| Background:In recent years,with the continuous development of economy and society,the change of people's lifestyle and the increasingly prominent problem of population aging,the prevalence of osteoporosis(OP)and obesity has been increasing.Studies have revealed that OP and obesity are interrelated metabolic derangements,which are serious and prevalent health issues.Traditionally,evidence suggests that obesity protects against bone loss and OP,it has been previously believed that body weight was benefit for bone formation.However,more and more animal studies have confirmed that high fat diet consumption can lead to bone strength reduction and trabecular microstructure damage,clinical studies have also found that excess fat mass is a risk factor for bone loss in human.Recent studies have revealed that adipose tissue is not merely an energy-storing organ,but is also an important endocrine organ,it secretes a variety of biologically active molecules,such as adiponectin,leptin,and resistin etc,which participate in the body's energy metabolism,immune responses and inflammation responses.It has been found that the above-mentioned adipocytokines exert extensive biological activities,including the regulation of glucose metabolism,lipid metabolism,insulin sensitivity,cardiovascular function,and bone metabolism.In vitro,it was showed that adiponectin could promote the osteogenic differentiation of bone mesenchymal stem cells,promote the proliferation and differentiation of osteoblasts,and inhibit the generation of osteoclasts.Leptin can promote the osteogenic differentiation of human bone marrow mesenchymal stem cells,stimulate the proliferation,differentiation and mineralization of human osteoblasts,inhibit the function of osteoclasts,and reduce the formation of osteoclasts.In vivo,animal experiments have confirmed that adiponectin-adenovirus treatment increased trabecular bone mass,accompanied by decreased number of osteoclasts and levels of plasma NTx,a bone-resorption marker,while subcutaneous injection of leptin could prevent bone loss in ovariectomy rats and promote bone formation in ob/ob mice,and intraperitoneal injection of leptin may have a positive effect on SD rat femur fracture healing.Thus,adipocytokine plays an important role in the regulation of bone metabolism,and the abnormal secreted adipocytokine may also be an important reason for the increased risk of fracture in obese patients.Therefore,the study on the correlation between adipocytokine and bone metabolism and the mechanism may provide a new pathway for the prevention and treatment of osteoporosis.As a newly discovered adipokine,visceral adipose tissue derived serine protease inhibitor(vaspin)was identified as a member of the serine protease inhibitor(serpin)family,which is highly expressed in visceral adipose tissue when obesity and insulin levels peak in Otsuka Long-Evans Tokushima Fatty(OLETF)rats.Studies have found that vaspin can improve glucose tolerance levels,ameliorate insulin resistance,reduce inflammatory response,and it is also closely related to the occurrence of cardiovascular and cerebrovascular events.Both central vaspin administration and peripheral vaspin administration can improve the glycogen metabolism and insulin sensitivity of SD rats which fed by high fat diet.Vaspin manipulation can not only prevent the development of monocrotaline-induced pulmonary arterial hypertension in rats,but also prevent elevation of blood pressure through inhibition of peripheral vascular remodeling in spontaneously hypertensive rats.Notably,emerging studies have found that vaspin is closely related to bone metabolism in vitro.Recent studies have shown that vaspin attenuates osteoclast formation and reduce the apoptosis of human osteoblasts in vitro.The clinical studies observed that there was a positive independent association between vaspin and the femoral neck BMD in postmenopausal women.Therefore,it is logical to hypothesize that vaspin exerts a positive effect on bone metabolism as a compensatory factor.However,the current studies on vaspin and bone metabolism were all in vitro,and whether in vivo administration of vaspin exert any effect on bone metabolism as adiponectin and leptin in SD rats remain unknown.The cell signaling pathway related to bone metabolism has been a hot topic in recent years,which can provide a new therapeutic target for the prevention and treatment of bone metabolism-related diseases.Recent in vitro studies have revealed that vaspin is involved in the regulation of bone metabolism through a variety of cellular signaling pathways,such as the RANK/RANKL pathway,the MAPK/ERK,and the Wnt/?