Effects Of Thyroid Stimulating Hormone On Bone Metabolism And Its Mechanism

Background:Osteoporosis(OP)is a systemic metabolic bone disease characterized by decreased bone strength,damaged bone microstructure,increased bone fragility,and easy fracture.With the aging of population,the incidence of osteoporosis is increasing year by year,which has become a global health problem.Changes in thyroid function are known to have profound effects on bone growth and maintenance of skeletal homeostasis in adults.The effect of thyroid dysfunction on bone has been attributed to changes in circulating thyroid hormones,but little attention has been paid to the simultaneous alteration of thyroid stimulating hormone(TSH).Subsequent studies have found a correlation between bone metabolism and serum TSH levels.These results indicate that TSH may directly regulate bone metabolism independent of thyroxine.In order to confirm the effect of TSH on bone metabolism,the previous study of our group found that with the increase of serum TSH concentration,male bone mineral density also increased,suggesting that the occurrence of male OP may be closely related to the change of serum TSH concentration.Compared with wild-type mice,mice with thyroid stimulating hormone receptor(TSHR)gene knockout had bone microstructure destruction,decreased bone mechanical performance,decreased femur deformation resistance and fracture resistance even under normal thyroid hormone level.In this study,based on the phenotype of TSHR systemic knockout mice,the mouse models of osteoclast,osteoblast and chondrocyte specific knockout TSHR were constructed respectively to explore the effects of TSH on osteoclast differentiation and bone metabolism from in vivo and in vitro experiments,which will contribute to osteoporosis.In particular,it provides a reference for the diagnosis and treatment of abnormal bone metabolism related to thyroid function,and provides a new idea for the development of anti-osteoporosis drugs.Objectives:1.To explore the causes of abnormal bone metabolism in systemic TSHR gene knockout mice2.To observe the effect of TSHR gene knockout on bone metabolism in mice3.To explore the effects of TSHR gene knockout on bone metabolism in osteogenic and cartilaginous precursor cells4.To study the effect of TSH on osteoclast differentiation in vitro5.To explore the mechanism of TSH inhibiting osteoclast differentiationMethods:1.Fluorescence flow cytometry was used to analyze the typing ratio of primary bone marrow cells of Tshr+/+ and Tshr-/-mice,and to detect the changes in the number of CD11b positive cells of osteoclast precursor cells after TSHR gene knockout.2.CD11b positive cells in primary bone marrow cells of Tshr+/+ and Tshr-/-mice were sorted by magnetic beads for transcriptome analysis to understand the changes of gene expression profile of osteoclast precursor cells by TSHR gene knockout.3.Tshr+/+ and Tshr-/-mouse bone marrow primary cells were induced to differentiate into osteoclasts in vitro.The effects of TSHR knockout on osteoclast differentiation in vitro were observed by tartrate resistant acid phosphatase(TRAP)staining.Acridine orange staining was used to observe the effect of TSHR knockout on the acid-secreting function of osteoclasts,i.e.bone resorption capacity.4.Myeloid specific TSHR gene knockout mice(Lyz2-Cre+Tshrflox/flox(TSHRMKO))were constructed,and the bone marrow primary cell typing ratio of myeloid specific TSHR gene knockout mice was analyzed by mass spectrometry flow cytometry to verify the changes of TSHR gene knockout on the number of CD11b positive cells in osteoclast precursor cells.The effects of TSHR gene knockout on bone strength,bone microstructure and bone morphology during osteoclast differentiation and development were analyzed by biomechanical experiments,micro CT bone scanning,HE staining and TRAP staining of femur slices.5.Mesodermal specific TSHR gene knockout mice(Twist2-Cre+Tshrflox/flox(TSHRCKO))were constructed,and the effects of TSHR gene knockout on bone metabolism during the differentiation and development of osteoblasts and chondrocytes were analyzed by biomechanical experiments,micro CT bone scanning,HE staining of femur sections and Runx2 immunohistochemical staining.6.RAW264.7 cell line was cultured and induced to differentiate into osteoclasts in vitro.The effects of TSH addition on osteoclast differentiation at different stages were observed,and the effects of TSH on phenotype,gene and protein expression of osteoclast differentiation were analyzed by TRAP staining,qPCR,western blot,transcriptomics and proteomics.Explore possible molecular and signaling pathways.7.Primary mouse bone marrow cells were cultured and induced to differentiate into osteoclasts in vitro.To explore the molecular mechanism of TSH’s effect on osteoclast differentiation,transcriptome analysis was conducted to analyze the differentially expressed genes and enrichment of bone marrow monocytes during TSH treatment in the process of osteoclast differentiation.Results:1.Compared with Tshr+/+ mice,the proportion of CD11b positive cells in primary bone marrow cells of Tshr-/-mice decreased,that is,the proportion and number of osteoclast precursor cells decreased.2.CD11b positive cells in bone marrow of Tshr+/+ mice and Tshr-/-mice were obtained by magnetic bead sorting.Differential genes were screened by transcriptome sequencing,and genes related to osteoclast differentiation and signaling pathway changes were enriched.3.Bone marrow primary cells of Tshr+/+ mice and Tshr-/-mice were cultured to induce osteoclast differentiation in vitro.The osteoclast differentiation and bone resorption capacity of Tshr-/-mice bone marrow primary cells were enhanced,in spite of decreasing number of precursor cells.4.By mass spectrometry flow cytometry analysis,CD11b positive cells were decreased in primary bone marrow cells of Lyz2-Cre+Tshrflox/flox(TSHRMKO)mice,and the proportion of CD11b positive cells was decreased,and the number of osteoclast precursor cells was decreased compared with that of the same-litter control Tshrflox/flox mice.The maximum load and stiffness of femur in TSHRMKO mice were decreased,cortical BMD,cancellous BMD,trabecular volume to total bone volume ratio(BV/TV)were significantly decreased,trabecular separation(Tb.Sp)had an increasing trend,cortical bone area(Ct.Ar)and cortical bone total area(Tt.Ar)had a decreasing trend.Staining of femur sections showed increased osteoclasts,enhanced bone resorption and decreased bone mass in TSHRMKO mice.5.Compared with control group Tshrflox/flox mice,Twist2-Cre+Tshrflox/flox(TSHRCKO)mice reduced body length,body weight and femur length,as well as the maximum load and stiffness of femur.Cortical bone mineral density,cancellous bone mineral density,BV/TV,trabecular number(Tb.N),trabecular thickness(Tb.Th)and trabecular connection density(Conn.D)decreased significantly,while Tb.Sp and structural model index(SMI)showed an increasing trend.Cortical bone thickness(Ct.Th),cortical bone area(Ct.Ar)and cortical bone total area(Tt.Ar)showed a decreasing trend.Staining of femur sections showed reduced osteoblasts,bone formation,and bone mass in TSHRCKO mice.6.TRAP staining and transcriptomics showed that TSH inhibited the differentiation of primary mouse bone marrow cells into osteoclasts.7.TSH inhibits the differentiation of RAW264.7 cell line into osteoclast,mainly at the initial stage of differentiation,activates AMPK signaling pathway,increases AMPK phosphorylation and inhibits mTOR phosphorylation,thereby inhibiting transcription factors NF-kB and NFATcl and inhibiting osteoclast differentiation related gene and protein expression.Conclusions:TSH inhibits osteoclast differentiation and bone resorption by activating AMPK signaling pathway,promotes osteoblast bone formation,increases bone mass and has a bone protective effect.