Effect Of ?-linolenic Acid On Bone Metabolism In Male SD Rats Fed On High-fat-diet And Its Mechanism

Background:In recent years,the prevalence of osteoporosis and osteoporotic fractures in men increased year by year.However,large epidemiological studies,clinical studies and animal experiments mostly focused on elderly women and ovariectomized animal models,and relatively few studies paid attention to bone metabolism of obese men induced by high-fat-diet(HFD).Diet is one of the risk factors for osteoporosis besides heredity,age,oestrogen deficiency,and calcium and phosphorus metabolism.Several studies including epidemiological studies and animal experiments had indicated that HFD had a negative impact on bone health.One middle-to-older-aged population study indicated that a high body fat percentage was a risk factor for osteoporosis with ageing.Several animal studies had reported that HFD consumption was associated with bone strength reduction and that adverse microstructure changes occurred in the cancellous bone compartment.Our previous experimental results showed that both HFD(60%fat)and high-cholesterol-diet can lead to the damage of trabecular structure and the decline of bone biomechanical indexes.Nutrition plays an important role in maintaining bone health.Studying the effects of dietary structure on bone metabolism can provide a theoretical basis for prevention of osteoporosis.Omega-3 unsaturated fatty acids(?-3 FA)are essential fatty acids commonly found in fish oil,flaxseed oil and comfrey oil.They included:eicosapentaenoic acid(EPA),docosahexaenoic acid(DHA)and a-linolenic acid(ALA),which can lower lipids,resist arteriosclerosis,reduce inflammatory factors,improve vascular endothelium,and have protective effects on cardiovascular and cerebrovascular diseases.Recent studies showed that ?-3 FA had protective effects on bone metabolism.ALA is a major component of plant-derived flaxseed oil(FO)and comfrey oil,which is cheaper and easier to obtain than DHA and EPA.However,the the effects of ALA on bone metabolism in different experimental animal models are inconsistent.FO contains approximately 60%ALA and represents a plant-derived ?-3 FA.A clinical study indicated that daily consumption of 6 g/d of FO may reduce bone resorption in haemodialysis patients.An animal study showed that ALA associated with calcium and protein provided by flaxseed flour contributed to bone quality in the post-partum period of dam rats.However,another animal study noted that feeding a 10%FO diet did not have effects on bone mass and bone strength compared with a 10%corn oil diet in growing mice.Whether FO can improve HFD-induced bone damage has not been reported at home and abroad.Therefore,this study aimed to evaluate whether totally replacing soybean oil and partly replacing lard with FO in HFD can improve HFD-induced bone damage in male SD rats.At present,the specific mechanism by FO on bone protection is unclear.The protective mechanisms of bone metabolism are mostly focused on the inhibition of OCs by ?-3 FA.There are less studies on OBs,and the findings are inconsistent and controversial.Therefore,we extracted rat primary OBs and applied palmitic acid to simulate high fat status in the body.We treated OBs with different concentrations of ALA in the presence or absence of PA to observe the effect of ALA on ALP activity,osteogenic-related gene and protein(?-catenin,RUNX2,osterix)expression and tried to explore the mechanism of ALA on bone protection.Objectives:1.To further determine the effects of HFD on bone mineral density,bone biomechanics,bone microstructure,bone formation markers and bone resorption markers in male SD rats.2.By replacing some of the ingredients in HFD with FO,we observe whether FO has a protective effect on bone damage induced by HFD.3.ALA alone or PA+ALA were used to treat rat primary OBs,and their effects on primary OBs were observed to determine the mechanism of ALA on bone protection.Methods:1.40 male SD rats aged 4 weeks,after being acclimated to the housing conditions for 1 week,they were randomly assigned to one of the four diets:(1)(NC group,n=10):normal control diet,fat provides 10%of energy;(2)(NY group,n=10):FO substitutes for all of the soybean oil and lard in the normal control diet,fat provides 10%of energy:(3)(HFD group.n=10):fat provides 60%of energy;and(4)(HY group.n=10):FO replaces all of the soybean oil and part of the lard in HF D,fat provides 60%of energy.Dynamically observe the effects of different dietary components on body composition and BMD in 12 weeks(adolescent)and 22 weeks(adult)of SD rats.At 12 weeks,the rats were draw blood in living body,and the serum was centrifuged at room temperature to measure the levels of the bone formation markers type 1 procollagen N-terminal propeptide(PINP)and Alkaline phosphatase(ALP),and the resorption marker type I collagen C-terminal peptide(CTX-1).After 22 weeks of intervention,the body composition and BMD were checked.After being starved for 12 hours,the rats were anesthetized and sacrificed.The serum was centrifuged at room temperature to detect PlNP,ALP and CTX-1.Three-point bending test was performed on the left femur to observe changes in bone strength.MicroCT examination was conducted on the right femur to observe changes in bone microstructure.The left tibias were stained with HE to observe the number of trabecular bone and bone marrow adipocytes.2.Enzymatic digestion and differential adherence were used to extract primary OBs.Alkaline phosphatase(ALP)and nuclear solid red staining were used to identify whether the cells were OBs.ALP staining was used to observe the effects of ALA and PA on early osteogenic function.The expression changes of osteogenic related genes and proteins(?