Preliminary Study On The Effect Of Sodium Dehydroacetate On Bone Metabolism In Chickens

Sodium dehydroacetate(Na-DHA)is widely used as a preservative and mildew preventing agent in food,feed and cosmetics.The long-term administration of Na-DHA can cause coagulation disorders in rats and broilers was reported,which the main reason is that Na-DHA inhibits VK epoxide reductase(VKOR)(VKORC1 and VKORC1L1 subunits)in the liver,causing VK circulation disorders and inhibiting VK-dependent γ-carboxylation of coagulation factors,resulting coagulation dysfunction.VK is closely related to bone health,and VK-dependent proteins such as bone metabolism-related protein osteocalcin(BGP)play an important role in bone metabolism.At present,the effects of Na-DHA on bone growth and bone metabolism have not been reported.In this study,we intend to investigate the effects of sodium dehydroacetate on bone metabolism in chickens and its possible mechanisms through feeding tests of broiler chickens with different doses of Na-DHA and in vitro culture tests of chicken osteoblasts.The results will provide a reference for the safe and rational use of Na-DHA as a feed additive and the effect of Na-DHA on bone metabolism of chickens.In the assay of Na-DHA on the chicken osteoblasts cultured in vitro,bone marrow stromal cells(BMSC)were isolated and cultured from chicken embryos.The chicken osteoblasts were from the induction culture of BMSC and identified according to cell morphology,the staining of alkaline phosphatase(ALP)and alizarin red.Chicken osteoblasts were treated with different concentrations(0.2,0.5,1.0,2.0,5.0 mmol/L)of Na-DHA for 24 hours,and the cell viability was measured by MTT assay;the alkaline phosphatase staining was used to measure the ALP activity;the alizarin red staining was used to measure the mineralization ability of cells.The expressions of BGP,OPG,RANKL,VKORC1 and VKORC1L1 proteins in the cells were detected by Western blot.The results showed that 2.0 and 5.0 mmol/L Na-DHA significantly inhibited the cell viability(P<0.001).The alkaline phosphatase activity and mineralization capacity were significantly decreased at 0.5 and 1.0 mmol/L Na-DHA concentrations(P<0.05).Western blot results showed that the protein expression of VKORC1 and VKORC1L1 was significantly decreased(P<0.05).The protein levels of BGP and OPG were significantly decreased(P<0.05),but the RANKL protein was significantly increased(P<0.05).Na-DHA inhibits BGP through VKORC1/VKORC1L1,which is one of the mechanisms affecting chicken osteoblasts and bone metabolism.In the feeding trial of Na-DHA to yellow-finned broiler chickens,four dose groups of 0,200,400 and 800 mg/kg were set up and added in feed for 10 weeks.The growth indexes of broiler tibiae were measured at different time periods,the serum levels of BGP,CTX and P1NP were detected by ELISA.The tibia was stained with HE and the number,thickness,area and separation of trabecular bone were statistically analyzed.The expression of bone metabolism-related proteins such as BGP,OPG,RANKL,VKORC1 and VKORC1L1 were detected by immunohistochemistry and Western blot.The results showed that the tibial bone growth of chickens in 800 mg/kg Na-DHA was significantly inhibited(P<0.05);the serum levels of bone formation markers BGP and P1NP were significantly decreased(P<0.05);and the content of bone resorption marker CTX was significantly increased(P<0.05).In the analysis of HE staining of chicken tibia,the arrangement of bone trabeculae was loose with the increase of dose of Na-DHA,and the number,thickness and area of bone trabeculae were changed significantly.In 800 mg/kg Na-DHA group,the number,thickness and area of bone trabeculae were significantly decreased(P<0.05),and the separation degree of bone trabeculae was significantly increased(P<0.05).In the immunohistochemical analysis,the positive expression of BGP and VKORC1 brown was significantly reduced in the 800 mg/kg Na-DHA group.In the 400 and 800 mg/kg of Na-DHA group,the protein expressions of VKORC1 and VKORC1L1 in tibia were significantly decreased(P<0.01),and the bone formation related proteins BGP and OPG were significantly decreased(P<0.01),but the bone resorption related protein was significantly increased(P<0.01).Na-DHA inhibits BGP through VKORC1/VKORCIL1,which is one of the mechanisms affecting bone growth and bone metabolism in chickens.In summary,Na-DHA has a clear effect on bone metabolism in chickens,causing a significant decrease in BGP,P1NP and OPG,indicators related to bone formation,and a significant increase in CTX and RANKL,indicators related to bone resorption,which can significantly inhibit bone growth in broilers.Presumed effects of Na-DHA on skeletal growth and bone metabolism in chickens through inhibition of BGP by means of VKORC1/VKORC1L1.