The Study On The Mechanism Of Gut Microbiome Dysbiosis And Decreased Bone Mineral Density Induced By Exposure To Tributyltin

ObjectivesTributyltin(TBT)is identified as an environmental obesogen,which could promote lipogenesis and induce obesity via activation of peroxisome proliferator activated receptor ?(PPAR?)at nanomole concentration.TBT was widely used as bactericide in agriculture and antiseptic paint for ocean steamships.This chemical substance has the characteristics of strong lipophilicity and long half-life reduction,so it can widely distributed in environmental media.Human exposed to it through water intake,seafood dietary and food wrap..Previous studies by our group have shown that exposure to environmental concentrations of TBT could cause metabolic syndrome in mice,i.e.bodyweight gain,dyslipidemia,impaired glucose tolerance,nonalcoholic fatty liver,and impaired insulin signaling pathway.Previous studies have pointed out that the development of obesity and metabolic abnormalities induced by TBT are related to islet cell apoptosis,adiponectin level disorder,epigenetic changes,inflammation and other factors,but the mechanism of TBT-induced obesity is complicated,which should be paid more attention.There are a vast ensemble of microbes inhabiting in human gut which contain approximately 1014 cells and over 100 times more genes than human genome.The gut microbiota provide us with important metabolic capabilitiesby involving in food digestion,absorption,metabolism as well as other physiological processes.In recent years,the role of gut microbiome dysbiosis in the progression of obesity is receiving increasing attention.At the same time,researchers have begun to pay attention to the phenomenon that the exposure of chemical substances in the environment affects the metabolic function of the body by affecting the steady state of the gut microbiome,which in turn causes the development of obesity.More and more studies have shown that exposure to TBT could induce obesity and metabolism disorder in vivo,however,whether TBT could lead to gut microbiome dysbiosis and the relationship of this change and obesity development was still unknown.So the topic project of the first part in this paper was aimed to explore the relationship among TBT exposure,gut microbiome disorder and obesity progression based on the following aspects:1)To clarify phenotypes of metabolic syndrome after exposure to environmental levels of TBT.2)Whether TBT exposure could induce gut microbiome dysbiosis(checked by 16s rRNA sequencing).3)Explore the relationship of gut microbiome dysbiosis and obesity development.After clarifying the association between gut microbiome dysbiosis induced by TBT and obesity in mice,we further explored the effects of low-dose TBT exposure on bone health.PPAR? is a key factor in the formation of adipocyte differentiation,and high expression of PPARy can promote the differentiation process of adipocytes.The mesenchymal stem cells in the bone marrow cavity are progenitor cells of osteocytes and adipocytes,and the directed differentiation of the two has an exclusive characteristic of either or both.Previous researches have confirmed that TBT exposure could reprogram bone marrow mesenchymal stem cells(BM-MSCs)towards adipogenesis while away from osteogenesis both in vitro and in vivo.The high differentiation of adipocytes in the marrow cavity is associated with a variety of bone metabolic diseases.However,whether TBT exposure affects bone health is currently not systematically studied.Dual-energy X-ray absorptiometry analyse(DXA)is a recognized "gold standard" for non-invasive detection of bone mineral density(BMD)and diagnosis of osteoporosis by WHO.Because the BMD of the mice was lower than the detection limit of the instrument used in this study,SD rats were used for bone health related experiments.The second part in this paper was designed to explore the mechanism and molecular basis of TBT-induced bone density decline:1)The effect of TBT exposure on the BMD and biomechanical properties tested by DXA and three-point bending test;2)Further observation for the morphological characteristics of the femur by HE staining and Oil Red O staining;3)The effect of TBT on the expression of the biomarkers of adipogenesis and osteogenesis detected by quantitative PCR;4)The effects of TBT exposure on biomarkers and their possible regulatory