The Effect Of Tributyltin On Bone Density And Structure And Study On Its Mechanism In Rats

BackgroundOsteoporosis is one of the most common bone diseases.It is a systemic bone disease characterized by reduced bone mass and damaged bone microstructure,resulting in increased bone brittleness and increased fracture risk.High concentration and high intensity of environmental pollution has been curbed,but the problem of environmental pollution is still serious.Mounting evidence obtained from studies on animal models and population studies revealed that exposure to endocrine disruptors negatively affected bone health.Tributyltin(TBT),a notorious ED,is widely used in wood preservatives,disinfection of industrial circulation water,antifouling coatings for ships and slime treatment in paper mills.Human exposure to TBT occurs mainly through the consumption of contaminated dietary sources.At nanomole concentration,TBT could promote lipogenesis and induce obesity via activation of peroxisome proliferator activated receptor γ(PPARγ).PPARγ is a key transcriptional regulator of adipogenesis,and high expression of PPARγ can effectively promote adipocyte differentiation.Adipocytes and osteoblasts are derived from the common progenitor mesenchymal stem cells(MSCs).MSCs,as the common progenitor cells of adipocytes and osteoblasts,are considered to have a negative correlation between the two,meaning that the extensive formation of adipocytes will lead to the reduction of osteoblastsOur previous studies on BMD in rats based on DXA measurements have shown that exposure to low doses of TBT leads to a decrease in BMD in the femur and a decrease in the biomechanical strength of the femur.Although measurement of BMD is an indispensable tool to identify individuals at high risk of injury,bone densitometry affords only a two dimensional areal view of the three dimensional mineralized mass of the skeleton.Micro-computed tomography(μCT)is the most common technique for the nondestructive assessment and analysis of the three-dimensional bone architecture Micro-computed tomography(μCT)has a higher resolution and can achieve direct visualization of bone structure.It is the most commonly used technique for nondestructive evaluation and analysis of three-dimensional bone structureThe complete hierarchical structure of the bones is maintained by the repetitive,and temporal coupling.This process is called "bone reconstruction".In previous studies,we found that TBT exposure can lead to a decrease in the number of osteoblasts,an increase in adipocytes,and an imbalance of adipo-osteogenesis in the bone marrow cavity.This study continued to study the effects of TBT exposure on osteogenesis and adipogenesis on the basis of previous studies,and explore the effects of changes in bone microenvironment on osteoclasts and related mechanisms.ObjectivesBased on the results of previous studies,this study used DXA to detect the effect of TBT exposure on femoral bone mineral density in rats.Using μCT to investigate the change of bone mineral density is caused by the influence of bone structure,calculated the moment of polar inertia of femur according to μCT results,and analyzed the effects of TBT exposure on the anti-torsional stiffness of femur in rats.The effects of TBT exposure on bone were further analyzed by detecting the contents of blood calcium,blood phosphorus and femoral ash,as well as the contents of bone minerals such as calcium,phosphorus and magnesium.Quantitative PCR and immunohistochemical techniques were used to investigate the mechanism of TBT exposure on femur microstructure.Methods1.Animals and Treatment21-day-old male SD rats acclimatizative three days,randomly assigned into four groups(n=10)based on body weight to achieve similar average weights in different groups.Rats were treated with corn oil or TBT(0.5,5 or 50 μg·kg-1)by gavage once every three days from ages of 24 d to 84 d,and were killed one day after the final gavage After reaching the surgical level of anesthesia,blood was obtained from the aorta ventralis.The serum was separated with 2500 rpm*10 min and stored at-80℃ for further analysis.Both femurs were isolated modestly,and then the muscular tissue was removed.All right femurs were weighed and measured.Cutting the femoral head and patellar surface off the left femur,25ml syringe was filled with phosphate buffer solution(PBS,pH=7.4)to drain out of the bone marrow,and 200 mesh cells were screened and filtered.RBC ruptured fluid was added and centrifuged.Stored at-80℃and other bones fixed in 4%(vol/vol)paraformaldehyde for further research.2.Bone density testThe bone mineral density of the femur of rats was analyzed using the axial dual-energy X-ray bone mineral density meter(DXA).Take out the rat femur in the buffer solution,dry the femur with absorbent paper,and place it in the working area of the measuring instrument.At the metaphysis and diaphysis end of the femur,a 0.3cm*0.3cm square area was selected as our study area.In the experiment,we set the scanning resolution to