| Part 1: TNF-? promotes osteoclast differentiation by up-regulating semaphorin3 D expression in estrogen-deficiency induced osteoporosisObjectivesOsteoporosis seriously affects the health of the elderly and is a global public health problem.Estrogen deficiency-induced osteoporosis is the most common metabolic bone disease,which is associated with estrogen deficiency,characterized by reduced systemic bone mass and destruction of the bone tissue microstructure,leading to increased bone fragility.However,the exact mechanisms of estrogen deficiency-induced osteoporosis were not fully elucidated.Estrogen deficiency-induced over-activation of osteoclasts is a leading cause of PMOP.Estrogen deficiency in postmenopausal women induce osteoblasts and T cells to produce cytokines,which can stimulate osteoclasts differentiation and activiation.It has been found that TNF-?,as a pre-inflammatory factor,is the first factor of many inflammatory factors and is also the critical factor for PMOP.TNF-?-induced osteoclasts differentiation is the main cause of increasing of osteoclasts number in PMOP.Given the complexity of the regulatory mechanisms of osteoclast regulation by TNF-?,its exact regulatory mechanism needs to be further explored.In recent years,more and more studies have shown that Semaphorins ? family of proteins is involved in the pathological process of bone remodeling and bone metabolism disorders.Semaphorin3A-deficient mice exhibit a phenotypically abnormal bone structure characterized by osteopenia.1,25-Dihydroxyvitamin D3 can promote the expression of Semaphorin3 B in osteoblasts,inducing the formation of osteoclasts and leading to the reduction of bone mass.However,it is unclear whether Semaphorins family proteins are also involved in the regulation of osteoclast differentiation by TNF-? in PMOP.In this study,molecular biology techniques,gene silencing and overexpression will be applied to screen the semaphorins ? family proteins involved in the induction of osteoclasts differentiation by TNF-? in PMOP.We will further clarify the role of semaphorins ?family proteins in TNF-? induced osteoclast differentiation and explore themolecular mechanism of TNF-? induced osteoclast differentiation through semaphorins family of proteins.This study provides experimental evidence for understanding the mechanism of postmenopausal osteoporosis and exploring new therapeutic targets.Methods1.The osteoclasts derived from normal mice were cultured.TRAP staining and qRT-PCR testing the expression of osteoclasts marker genes(TRAP,c-Fos and NFATc-1)were applied to clarify the effect of TNF-? on osteoclast differentiation.2.The expression of Sema ? family proteins(Sema3A,Sema3 B,Sema3C,Sema3 D and Sema3E)and Sema ? family protein receptor(Plx-A1,Plx-A2,Plx-A3,Plx-A4,NPR1 and NPR2)were detected by qRT-PCR during TNF-?-increased RANKL-induced osteoclast differentiation.3.Sham mice,ovariectomized(OVX)mice,OVX + PBS mice and OVX + TNF-?neutralizing antibody(anti-TNF-?)mice were constructed.The levels of TNF-? in in each group mice were detected by enzyme-linked immunosorbent assay(Elisa),and the model was determined by micro-computed tomography(micro-CT).4.The number of osteoclasts in the femoral metaphysis from Sham mice,OVX mice,OVX + PBS mice and OVX + anti-TNF-? mice were observed by TRAP staining.5.The bone marrow-derived macrophages(BMMs)from femur and tibia in each group mice were acquired and differentiated into mature osteoclasts in vitro.The expression of sema3 D in these osteoclasts were detected by qRT-PCR and Western blot.6.Sema3 D overexpression and shRNA lentiviral vector were used to infect osteoclast precursor cells.TRAP staining and qRT-PCR detecting the expression of osteoclast-forming marker genes(TRAP,c-Fos and NFATc-1)were used to observe the effect of Sema3 D on RANKL-induced osteoclasts differentiation.In addition,and the effect of overexpressing/gene silencing of Sema3 D on TNF-?