oc.N/B.Pm,Oc.S/BS,and ES/BS among different groups.To investigate whether IFNy is involved in the fluoride-inducing OP,we analyzed serum fluoride,IFNy,and BMD levels.We also measured the levels of IFNy mRNA and protein in BMMs harvested from sham-operated and Ovx WT mice with or without F ion-containing water for 8 weeks after surgery.In addition,To investigate the role of IFNy in estrogen-and/or fluoride-dependent bone loss,we used IFNy-knock-out(KO)mice with either sham-or Ovx-operation,and made them drink deionized water with or without F ions after surgery to analyze BMD,serum CT,and bone morphometric parameters.To eliminate the interference of autoimmunity of C57BL/6 mice and investigate the contribution of IFNy signaling in T cells to the bone loss induced by E deficiency and/or fluoride,Balb/c nude mice were reconstituted using purified WT and IFNγ-/-spleen T cells.To confirm whether fluoride-enhanced bone loss was mediated by ERs in vitro,Raw264.7 cells were treated with estrogen or NaF.And detected the IFNy in both mRNA and protein levels.Results In vivo study,compared with Ovx group,the Ovx + F group exhibited a more powerful effect on these parameters than that in Ovx mice.The results confirmed that fluoride mediated bone loss.Compared with sham-operated mice,IFNy mRNA and protein in Ovx BMMs were both increased(0.93-fold and 0.98-fold,respectively).Ovx + F significantly enhanced the mRNA and protein levels of IFNy compared with Ovx(0.35-fold and 0.42-fold,respectively),which imply that IFNy may be involved in fluoride-induced OP in Ovx-induced mice.The further study which used IFNγ-KO mice shown that BMD and serum CTx were not influenced in either Ovx-IFNγ-/-or Ovx + F-operated nude mice.However,BMD was significantly decreased in Ovx nude mice compared with sham nude mice following adoptively transferred T cells from WT mice.In vitro study,we found that estrogen could inhibit IFNy in both mRNA and protein levels;however,IFNγ expression was increased by NaF significantly.Subsequently,we used shRNA blocked ERa and ERβ,respectively.We found that NaF-inducing IFNy was mediated by ERa,but not ERβ.Conclusion In this study,we found that fluoride increasing the risk of postmenopausal OP by stimulating IFNy.When mice showed specific blockades of IFNy signaling,both estrogen deficiency(ovx-operation)and fluorine failed to induce bone loss.Furthermore,this study found that fluorine increased the expression of IFNy and resulted in bone loss,which was mediated by ERα,but not ERβ.All these results suggest that suppression of IFNy levels may constitute a new therapeutic approach for preventing bone loss.Part III:Imaging features of osteoporosis in postmenopausal women Objective To evaluation the imaging and features of clinical imaging of osteoporosis in postmenopausal women.Methods 45 healthy women of childbearing and osteoporosis in postmenopausal women suffering osteoporosis among 10-12 years.Healthy women were selected as normal control group and the postmenopausal osteoporosis women were selected as research group.All subjects are determination of follicle stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E2),Forearm AP&LAT,Tibia Fibula AP&LAT,Pelvis AP,and L-Spine PA&LAT were scaned by CT and MRI.The mean CT values of L1-L4 vertebral cancellous bone were measured at the same time.The bone density of L1-L4 vertebrae was measured by dual energy X-ray absorptiometry.The bone mineral density,individual bone mineral density and T value were recorded.The BMD of L1-4 were calculated by DEXA and analyzed by SPSS 17.0 software.All data were analyzed by two independent samples t test,among them,the number of positive CT values in the osteoporosis group was compared with that of DXA measured by the McNemar chi-square test,when the p-value was lower than 0.05 that there were significant differences.Results Compared with control group,the estradiol level in postmenopausal osteoporosis group was significantly decreased,by contrast,the follicle stimulating hormone(FSH)and luteinizing hormone(LH)were significantly increased,and the T value was-2.75 ± 0.94.BMD was 0.631 ± 0.197.And 45 patients with osteoporosis,only 39 were detected in DXA.All cases showed different degrees of low bone mineral density,thinning of the bone cortex,sparse trabecular bone.X-ray plain film was detected in 26 cases,and 45 cases were detected by CT.The MRI in the T1WI sequence showed a patchy or homogeneous signal in the vertebral body,and 37 cases were detected.Conclusion After menopause,estrogen deficiency can inhibit the activity of osteoblasts,and at the same time,increase the activity of osteoclasts,which eventually leads to osteoporosis.Dual energy X-ray absorptiometry can be used to measure bone mineral density,CT has higher density resolution,and the two is more sensitive to the evaluation of osteoporosis.The ability to selectively measure the CT value of the cancellous bone of the vertebral body is more sensitive to the diagnosis and evaluation of osteoporosis.Part IV:The imaging changes of fluorosis on the of osteoporosis in postmenopausal womenObjective To study the changes of imaging manifestations and biochemical indexes of fluorosis on postmenopausal women with osteoporosis.Methods 35 healthy women of childbearing and osteoporosis in postmenopausal women suffering osteoporosis among 10-12 years.Healthy women were selected as normal control group and the postmenopausal osteoporosis women were selected as research group.All subjects are determination of blood and urine fluoride,cathepsin K(Cathepsin K,Cat K),tartrate resistant acid phosphatase(TRAP-5b),alkaline phosphatase(ALP),calcitonin(Calcitonin,CT),osteoprotegerin(osteoprotegerin,OPG),RANKL(receptor activator of nuclear factor with kappa B ligand,receptor activator of nuclear factor kappa B ligand.Forearm AP&LAT,Tibia Fibula AP&LAT,Pelvis AP,and L-Spine PA&LAT were scaned by CT and MRI.The BMD of L1-4 were calculated by DEXA and analyzed by SPSS 17.0 software.All data were analyzed by two independent samples t test,when the p-value was above 0.05 that there were significant differences.Results The levels of blood fluorine,urine Fluorine,RANKL,Cathepsin K,TRACP-5b,Calcitonin in postmenopausal osteoporosis women with fluorosis were increased,and OPG,ALP were decreased.and.The average CT value of L1-L4 vertebral,T value and BMD value of body were decreased.X-ray and CT findings of all cases showed that the bone texture was thick and sparse,and the density was reduced.MRI mixed in T1WI sequence list signal.Conclusion Fluorosis can aggravate the degree of osteoporosis in postmenopausal women.