networks by immunohistochemistry and immunofluorescence.Methods1.Establishment and identification of obesity model induced by TBT exposure in miceAfter 3 days of adaptive feeding,21-day-old male SPF ICR mice were randomly divided into 2 groups according to body weight,with 10 rats in each group.Mice were intraperntoneally injected every 3 days for a total of 10 exposures.The injection was TBTCl dissolved in corn oil,and the dose was 0,50 ?g/kg(injected at 5 ml/kg·bw).The body weight was weighed and recorded before each intraperitoneal injection,and the amount of injection was adjusted according to the weight of the day.After the last TBTCl exposure,the mice were given normal rearing for 30 days.At the end of the experiment,four mice were selected randomly in each group to sterilely collect their faeces before sacrifice,and all mice were weighed.After light anesthesia,blood was collected from the retro-orbital venous plexus of the mouse,stored at room temperature for 2h.Serum was separated at 3000g for 15 minutes and stored at-80? for detection of serum triglyceride(TG),total cholesterol(TC)and low-density lipoprotein(LDL).After dissection of the cervical spine,the epididymis and perirenal adipose tissue were dissected and weighed.2.16s rRNA sequencing and data analysisAfter extracting total DNA from mice feces,PCR amplification and product purification were performed.After the quality-checked library was mixed,it was added to the Illumina MiSeq sequencing platform for high-throughput parallel sequencing.After filtering the data,the Alpha diversity and Beta diversity of the gut microbiome were analyzed and the difference was tested.3.Correlation analysis between gut microbiome dysbiosis and metabolic abnormality caused by TBTThe association among TBT exposure,gut microbiome dysbiosis and bodyweight gain was established by analyzing the linear correlation between weight gain and Shannon's index,Simpson's index,the abundance of Proteobacteria and the abundance of Lactobacillaceae.4.Establishment of a rat model of BMD induced by TBT exposureAfter three days for acclimatization,21-day-old male SPF SD rats wer divided into 4 groups according to body weight(n=10).Rats were treated by gavage every 3 days for 60 days.The injection was TBTCl dissolved in corn oil,and the dose was 0.5,5,50 ?g/kg(injected at 5 ml/kg·bw).The body weight was weighed and recorded before each gavage,and the amount of injection was adjusted according to the weight of the day.The rats were sacrificed ten days after the last TBTCl exposure(PNDs 94).5.Evaluation of BMD and biomechanical properties in ratsAfter the rats were sacrificed,the right femur was taken,with the muscle tissue and connective tissue removed.And the weight and length were measured.The DXA method was used to measure the BMD at the metaphysis and the diaphysis of the femur,followed by a three-point bending test.The maximum load and stiffness coefficient of the bone were obtained by displacement-stress curve analysis.6.Bone histomorphometryAfter fixed in 4%paraformaldehyde for 4 days,5 samples per group were decalcified by 10%(w/v)EDTA solution(pH=7.4)for four weeks.After decalcification was completed,bones were cut off just proximal to the knee-joint.Three demineralized femurs were embedded in paraffin and sectioned serially at 5 ?m intervals.Hematoxylin and eosin(HE)staining and Oil Red O staining were using standard procedures,in order to observe the morphological structure and lipid accumulation of the femur.7.Immunohistochemical and immunofluorescence assayTo investigate the effects of TBT exposure on adipogenic osteogenesis in bone marrow and its possible regulatory network,immunohistochemical was used to detect the expression of adipose-forming markers(PPAR?),bone formation markers(Runx2)and Wnt signaling pathway key molecules(GSK3?)in rat femur.Another key molecules of Wnt signaling pathway(?