-enhanced RANKL-induced osteoclast differentiation were also observed.7.The effect of overexpressing/gene silencing of Sema3 D on TNF-? induced osteoclast precursors proliferation was observed by cell viability assay(CCK-8)assay,EdU fluorescence staining assay and CFSE fluorescence intensity assay.8.Western Blot was used to examine the effects of TNF-? on the activation of p38,p65 and JNK signal pathways at different time points(5 min,15 min,30 min,60 min)in RANKL-induced osteoclasts differentiation.9.After blocking p38,p65 and JNK signal pathways with SB203580?BAY 11-7082?SP600125 respectively,western Blot and qRT-PCR were used to observed the effect of TNF-? on Sema3 D expression in RANKL-induced osteoclasts differentiation.10.TRAP staining and qRT-PCR testing the expression of osteoclasts marker genes(TRAP,c-Fos and NFATc-1)were applied to observe the effect of blocking JNK signal pathways after overexpressing Sema3 D on osteoclast differentiation.11.Nanoparticles of chitosan-coated siRNA-Sema3 D were constructed,and then OVX mice were injected via the tail vein.The morphological changes of bone in Sham mice,OVX mice,OVX + siRNA-Sema3 D mice and OVX + siRNA-NC mice were observed by micro-CT.Results1.TNF-? could promote osteoclast formation in vitro.In addition,TNF-? could significantly increase osteoclasts formation at early stage(day 0,1 and 2),whereas adding TNF-? to osteoclastic precursor cells at late stage(day 3 and 4)could not affect osteoclasts formation.2.TNF-? significantly increased the expression of sema3 D during the process of osteoclast formation.Furthermore,TNF-? upregulated the expression of sema3 D in a time-dependent manner.In addition,we found that TNF-? significantly decreased the expression of NPR1 and decreased the expression of Plx-A3 during osteoclastogenesis.3.Overexpression of Sema3 D significantly enhanced RANKL-induced osteoclastogenesis,whereas silencing of Sema3 D obviously reduced RANKL-induced osteoclast differentiation.In addition,overexpression of Sema3 D can significantly promote the TNF-?-positive regulation of RANKL-induced osteoclast formation,while blockade of Sema3 D can alleviate TNF-?-induced osteoclast formation,suggesting that Sema3 D was involved in TNF-?-induced osteoclast formation.4.TNF-? can significantly promote the proliferation of osteoclast precursor cells.Silencing of Sema3 D can significantly relieve TNF-?-induced osteoclast precursor cell proliferation.These results suggested that Sema3 D was involved in TNF-?-mediated the proliferation of osteoclasts precursors.Sema3 D overexpression or gene silencing did not affect cells apoptosis.5.The p38,p65 and JNK signaling pathways were further activated in TNF-?-induced osteoclast differentiation.Blocking of JNK signaling pathway with SP600125 cansignificantly alleviate the induction of sema3 D expression by TNF-?.In addition,blocking JNK signaling pathway can obviously alleviate TNF-?-induced osteoclastogenesis,whereas blocking the JNK signaling pathway followed by increasing expression of sema3 D can promote the formation of osteoclasts.6.Depletion of Sema3 D in vivo significantly alleviated OVX-induced osteoporosis and decreased the level of CTX-1,a marker of bone resorption,in the mouse serum.Conclusions1.Estrogen deficiency leads to the elevated levels of TNF-?.TNF-? is an important factor for PMOP,which could increase osteoclast differentiation and bone resorption.TNF-? could significantly increase osteoclasts formation at early stage.2.The expression of sema3 D was significantly increased during TNF-?-induced osteoclast differentiation.On the one hand,sema3 D increases the number of osteoclasts by promoting the proliferation of osteoclast precursor cells.On the other hand,sema3 D influences the formation of osteoclasts by enhancing RANKL-induced osteoclast differentiation.3.TNF-? increased sema3 D expression through activation of JNK pathway.Silencing of Sema3 D can effectively alleviate OVX-induced osteoporosis in mice.Part 2: TNF-? inhibits osteoblast differentiation by down-regulating semaphorin3 B expression in estrogen-deficiency induced osteoporosisObjectivesOsteoporosis seriously affects the health of the elderly and is a global public health problem.Estrogen deficiency-induced osteoporosis is the most common metabolic bone disease,characterized by enhanced bone turnover with increased bone formation and even greater rates of bone resorption,leading to a net bone loss.Osteoporosis affects 40 % to50 % of women over 60 years of old,about 30 % to 50 % of them will suffer osteoporosis-related fractures that severely affect the health and quality of old women and even shorten their life span.However,the exact mechanisms of estrogen deficiency-induced osteoporosis were not fully elucidated.Normal bone remodeling maintains constant bone mass by an orchestrated balance between the resorption of old bone by osteoclasts and the formation of new bone by osteoblasts.Previous studies showed that the number and activity of osteoclasts were increased due to estrogen deficiency in postmenopausal women.However,bone loss caused by increasing of osteoclasts was not fully compensated by osteoblasts proliferation.Thus,the