-catenin signaling pathway.The Smad2/3-Runx2 signaling pathway is an important signaling pathway involved in bone formation,which is also a research hotspot in the correlation between cell signaling pathway and bone metabolism.Currently,it has not been confirmed that whether vaspin can promote osteoblastic differentiation in rat osteoblasts through Smad2/3-Runx2 signaling pathway.In our preliminary experiments,different concentrations of vaspin were used to treat osteoblasts(OB),the RT-PCR results showed that the mRNA expression levels of Smad2/3 and runt related transcription factor 2(Runx2)increased significantly,which suggested that vaspin may regulate the the osteoblastic differentiation of OB through the Smad2/3-Runx2 signaling pathway.Therefore,in this study,we hypothesized that vaspin may promote the osteogenic differentiation of OB by activating the Smad2/3-Runx2 signaling pathway.Our group previous studies have confirmed that high-fat feeding can result in loss of SD rat bone mass,in this study,we used SD rats to study the correlation between vaspin and bone metabolism,demonstrate the potential mechanism,thus aimed to clarify he biological roles of vaspin in the high fat-induced bone loss.And at the same time,different concentrations of vaspin were used to treat with rat OB in vitro,in order to investigate whether vaspin is involved in regulating the osteoblastic differentiation of OB and study the possible mechanisms,provide more theoretical basis and new therapeutic directions for the prevention and treatment of OP.Objectives:1.To determine the effects of high fat diet and vaspin intervention on body weight,food intake,the whole body metabolic status,bone biomechanics,bone microstructure and bone turnover maker and the osteogenic related gene expression levels in male SD rats.2.The primary OB of SD rats was extracted,and the proliferation of OB was observed at different concentrations of vaspin to determine the optimal experimental concentration.3.Different concentrations of vaspin(10,50,100 ng/mL)were used to intervene OB to explore whether vaspin could promote osteogenic differentiation of OB and study the possible mechanism.Methods:1.Animals and treatment:This study chose 40 male SD rats aged 4 weeks,after being acclimated to the housing conditions for 1 week,they were randomly divided into normal diet group and high fat diet group,respectively fed by normal diet or high fat diet for 12 weeks,then the normal diet group were randomly divided into the ND group(n=10)and the normal diet+vaspin intervention group(ND+vaspin)(n=10),the high fat diet group were randomly divided into the HFD group(n=10)and the high fat diet+vaspin intervention group(HFD+vaspin)(n=10).They were continue to be fed either normal diet or high fat diet,the vaspin intervention group was treated with intraperitoneal injection of vaspin(l?g/kg body weight,once a day)for 10 weeks,while the control group was intraperitoneally injected with the same dose of saline for 10 weeks.2.Metabolic assessment:The body metabolism of rats were measured by metabolic chambers,including:volume of oxygen consumption(VO2),production of carbon dioxide(VCO2),respiratory exchange ratio(RER),heat production(H)and physical activity.3.At the end of the study,the rats were anesthetized and sacrificed,and the venous blood samples were collected.Serum calcium(Ca),inorganic phosphorus(P),total cholesterol(TC),triacylglycerol(TG),blood glucose,high-density lipoprotein cholesterol(HDL)and low density lipoprotein cholesterol(LDL)concentrations were measured by automatic biochemical analyzer.The serum levels of type 1 collagen C-terminal peptide(CTX),alkaline phosphatase(ALP)and procollagen I N-terminal peptide(PINP),C-telopeptide of type I collagen(CTX)were detected by ELISA kits.4.Three-point bending test was performed on the right femur to access bone strength.Microcomputed tomography(MicroCT)examination was performed on the left femur was to observe the changes of bone microstructure.The mRNA expression levels of Runx2 and Osterix(OSX)in the tibia of SD rats were measured by RT-PCR.5.Enzymatic digestion and differential adherence were used to extract primary OB.Alkaline phosphatase(ALP)and HE staining were used to identify whether the cells were OB.The effect of different concentrations of vaspin on the proliferation of primary OB was examined by CCK-8.6.The ALP activity determination kit detects the effect of vaspin at different concentrations on the ALP activity of OB.The effect of vaspin at different concentrations on the regulation of bone-related genes such as alphal Type ? Collagen,Colla1(Colla1),Runx2 and OSX were detected by RT-PCR.7.Western Blotting was used to detect the effects of vaspin of different concentrations on the expression levels of bone-related proteins,such as catenin beta1(?-catenin),Runx2,OSX,Smad2,p-smad2,Smad3,p-smad3,and the internal reference protein GAPDH.8.The effects of different concentrations of vaspin on the protein expression of Runx2 were detected by immunofluorescence.Results:1.The administration of vaspin for 10 weeks can significantly decrease the body weight gain in high fat diet-fed rats,while it shows no significant effect on the food intake.After rats were fed with high fat for 12 weeks,the body weight of high fat diet-fed group was significantly higher than that of ND-fed group(P<0.05).However,after vaspin intervention for 10 weeks,the final body weight of HFD+vaspin group was significantly reduced compared to the HFD group(P<0.05).There was no difference in body weight among the ND,ND+vaspin and HFD+vaspin group(P>0.05).On the other hand,in the tested vaspin dose,neither the ND+vaspin group nor the HFD+vaspin group showed any significant difference on food intake,compared to the ND group and the HFD group respectively(P>0.05).Taken together,these data indicated that the administration of vaspin for 10 weeks can significantly decrease the body weight gain in high fat diet-fed rats,while it shows no significant effect on the food intake.2.Vaspin intervention can significantly improve