-catenin)were detected by immunofluorescence method.8.Real-Time quantitative RT-PCRTrizol was used to extract bone marrow total RNA,and quantitative RT-PCR analysis was used to analyze the expression of biomarkers of osteogenesis(Runx2?alkaline phosphatase.?osteocalcin)and adipogenesis(PPAR?,FABP4 and so on).GAPDH was used as loading control,the melting curve of each target gene was examined,and the relative expression of each target gene was analyzed.We investigated the effect of TBT exposure on the expression of major regulators of lipogenesis and osteogenesis in bone marrow.Results1.TBT exposure increased body weight and epididymal adipose tissue mass in miceDuring the experimental period,the mice in each group had normal activities and mental status.There was no armpit hairiness between the groups.At the end of the experiment,the bodyweight of mice in the 50 ?g/kg TBTCl group increased by 7.7%compared with the control group(P<0.01).The anatomical results showed that the mass of epididymal adipose tissue was also significantly increased in 50 ?g/kg TBTCl mice(P<0.01).2.Tributyltin exposure induces gut microbiome dysbiosisCompared with the control group,the numbers of observed bacterial phylotypes decreased by 18.5%in TBT exposure mice(P<0.05),accompanied by a higher Simpson's index(P<0.05)and a lower Shannon s index(P<0.01),indicating that TBT induced a significant decline in the gut microbiome richness of mice.At the same time,The taxonomic assignments and fold changes of gut microbial communities were significantly changed.At phylum level,the abundances of Proteobacteria and Deferribacteres both increased significantly after TBT exposure(P<0.05 and P<0.01,respectively),while the level of Firmicutes declined(P<0.05).And at Family level,Lactobacillaceae and Bifidobacteriaceae reduced by 4.84 and3.24 fold,respectively,in TBT-exposure group,while Helicobacter increased by 6.78 fold,compared with the control group.3.Correlation between gut microbiota and body weight.Body weight was negatively correlated with Shannon's index(R2=0.7974;P<0.01).While it was positively related to Simpson's index(R2=0.6947;P<0.05),so did to the abundance of Proteobacteria(R2=0.7877;P<0.01).In addition,a clear negative correlation was identified between the body weight and the abundance of Lactobacillaceae(R2=0.6048;P<0.05).4.Exposure to TBT reduces diaphysial BMD in rats.50 ?g/kg TBT-exposure resulted in a significantly decreased BMD at the femur diaphysis region(-18.75%;P<0.01).However,there were no difference in in femoral length,weight and metaphyseal BMD between TBT treatment rats and control rats.Although TBT treatment resulted in a dose-response relationship reduction in maximum load and stiffness through the displacement-stress curve of rat femurs,the 11.3%reduction in maximum load and 28%reduction in maximum stiffness in rat exposed to 50 ?g/kg TBTCl compared to control,did not reach statistical significance(P=0.19 for maximum load and P=0.10 for maximum stiffness).5.TBT promoted adipogenesis while inhibited osteogenesis in vivo.In HE staining,although the adipocyte diameter did not show statistical differences among the groups,we found that the number of fat cavities increased with increasing dose.In the Oil Red O staining,only a small area of the oil red O colored region was present in the control group,and a large amount of fat accumulation appeared in each group exposed by TBTCl.The area of the Oil Red O staining area was significantly different(respectively increased by 2.94 3.45 4.62 compared to the control).At the same time,serum alkaline phosphatase activity decreased,suggesting that bone formation activity was weakened.The PCR results further revealed that the expression levels of the osteogenic markers Runx2 and alkaline phosphatase in the bone marrow decreased.Finally,immunohistochemistry confirmed that the expression of the adipogenic marker PPARy was elevated in the bone marrow.Conversely,the expression of the osteogenic marker Runx2 was decreased.6.Wnt signal pathway was suppressed in TBT-treated group.Immunohistochemistry and immunofluorescence showed that the key molecules of Wnt signaling pathway in bone marrow had different expression levels in different groups:50p.g/kg TBTCl exposed group showed obvious lower expression of ?-catenin in both the diaphysis and metaphysis.The decline suggests that TBT exposure can negatively regulate the expression of the Wnt signaling pathway in the bone marrow.Conclusions1.Exposure to environmental levels of TBT in adolescent induces gut microbiome dysbiosis in mice,which is closed to obesity development.2.Exposure to environmental levels of TBT resulted in decreased BMD in femur and abnormal accumulation of lipids in the bone marrow.3.TBT exposure may enhance the adipogenic differentiation of bone marrow mesenchymal stem cells by negatively regulating Wnt signaling pathway.