balance of bone formation-bone resorption was disturbed,resulting in bone loss and increased fracture risk.Recently studies showed that in the early PMOP,osteoclast activity was increased and osteogenesis was also compensatory increase.However,as the disease progresses,osteogenic activity was inhibited,leading to disturb the balance of bone resorption and bone formation,resuling in bone loss.It was been confirmed that estrogen deficiency in postmenopausal women induce osteoblasts and T cells to produce cytokines.It has been found that TNF-?,as a pre-inflammatory factor,is the first factor of many inflammatory factors and is also the critical factor for PMOP.Osteoblasts are differentiated from MSCs,and TNF-? can inhibit the osteogenic differentiation of MSCs through bonding to its receptor TNFR1.The inhibitory effect of TNF-? on osteogenic differentiation of MSCs is an important reason for the reduction of bone formation in PMOP.However,the exact mechanism by which TNF-?inhibits the differentiation of MSCs into osteoblasts has not been fully elucidated.In recent years,more and more studies have shown that Semaphorins ? family proteins is involved in the pathological process of bone remodeling and bone metabolism disorders.Sema3 A can promote the differentiation of osteoblasts and inhibit the formation of osteoclasts.Sema3 E can inhibit mouse osteoblast migration and inhibit osteoclast formation.In addition,osteoblast-specific transgenic mice expressing Sema3 B significantly enhanced bone mineralization.Although these studies confirm that the Sema III family proteins is closely related to bone metabolism,it is unclear whether Sema III family proteins are also involved in the inhibition of TNF-?-induced MSCs differentiation into osteoblasts in PMOP.PurposeIn this study,molecular biology techniques,gene silencing and overexpression will be applied to screen the semaphorins ? family proteins involved in the inhibition of osteoblasts differentiation by TNF-? in PMOP.We will further clarify the role of semaphorins ?family proteins in TNF-? inhibited osteoblast differentiation and explorethe molecular mechanism of TNF-? inhibited osteoclast differentiation through semaphorins family of proteins.This study provides experimental evidence for understanding the mechanism of postmenopausal osteoporosis and exploring new therapeutic targets.Methods1.The BMMSCs derived from normal mice were cultured.The expression of Sema? family proteins(Sema3A,Sema3 B,Sema3C,Sema3 D and Sema3E)and Sema ?family protein receptor(Plx-A1,Plx-A2,Plx-A3,Plx-A4,NPR1 and NPR2)were detected by qRT-PCR during TNF-?-decreased differentiation of BMMSCs into osteoblasts.2.Sham mice,ovariectomized(OVX)mice,OVX + PBS mice and OVX + TNF-?neutralizing antibody(anti-TNF-?)mice were constructed.The levels of TNF-? in in each group mice were detected by enzyme-linked immunosorbent assay(Elisa),and the model was determined by micro-computed tomography(micro-CT).Immunohistochemistry was used to test the expression of TNF-? in the metaphysis of femur in mouse model.3.A mouse model with TNF-? neutralizing antibody to inhibit OVX-induced osteoporosis was conducted.The expression of Sema3 B in the metaphysis of femur was observed by immunohistochemistry.The expression of Sema? family protein receptor(Plx-A1,Plx-A2,Plx-A3,Plx-A4,NPR1 and NPR2)were detected by qRT-PCR.4.The BMMSCs from femur and tibia in each group mice were isolated.The expression of Sema3 B was measured by qRT-PCR.5.The BMMSCs from each group mice were isolated and cultured with osteogenic inducing medium for different time points(7 days,14 days).QRT-PCR and western blot were used to detect the expression of Sema3 B.Elisa assay was used to detect the content of TNF-? in the cell culture medium.6.Sema3 B overexpression and shRNA lentiviral vector were used to infect BMMSCs.ALP staining,ALP activity assay,Alizarin red staining and qRT-PCR detecting the expression of osteoblast-forming marker genes(Runx2,Osterix and Osteocalcin)were used to observe the effect of Sema3 B on the differentiation of BMMSCs into osteoblasts.In addition,the effect of overexpressing/gene silencing of Sema3 B on TNF-?-decreased osteoblasts differentiation were also observed.7.The effect of overexpressing/gene silencing of Sema3 B on TNF-?-decreased BMMSCs proliferation was observed by cell viability assay(CCK-8)assay,EdUfluorescence staining assay and CFSE fluorescence intensity assay.8.Effect of TNF-? on the expressions of DKK-1,GSK-3? and ?-catenin,which were key proteins in Wnt/?