| Subject We aim to explore the effect of active vitamin D deficiency caused by Cyp27b1knockout on the growth and development of placenta and fetal mice.Methods Cyp27b1 heterozygous(Cyp27b1+/-)mice in C57BL/6J background were generated using Cas9 nuclease-mediated DNA cleavage by mouse Cyp27b1gene-specific sg RNA.Cyp27b1+/+and Cyp27b1+/-female mice were produced from Cyp27b1+/-females bred with Cyp27b1+/-males.Mice were raised in clean mice room,with free diet and water,suitable humidity and temperature,and keeping 12 hours of circadian rhythm,To evaluate fetal development,Cyp27b1+/+and Cyp27b1+/-females were housed overnight with Cyp27b1+/+males and the presence of a vaginal plug was designated as gestational day?GD?0.Pregnant mice were divided into Cyp27b1+/+group,Cyp27b1+/-group and 1?,25?OH?2D3+Cyp27b1+/-group.Each group had 10pregnant mice.In the 1?,25?OH?2D3+Cyp27b1+/-group,pregnant mice were subcutaneously injected with 1?,25?OH?2D3?500 pg/kg?once a day throughout pregnancy.In the Cyp27b1+/+and Cyp27b1+/-groups,pregnant mice were subcutaneously injected with vehicle once a day throughout pregnancy.Maternal food intake and weight gain were monitored.Pregnant mice were sacrificed at GD18 after anesthetizing with phenobarbital sodium?50 mg/kg?.The number of live fetus,dead fetus and resorption site in each litter were counted.Maternal sera and placentas were collected.Fetal weight,fetal crown-rump length,placental weight and placental diameter were measured.To explore the effect of vitamin D deficiency on the development of fetus.We analyzed the fetal weight,fetal crown-rump length and IUGR rate.In order to explore the effect of vitamin D deficiency on the development and function of placenta,the changes of placental structure were observed by hematoxylin eosin staining.The placenta cell apoptosis was detected using TUNEL kit.The expression and distribution of Ki67 were detected by IHC,and the expression of placental Glut-1 and Fatp-4 m RNAs were detected by real-time RT-PCR.In order to investigate the effect of vitamin D deficiency on placental inflammation,the expression of placental Tnf-?m RNA,a proinflammatory gene,and placental Mcp-1 and Kc m RNAs,two inflammatory chemokine genes,were detected by real-time RT-PCR.The expression of placental NF-?B p65 was detected by western blotting.In addition,59pregnant women were recruited from the First Affiliated Hospital of Anhui Medical University to explore the effect of vitamin D deficiency on the development of fetus and placenta.The sera and placentas of pregnant women were collected.Fetal weight,placenta diameter and thickness were measured.The level of serum TNF-?protein in pregnant women were detected by enzyme-linked immunosorbent assay,the expression of placental TNF-?,MCP-1 and KC m RNAs were detected by real-time RT-PCR.The expression and distribution of placental NF-?B p65 were detected by Western blotting and immunohistochemistry.Results There is no significant difference in food intake and weight gain among the different groups.Cyp27b1+/-reduced Cyp27b1 and Cyp24a1 m RNAs level.Fetal weight of Cyp27b1+/+group,Cyp27b1+/+group and 1?,25?OH?2D3+Cyp27b1+/-group were?1.09±0.09?g,?0.98±0.09?g and?1.08±0.07?g,and the fetal crown-rump length was?21.91±0.83?mm,?20.24±0.98?mm and?21.62±0.84?mm,respectively.Compared with the Cyp27b1+/+group,fetal weight and the crown-rump length of the Cyp27b1+/+group were significantly reduced.Interestingly,1?,25?OH?2D3supplementation significantly evaluated fetal weight and crown-rump length compared to those of the Cyp27b1+/-mice.Correspondingly,IUGR rate was higher in the Cyp27b1+/-group than in the Cyp27b1+/+group,and supplementation with1 ?,25?OH?2D3attenuated elevation of IUGR rate in Cyp27b1+/-mice.The effect of active vitamin D deficiency on placental development was evaluated.Placenta weights were lower in Cyp27b1+/-mice than those of Cyp27b1+/+mice?0.076±0.017 vs 0.086±0.029?.Moreover,placenta diameters were lower in Cyp27b1+/-mice?7.46±0.52 vs8.24±0.77?.Interestingly,1?,25?OH?2D3supplementation prevented the reduction of placenta weights?0.076±0.017 vs 0.083±0.004?and diameters?7.46±0.52 vs 8.03±0.29?in Cyp27b1+/-mice.Placental cell proliferation was detected using IHC.Placenta cell apoptosis was detected using TUNEL kit.No significant statistical difference was observed among different groups.Placenta Ki67-positive cells were reduced in Cyp27b1+/-mice than those of Cyp27b1+/+mice,and 1?,25?OH?2D3supplementation evaluated the Ki67-positive cells in Cyp27b1+/-mice.In addition,Blood sinusoids area and CD34-positive vessel were decreased in placentas from Cyp27b1+/-mice,and 1 ?,25?OH?2D3supplementation evaluated the blood sinusoids area and CD34-positive vessel in Cyp27b1+/-mice.Placental nutrient transport was analyzed using real-time RT-PCR.Placental Glut-1 and Fatp-4 m RNAs were downregulated in the Cyp27b1+/-group,and these m RNAs were upregulated in the 1?,25?OH?2D3+Cyp27b1+/-group.The effect of gestational active vitamin D deficiency on placental inflammation was analyzed.Placental Tnf-?,Mcp-1 and Kc m RNAs were upregulated in Cyp27b1+/-group.NF-?B p65 subunit was then measured in placentas of Cyp27b1+/-mice.NF-?B p65subunit was increased in placentas of Cyp27b1+/-mice.As expect,1?,25?OH?2D3supplementation not only downregulated placental Tnf-?,Mcp-1 and Kc m RNAs level but also reduced placental NF-?B p65 expression of Cyp27b1+/-mice.As for human subject,we found that birth weight was much lower in VDD subjects than controls?3002±470 vs 3296±505?.Despite of no significant difference on placenta thickness,placenta diameter in VDD subjects was smaller than those of controls?19.00±1.44 vs20.29±2.48,n=19?.Mechanistically,several inflammatory cytokines were upregulated and placental NF-?B was activated not only in VDD diet-fed mice but also in VDD pregnant women.Conclusion These results suggest that gestational vitamin D deficiency causes placental insufficiency and fetal intrauterine growth restriction partially through inducing placental inflammation. |
PDF Full Text Request