Effects Of Sodium Dehydroacetate Exposure During Pregnancy On Bone Metabolism And Its Mechanism In Pregnant And Fetal Rat

Sodium dehydroacetate(DHA-S)was widely used in food,feed and cosmetics because of its antibacterial effect.Our previous studies have shown that DHA-S can inhibit Vitamin K 2,3 epoxide reductase subunit 1(VKORC1)and VKORC1-like protein 1(VKORC1L1)to cause abnormal VK circulation,which reduces the production of VK and VK-dependent coagulation factors,thus leading to coagulation disorders.VK-dependent proteins,including osteocalcin and periostein,play important roles in the process of bone and blood vessel calcification.DHA-S can inhibit VK-dependent proteins via VKORC1/L1,but its effect on bone growth has been less reported.In this study,DHA-S was given to rats by gavage during pregnancy.We detect the DHA-S concentration in blood,the serum or bone metabolism related indexes and the expression levels of VKORC1 and VKORC1L1 in pregnant and fetal rats to understand the relationship between the blood concentration of DHA-S in pregnant and fetal rats and the influence of DHA-S on the growth and metabolism of femail in bone of pregnant and fetal rats,which will be useful to explore the mechanism of DHA-S on bone metabolism in pregnant and fetal rats.Wistar rats were orally given 10 mg/kg,30 mg/kg and 50 mg/kg DHA-S for 21 consecutive days during pregnancy,and set up control group.At the 20th day of gestation,the serum and femur tissues of pregnant and fetal rats were collected,with measuring the weight,body length,femur length,skull diameter of the fetus.The HE staining and Von Kossa staining of their femur were made.HPLC method was used to determine the blood concentration of DHA-S in pregnant and fetal rats to preliminarily understand the relationship between the blood concentration of DHA-S in pregnant and fetal rats.Serum ELASA method was used to determine osteocalcin(BGP),osteoprotectin(OPG),N-terminal propeptide of type I procollagen(PINP),alkaline phosphatase(ALP),Receptor activator of nuclear factor kappa B ligand(RANKL),C-terminal cross-linked terminal peptide of type I collagen(CTXI)and tartrate-resistant acid phosphatase(TRAP).The expression levels of bone metabolism-related proteins,VKORC1 and VKORC1L1 in pregnant and fetal bone were detected by Western Blot or immunofluorescence.The correlation between blood DHA-S concentration and bone metabolism indexes of pregnant or fetal rats were analyzed.Among the bone formation indexes in serum of pregnant rats,BGP,OPG,PINP and ALP in 10 mg/kg dose DHA-S,and PINP and ALP in 30 mg/kg group were found no significant effects compared to the pregnant rats in control(P>0.05).The BGP,OPG,PINP and ALP(P<0.05 or P<0.01)in 50 mg/kg appeared significantly decreased(P<0.05).The bone resorption indexes RANKL,CTX-I and TRAP in 10 mg/kg and 30 mg/kg dose groups had no significant effects(P>0.05),were significantly increased in 50 mg/kg group(P<0.05 or P<0.01).The correlation analysis showed that DHA-S concentration was negatively correlated with bone formation indexes BGP,OPG,PINP and ALP,while positively correlated with bone absorption indexes RANKL,CTX-I and TRAP,respectively.The BGP,OPG,RANKL in femur of pregnant rats showed no significant effects on(P>0.05)in the 10 mg/kg dose group,and appeared significant or extremely significant difference in 30 mg/kg and 50 mg/kg pregnant rats(P<0.05 or P<0.01).The serum VK content of pregnant rats was no significant differennt in 10 mg/kg dose group compared to the control,but decreased significantly(P<0.05)in 30 mg/kg and 50 mg/kg dose groups.The protein levels of VKORC1 and VKORC1L1 in pregnant rat were significant or extremely significant effects on VKORC1 and VKORC1L1 in pregnant rat(P<0.05 or P<0.01)in both 30 mg/kg and 50 mg/kg dose groups except 10 mg/kg dose group.In the fetal rats,DHA-S significantly inhibited fetal bone development through significant effect the body length,femur length and bone microstructure.The body weight,body length,femur length and skull diameter of fetus in 30 mg/kg and 50 mg/kg dose groups were significantly lower than control group(P<0.05).The bone formation indexes BGP,OPG and PINP in 10 mg/kg dose fetal rat serum were no significant difference compared to the control(P>0.05),others in 30 mg/kg and 50 mg/kg groups had significant or extremely significant difference(P<0.05 or P<0.01).The serum bone resorption indexes RANKL,CTXI and TRAP in 10 mg/kg and 30 mg/kg dose groups of fetal rats showed no significant difference(P>0.05),but these in 50 mg/kg were significantly higher than that in the control(P<0.05 or P<0.01).The blood DHA-S concentration in fetal rats was negatively correlated with the serum bone formation indexes BGP,OPG,PINP and ALP,while was positively correlated with serum bone absorption indexes RANKL,CTX-I and TRAP,respectively.Western Blot analysis showed the OPG and RANKL expression levels in fetal femur had no significant difference(P>0.05),the BGP was significantly decreased(P<0.05)in 10 mg/kg.But the BGP and OPG were significantly decreased,and the RANKL significantly increased in 30 and 50 mg/kg dose groups(P<0.05 or P<0.01).The mRNA levels of BGP,OPG and RANKL in femur were not significantly changed in DHA-S treatment groups.Serum VK content of fetal rats was significantly decreased in all DHA-S treatment groups(P<0.05).The VKORC1 and VKORC1L1 protein in the 10 mg/kg dose group showed no significant difference,but that in fetal femur of 30 mg/kg and 50 mg/kg dose groups were significantly decreased(P<0.05 or P<0.01)by immunofluorescence analysis.The mRNA levels of Vkorcl and Vkorclll in DHA-S treatments were not significant different from that in the control(P>0.05).In conclusion,30 and 50 mg/kg DHA-S treatments was found significantly inhibition on the bone development of fetal rats,in which significant affects the body length,femur length and bone microstructure of fetal rats.DHA-S appeared significantly inhibited the bone formation related indexes such as BGP,OPG,PINP,ALP,and significantly increase bone resorption indexes such as RANKL,CTX-I,TRAP,etc.DHA-S can significantly reduce the serum VK content and the levels of VKORC1 and VKORC1L1 in bone tissue in pregnant and fetal rats.It is showed that the inhibition of DHA-S on VKORC1/VKORC1L1-VK is one of the mechanisms of the effect of DHA-S on bone formation and bone metabolism.