the whole body metabolic parameters.After 10 weeks of vaspin or saline intervention,the V02,VC02,RER,H,physical activity and the area under the curve(AUC)were reduced in the HFD rats as compared to the ND rats(P<0.05).VO2,VCO2,RER,H,physical activity and AUC of the HFD+vaspin group were significantly increased compared with that of the HFD group(P<0.05),and there was no statistical difference compared with that of the ND group(P>0.05).Above results suggested that the whole body metabolism of rats was significantly decreased in the HFD rats in comparison to the ND rats,while vaspin can improve the whole body metabolic parameters.3.HFD significantly reduced serum ALP and P1NP levels,and increased serum CTX and blood glucose levels,while vaspin supplementation can antagonize the effect of high fat diet on the serum biochemical markers.Compared with ND group,ND+Vaspin group and HFD+vaspin group,the serum ALP level and P1NP level in HFD group were significantly decreased(P<0.05),while the serum level CTX was significantly increased(P<0.05).Compared with the HFD group,the serum ALP level and P1NP level in the HFD+vaspin group were significantly increased,while the serum CTX was significantly decreased(P<0.05).The above experimental results confirmed that high-fat feeding can reduce the serum levels of ALP and P1NP and increase the serum level of CTX,while vaspin intervention can antagonize the effect of high fat diet on the above bone turnover markers.4.Vaspin intervention can improve the bone biomechanical damage induced by high fat diet.The maximum load,maximum fracture load,stiffness,energy absorption,elastic modulus,and the maximum strength of the HFD group were significantly decreased compared to ND,ND+vaspin,HFD+vaspin groups(P<0.05).However,there were no differences among the ND,ND+vaspin,HFD+vaspin groups(P>0.05).The results indicate that bone strength is significantly reduced in HFD rats relative to ND rats,while vaspin treatment can improve bone strength-related parameters.5.Vaspin intervention prevented high fat diet induced bone loss and microarchitecture deterioration.Compared with the ND group,HFD diet caused a significant decrease in trabecular bone parameters including Tb.vBMD,Tb.N,Tb.Th,and Tb.BV/TV,and an increase in Tb.Sp and SMI(P<0.05).Conversely,10 weeks of vaspin intervention prevented HFD-induced bone loss and microarchitecture deterioration,as evidenced by the increased Tb.vBMD,Tb.N,Tb.Th,and Tb.BV/TV,the decreased Tb.Sp and SMI compared with the HFD group(P<0.05).6.Vaspin intervention significantly upregulated mRNA expression levels of osteogenic related genes in SD rats.It was found that the mRNA levels of Runx2 and OSX were down-regulated in HFD group(P<0.05),while the changes in HFD+vaspin group were not significant difference compared to the ND group(P>0.05).Furthermore,the ND+vaspin rats demonstrated higher mRNA expressions of Runx2 and OSX than those in ND rats,suggesting that vaspin might promote bone formation in vivo.7.Vaspin at different concentrations had no significant effect on OB proliferation.The results of CCK-8 activity assay showed that vaspin exerted no significantly cytotoxic effects,even concentrations up to 100 ng/mL at 12 h,24 h,48 h or 72 h compared with the control group(P>0.05).The results indicated that vaspin at different concentrations can not affect osteoblast proliferation.8.Vaspin significantly increased the ALP activity of OB,and significantly upregulated osteogenic related genes in a dose-dependent manner.OB treated with 10 ng/mL vaspin exhibited no significant increase in the ALP activity,nor the expression levels of Runx2,Osx and Collal compared with the control(P>0.05),while a significant promotion was first observed in 50 ng/mL vaspin-treated cells and the promotion effects peaked in 100 ng/mL vaspin-treated cells.Then,OB were treated with 50 ng/mL vaspin for different times(12 h,24 h,48 h)(P<0.05).Compared with the control without vaspin stimulation,the mRNA levels of Runx2,OSX and Collal were all up-regulated at 12 h,24 h and 48 h.However,there was no significant difference in the mRNA levels of the above genes among the three groups(P>0.05).The results suggest that vaspin may enhance the differentiation of osteoblasts dose-dependently but not time-dependently.9.Vaspin significantly increased the protein expression levels of P-catenin,Runx2,OSX,p-smad2,p-smad3,p-smad2/Smad2,and p-smad3/Smad3.The protein expression of Smad2,Smad3,p-Smad2,p-Smad3,Runx2,OSX and ?-catenin in primary rat osteoblasts cells OB was determined using Western blot analysis after treatment with vaspin(10-100 ng/mL)for 48 h.The results showed that vaspin dose-dependently increased the protein levels of Runx2,OSX,?-catenin,p-Smad2,p-Smad3,the ratio of p-Smad2/Smad2 and p-Smad3/Smad3.10.Vaspin significantly increased the number of Runx2 fluorescent cells.Runx2 was also examined by immunofluorescence.It was shown that treatment with 50 ng/mL vaspin resulted in an increase in the number of Runx2 fluorescent cells and peaked at 100 ng/ml compared with the control cells.Conclusions:Vaspin can improve the weight gain,improve the overall metabolic level of the body,and reverse the bone damage caused by high fat diet.Vaspin can promote osteogenic differentiation of primary OB in SD rats by activating Smad2/3-Runx2 signaling pathway. |
PDF Full Text Request