-catenin,RUNX2 and osterix)were detected by real time-qPCR and Western Blotting,respectively.Results:1.FO reduced body weight,whole body and abdominal fat accumulation,improved spinal BMD in male SD rats induced by HFD,but had no effect on whole body BMD and femur BMD.After 12 weeks of intervention,the HY group can improve the weight gain,reduce the increase of body fat caused by HFD in male SD rats compared with the HFD group,this indicates that FO can reduce body weight,alleviate the accumulation of whole body and abdominal fat caused by HFD.Whether or not the body weight was adjusted,the addition of FO for 12 weeks had no significant effect on whole body BMD,femur BMD,and spinal BMD.The effect of FO on weight and body composition persisted for 22 weeks.After 22 weeks of intervention,adjusting weight effects,the HY group can improve spinal BMD compared with the HFD group(P<0.05),but there was no significant improvement in the whole body and femoral BMD.2.Effects of FO on bone formation markers P1NP,ALP and bone resorption marker CTX-1.After different dietary interventions for 12 weeks,the bone formation markers P1NP and ALP were not changed among four groups.Compared with the NC group,the HFD group had an increase in the bone resorption marker CTX-1(P<0.01),and the HY group can reduce the increase of CTX-1 induced by HFD compared with the HFD group(P<0.01).Compared with the NC group,P1NP and ALP were decreased(both P<0.01),and CTX-1 was increased(P<0.01)in HFD group after 22 weeks of intervention.The HY group can improve the decreases of P1NP(P<0.01)and ALP(P<0.05)and decrease the increase of CTX-1 compared with the HFD group(P<0.01).However,compared with the NC group,the NY group had no difference in the above indicators in 12 weeks or 22 weeks.3.FO improved bone biomechanical properties in HFD-induced bone damage.The biomechanical properties,such as the maximum load,maximum fracture load,ultimate tensile strength,stiffness,energy absorption,and elastic modulus were decreased(P<0.01 or P<0.05)in the HFD group compared with the NC group after four different diets for 22 weeks.The maximum load,maximum fracture load,and ultimate tensile strength were significantly enhanced(P<0.01 or P<0.05)in the HY group compared with the HFD group,but the stiffness,energy absorption,and elastic modulus showed no significant differences between the HY group and the HFD group.Moreover,compared with the NC group,the NY group had no difference in the above indicators.4.FO ameliorated trabecular bone damage in HFD-induced bone loss Consistent with the results of the three-point bending test,the trabecular volume bone mineral density(Tb.vBMD),trabecular bone volume/total volume(Tb.BV/TV),trabecular number(Tb.N),and trabecular thickness(Tb.Th)were decreased(P<0.01 or P<0.05)and the trabecular separation(Tb.Sp)and structure model index(SMI)were increased(P<0.01 or P<0.05)in the HFD group compared with the NC group after four different diet treatments for 22 weeks,and all of the above parameters were markedly improved(P<0.01 or P<0.05)in the HY group compared to the HFD group.However,compared with the NC group,the NY group had no difference in the above indicators.Meanwhile,the cortical volume bone mineral density(Ct.vBMD),cortical bone volume/total volume(Ct.BV/TV)and cortical bone thickness(Ct.Th)had no significant differences among the four groups.5.FO increased the trabecular number and reduced the number of bone marrow adipocytes.The results of HE staining in the right tibia samples showed that the trabecular number was decreased in the HFD group compared with the NC group,while the HY group markedly increased the trabecular number compared with the HFD group(all P<0.01).We also found that the number of bone marrow adipocytes in the HFD group was increased compared with the NC group,while it was dramatically decreased in the HY group compared with the HFD group(all P<0.01).Similarly,there were no significant differences between the NC and NY groups.6.ALA alone had no effect on ALP staining of primary OBs,but ALA could improve the inhibition of PA on ALP staining.Primary OBs were treated with different concentrations of ALA for 5 days,and there were no significant changes in ALP staining compared with the control group.OBs were treated with PA0.2mM for 5 days,ALP staining activity was significantly reduced compared with the control group,which suggested that PA0.2mM can inhibit the early osteogenesis of OBs.However,simultaneous application of PA 0.2 mM and different concentrations of ALA to OBs for 5 days,ALA can alleviate the inhibition of PA on ALP activity in a concentration-dependent manner.7.ALA can promote the expression of osteogenic related genes and proteins(P-catenin,RUNX2,osterix),PA inhibits the expression of these genes and proteins,and ALA can improve the inhibition of PA.RT-qPCR and Western blotting revealed that OBs treated with ALA showed higher gene and protein expression(?-catenin,RUNX2,and osterix)compared with the vehicle group.The results suggested that ALA can promote osteoblastic differentiation by promoting the expression of osteogenic related genes and proteins.Consistent with ALP staining,significant decreases in osteogenic relative gene and protein expression were observed in PA treatment group compared with the vehicle group(all p<0.0001),and ALA can mitigate the decreases in osteogenic relative gene and protein expression induced by PA in a concentration-dependent manner(all p<0.01).Conclusions:1.HFD can increase body weight,whole body and abdominal fat accumulation,but has no significant effect on cortical BMD and cortical bone microstructure parameters.HFD can reduce bone formation markers,increase bone resorption marker,reduce bone strength,destroy trabecular structure,and increase the number of bone marrow adipocytes.2.FO replaces some components of HFD,which can reduce body weight,reduce whole body fat and abdominal fat accumulation,increase bone formation markers,reduce bone resorption marker,improve bone strength and trabecular structure parameters,and reduce the number of bone marrow adipocytes.Therefore,FO can be used as a potential therapeutic substance for the treatment of HFD-induced bone damage.3.The mechanism by which FO exerts bone protection may be through its active ingredient ALA to improve ALP activity and promote the expression of osteogenesis-related genes and proteins(?-catenin,RUNX2,osterix).