-catenin Signaling Pathway,was examined in BMMSCs by qRT-PCR.9.Wnt/?-catenin signaling in BMMSCs was activated with Licl(20 mM)and BIO(5?M),respectively,followed by treating with 10 ng/ml TNF-? for 24 h.Then the expression of Sema3 B in BMMSCs was examined by western blot.Results1.The expression of sema3 A,sema3C,sema3 D and sema3 E was not significantly change in TNF-?-inhibited the differentiation of BMMSCs into osteoblasts,while the expression of sema3 B was significantly reduced in a time-dependent manner.2.The bone mineral density(BMD)and static microstructure parameters(Tb.Th,BV /TV and Tb.N)in OVX mice were significantly lower than those in sham mice,while those in OVX + anti-TNF-? mice obviously increase.Compared with Sham control mice,the number of trabecular bone was decreased in OVX mice.Neutralizing antibody of TNF-?could obvious alleviate the change of rabecular bone structure of metaphyseal femur in OVX mice.3.The level of TNF-? in OVX mice was significantly higher than that in Sham mice,but TNF-? neutralizing antibody could obviously reduce the level of TNF-? in OVX mice.Compared with sham mice,TNF-? expression was significantly increased in OVX mice metaphyseal,and while the application of TNF-? neutralizing antibody can significantly attenuate it.4.The expression of Sema3 B in OVX mice was significantly decreased compared with that in sham mice,while the expression of Sema3 B was significantly increased in OVX mice treated with TNF-? neutralizing antibody.Compared with Sham-MSCs,the expression of Sema3 B in OVX-MSCs was significantly decreased,while the neutralizing antibody of TNF-? significantly increased Sema3 B expression in BMMSCs from OVX mice.5.Sham-MSCs,OVX-MSCs,OVX+anti-TNF-?-MSCs were cultured with osteogenic inducing medium in vitro for 7 days and 14 days respectively.The results showed that the expression of Sema3 B in OVX-MSCs was significantly less than that in Sham-MSCs.However,the expression of Sema3 B in OVX + anti-TNF-?-MSCs was significantlyincreased compared with OVX-MSCs.Compared with Sham-MSCs,the level of TNF-? in cells culture medium of OVX-MSCs was significantly increased,while the level of TNF-?in OVX-anti-TNF-?-MSCs was significantly less than that in OVX-MSCs.6.The expression of Sema? family protein receptors(NRP1,Plx-A1 and Plx-A4)were significantly decreased in OVX-MSCs,while the expression of Plx-A3 was significantly increased.7.Overexpression of Sema3 B significantly promoted the osteogenic differentiation of BMMSCs,while silencing of Sema3 D significantly inhibited the osteogenic differentiation of BMMSCs.In addition,overexpression of Sema3 B can significantly decreased the TNF-?-negative regulation of the differentiation of BMMSCs into osteoblasts,while blockade of Sema3 B can promote the inhibitory effect of TNF on osteogenic differentiation,suggesting that reduction of Sema3 B was involved in TNF-?-inhibited the differentiation of BMMSCs into osteoblasts.8.TNF-? can significantly inhibit the proliferation of BMMSCs,and overexpression of Sema3 B can significantly relieve the inhibitory effect of TNF-? on the proliferation of BMMSCs.9.TNF-? inhibited the expression of DKK-1,GSK-3? and ?-catenin in the differentiation of BMMSCs into osteoblasts,indicating that TNF-? can significantly inhibit the Wnt / ?-catenin signaling pathway in BMMSCs.10.Activation of Wnt / ?-catenin signaling pathway can significantly relieve the inhibitory effect of TNF-? on Sema3 B expression in BMMSCs,suggesting that TNF-?negatively regulates the expression of Sema3 B by inhibiting the Wnt / ?-catenin signaling pathway.Conclusions1.Estrogen deficiency leads to the elevated levels of TNF-?.TNF-? is an important factor for PMOP,which could decrease the differentiation of BMMSCs into osteoblast,resulting in reduced bone formation.The expression of sema3 B was significantly decreased during TNF-?-mediated osteoblast differentiation.TNF-? inhibited the proliferation of BMMSCs and the differentiation of BMMSCs into osteoblasts through decreasing the expression of sema3 B.2.TNF-? inhibited the expression of DKK-1,GSK-3? and ?-catenin in the differentiation of BMMSCs into osteoblasts,indicating that TNF-? can significantly inhibitthe Wnt / ?-catenin signaling pathway in BMMSCs.The activation of Wnt / ?-catenin signaling pathway can significantly relieve the inhibitory effect of TNF-? on the expression of Sema3 B in MSCs,suggesting that TNF-? negatively regulates the expression of Sema3 B by inhibiting Wnt / ?-catenin signaling pathway. |
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