Effect Of Hypercholesterolaemia On Male Bone Metabolism And Mechanism Research

Background and ObjectivesThe prevalence of OP and the lifetime risk of osteoporotic fractures in men has gradually increased,so as the incidences of hypercholesterolaemia.Previous clinical studies have examined the relationship between hypercholesterolaemia and BMD but yielded contradictory results.Most of those studies have focused on female subjects as opposed to male subjects.therefore,we aimed to investigate the effects of hypercholesterolaemia on bone in men and to elucidate the mechanisms underlying these effects.MethodsA total of 216 men aged more than 18 years from FeiCuiJun Community were recruited for this cross-sectional study.Serum lipid profiles were measured using by the BECKMAN Chemistry Analyzer AU5800 System 195(Beckman Coulter,Tokyo,Japan),and serum Beta-CTX and PINP concentrations were determined determined by electrochemiluminescence immunoassay(cobas 8000 analyser,Switzerland).We designed tow male animal models.The first model involved male Sprague-Dawley rats,which we fed a high-cholesterol diet(HCD)(2%cholesterol and 98%standard rodent chow)for 24 weeks to induce exogenous hypercholesterolaemia,after which we fed the rats a normal control diet(NCD;dietary modification)or the aforementioned HCD(exogenous hypercholesterolaemia)for another 12 weeks.The second model involved male wild-type(Apoe+/+)rats and male Apoe-ko(Apoe-/-)rats(congenital hypercholesterolaemia model),which were a fed normal diet of 100%standard rodent chow for 20 weeks.After the rats were sacrificed,we measured their serum lipid profiles using an automatic biochemical analyser(Olympus AU5400,Japan)and determined their serum BGP,ALPL,PINP,CTX-I and TRAP levels using ELISA kits(Uscn Life Science,Wuhan,China).Their left femurs were collected for micro-CT(mCT)analysis and biomechanical analysis.Primary osteoblasts isolated from the calvaria of neonatal rat pups were used for the in vitro study.The primary osteoblasts were starved of serum for 2 hours and then treated with cholesterols(Sigma-Aldrich,USA,0,20?g/mL and 10?g/mL)for 12hours,36hours and 48 hours.We subsequently analysed osteoblast function by measuring BGP,ALPL,and collagen I mRNA expression levels and BGP and ALPL protein expression by using quantitative real-time PCR(qRT-PCR)and ELISA(Uscn Life Science,Wuhan,China).ResultsBone mineral density(BMD)was lower,and bone resorption marker(Beta-CTX)and bone formation marker(PINP)levels were higher in men with hypercholesterolaemia than in control subjects.The results of partial correlation analysis showed that TC and LDL-C levels were inversely related to BMD,positively related to beta-CTX and PINP levels after adjustment for age and that BMI and FPG,HbA1c,To,FT4,TSH,25(OH)VD and TG,but HDL-C levels is not.Multiple linear regression analysis showed beta-CTX and PINP served as dependent variables,and other factors erved as independent variables,TC was a significant independent predictor of BMD,beta-CTX and PINP,and was negatively correlated with BMD and positively correlated with beta-CTX and PINP.LDL-C was positively correlated with beta-CTX levels but was not significantly correlated with BMD or PINP levels.In first model,HCD administration caused significant increases in serum TC levels,decreases in BMD,increases in both bone resorption marker levels(CTX-I,TRAP)and bone formation marker levels(PINP,BGP,and ALPL)compared with NCD administration.HCD administration damaged bone microstructures(as shown via bone mCT),reduced bone strength(as shown by our biomechanical parameter results).Dietary modification facilitated by withdrawal of the HCD for 12 weeks decreased serum cholesterol levels,slightly attenuated the changes of bone including bone resorption and bone formation markers,bone microstructures and bone strength induced by the HCD.In second model,Apoe-/-rats with congenital hypercholesterolaemia displayed decreased BMD and increased bone resorption marker levels(CTX-I,TRAP)and bone formation marker levels(PINP,BGP,ALPL)compared with Apoe+/+ rats.Apoe-/-rats with congenital hypercholesterolaemia damage bone microstructural(as shown by bone mCT),as well as reduced bone strength(as shown by our biomechanical parameter results).In cell experiment,primary osteoblasts isolated from the calvaria of neonatal rat pups are identified as ideal experimental osteoblast model cells by HE-staining,ALP staining,Alizarin red and Von Kossa staining.RT-PCR results showed with increasing cholesterol doses after cholesterol treatment for 24 hours,BGP,ALPL and collagen I mRNA expression levels gradually increased in the treated groups compared with the control group.There were no differences osteoblast functional gene mRNA expression levels among the different times(12 h,24 h,48 h)after 10 ?g/mL cholesterol treatment.ELLISA results showed with increasing cholesterol doses after cholesterol treatment for 24 hours,BGP and ALPL protein expression levels gradually increased in the treated groups compared with the control group.There were no differences in osteoblast functional gene protein expression levels among the different times(12 h,24 h,48 h)after 10 ?g/mL cholesterol treatment.ConclusionHypercholesterolaemia increases the risk of osteopenia or OP in men at least in part by promoting both bone resorption and bone formation.In addition,hypercholesterolaemia damages bone microstructure,resulting in osteopenia or OP and reduced bone strength,leading to a higher risk of fracture.Dietary modification slightly attenuated the changes of bone induced by the HCD.