and the scanning speed to 60mm/s.Repeat the experiment twice for each area,and take the average of the two measurements as the final value of the study area3.Micro-computed tomography(μCT)The fixed femurs were scanned with a Scanco μCT100 scanner.For cortical bone analysis,the femur specimens were scanned similarly.1mm thick sections immediately distal to the mid-diaphysis were used as ROI.In addition,total cross sectional area(TCS.Ar),cortical cross sectional area(Ct.Ar),cortical thickness(Ct.th),periosteal circumference(Ps.Cf),endocortical circumference(Ec.Cf)and marrow area(Ma.Ar)were obtained from the analysis.4.The polar moment of inertia(J0)Jo was calculated as the medio-lateral(IML)+antero-posterior(IAP)axes.5.RNA isolation and quantitative real-time reverse transcription-polymerase chain reaction(QPCR).Bone marrow(BM)was flushed from the left femur(n=5 per group).Glyceraldehyde-3-phosphate dehydrogenase was used as internal reference to examine the dissolution curves of each target gene and analyze the relative expression of each target gene.To investigate the effects of TBT exposure on the expression of transcription factors regulating adipogenesis and bone formation in bone marrow.6.Bone histomorphometryAfter fixed in 4%paraformaldehyde for 4 days,samples were decalcified by 10%(w/v)EDTA solution.After decalcification,hematoxylin and eosin(HE)staining and Trap staining were using standard procedures,in order to observe the osteoclast activity and lipid accumulation of the femur.7.Serum ParametersSerum Ca2+and Pi levels were detected by colorimetry,following the manufacturer’s instructions.8.Ash contentFive left femora per group were used to detect the ash content.The dried,and ash weights were determined as described previously.Ash content was determined as the ash weight normalized for hydrated weight.9.Determination of bone mineral contentFive right femurs were selected,use ICP to detect the content of calcium,phosphorus,magnesium and other elements in the rat femur,and further determine the changes in the content of various minerals in the rat bones.10.Immunohistochemical assayOPG,also called osteoclast inhibitor,can inhibit the activation of osteoclasts,promote osteoclast apoptosis,and play a very important role in the process of bone formation and bone resorption.SOX2 is one of the initial factors of induced pluripotent stem cells differentiation,and detection of its expression can reflect the effect of TBT exposure on the number of stem cells in the bone marrow cavity.Results1.Effect of TBT on body weight and femurRat body weights increased with prolonged feeding time,however,exposure to TBT caused no significant effects on the body weights at all time points observed compared to the control.The bone mineral density of the femoral diaphysis exposed to 50μg·kg-1 TBT decreased significantly.2.Effect of TBT on microstructure of femurIn contrast,the analysis of the mid-diaphysis showed that TBT-treated rats had a smaller Ct.Ar,Th.Ar and Ps.Cf than those of their control counterparts(P<0.05),but had no effects on other indexes.3.Effect of TBT on JoJ0 was significantly reduced in 50 μg·kg-1 TBT-treated rats by 26.28%,compared with control rats(P<0.05).4.Effect of TBT on the mRNA expression of genes involved in adipogenesis and osteogenesis in BMAnalysis with QPCR analysis showed no notable TBT-related changes in the expression of PPARy,Fabp4,ALP and OC at 84 d.The expression of Angptl4 showed a significant decrease in the 0.5 μg·kg-1 and 5 μg·kg-1 TBT groups compared with the control(P<0.05).5.Effect of TBT on the accumulation of bone fatThe results of HE staining of rat femurs showed that the number of femoral fat cavities increased significantly with the increase of the exposure dose(P<0.05).6.Effect of TBT on bone metabolismWe detected the bone metabolism biochemical markers in the serum of 84-day-old rats.The activity of TRAP showed a dose-dependent decrease on PND 84.The activity of TRAP was reduced by 19.96%(P<0.05),29.30%(P<0.01)and 53%(P<0.001),respectively,in rat treated with 0.5,5 and 50 μg·kg-1 TBT in comparison with the control group.There were no significant changes in serum levels of Ca2+.and Pi between the control and TBT treated rats.7.Effect of TBT on ash contentThere was no significant difference in ash content between the TBT groups and the control group.The tin ion in bone is below the detection limit8.Immunohistochemical assayImmunohistochemical results showed that OPG showed a weak positive expression under 50μg·kg-1 TBT exposure,and SOX2 did not change significantly after TBT exposureConclusion1.In conclusion,treatment with 50 μg·kg-1 TBT caused a significant decrease in femoral Ct.Ar,Th.Ar and J0 of rat.This results in a decrease in torsional stiffness of the femur and increases the risk of fracture.2.By the end of this study,rats had compensated for the effect of TBT on bone structure,which was manifested as reduced osteoclast activity and compensated for the effect of TBT on MSCs differentiation at the transcriptional level.