| Preeclampsia is a pregnancy-specific disease with an incidence of about 3-5%of all pregnancies,causing maternal hypertension,proteinuria,edema,fetal intrauterine growth restriction,and asphyxia.Preeclampsia remains a leading cause of maternal and perinatal morbidity and mortality.Despite years of research on preeclampsia,the pathogenesis has not been fully elucidated,and prevention and prediction are still not possible.The only cure for preeclampsia is removal of the faulty placenta,i.e.,delivery.Data from observational studies has shown that there was a decrease of plasma 25(OH)D in patients with preeclampsia and an increase of preeclampsia risk in patients with 25(OH)D deficiency.The risk is the highest in the winter months as the production of vitamin D by sunlight is lowest.Low plasma vitamin D in pregnant mice may lead to symptoms of preeclampsia,including elevated blood pressure and deregulated placental development.Vitamin D acts as a pleiotropic secosteroid hormone instead of a true vitamin via both genomic response and non-genomic response.Vitamin D is involved in not only calcium homeostasis but also in immune function,disease prevention,cancer treatment and so on.However,there is no proof that vitamin D supplementation during early pregnancy will relieve any adverse outcomes in preeclampsia.It is unknown that whether these improvement relate to any changes in the activity of biological macromolecules.Reduction in uteroplacental perfusion(RUPP)in rat model is verified as mimicking the central role of reduced placental perfusion and expressing the same phenotypic characteristics with preeclampsia in human.Therefore,we used the rat model of reduced uteroplacental perfusion pressure(RUPP)to observe whether 1,25(OH)2D have any effect on alleviating symptoms of preeclampsia,including maternal blood pressure,renal and heart function as well as placenta and offspring development.In addition we also observed what kind of biochemical changes the effects were related to.Our results would provide important experimental basis for clinical application of vitamin D in preeclampsia.Part I Levels of serum 25(OH)D in patients with preeclampsia and expression of vitamin D receptor in placentaObjective:We compared expression of vitamin D receptor in placenta between preeclampsia and normal pregnant pregnancy.We observed the relationship between metabolism of vitamin D with maternal and fetal outcome.Methods:Third-trimester placentas from 32 normotensive(NT group)and 25 preeclamptic(PE group)pregnant women were collected immediately after delivery.We measured serum total calcium,phosphorus and alkaline phosphatase,maternal and umbilical cord serum 25(OH)D concentrations.VDR,CYP27B1,CYP24A1 expressions were studied in placenta between preeclampsia and normal pregnant pregnancy.Correlation between VDR mRNA with umbilical cord serum vitamin D,placenta weight,newborn birth weight and newborn birth length was determined with the spearman correlation test respectively.Results:1 Clinical characteristicsIn two groups maternal and gestational ages were similar.Maternal blood pressure and 24h proteinuria were significantly higher in PE group(P<0.01).On the contrary,newborn birth weight and lengths were significantly lower(P<0.01)in PE group,as well as placenta weight was lighter(P<0.05)in PE group.Maternal serum level of total calcium was slighter lower in PE group with no significant difference.No differences were found in maternal serum of phosphorus and alkaline phosphatase between the two groups.2 Low serum concentrations of 25(OH)D in maternal and umbilical cord blood were detected in PE groupIn PE group,serum levels of 25(OH)D in maternal and umbilical cord blood were significantly lower(P<0.05)compared with the NT group,what is more,maternal serum 25(OH)D concentrations correlated significantly with umbilical cord serum 25(OH)D(r= 0.825,P<0.01).3 Placental gene expression of CYP27B1,CYP24A1 and VEGF in PE groupMetabolism enzyme gene of vitamin D containing CYP27B1 and CYP24A1 expressed lower as compared to NT group without significant difference.Placenta tissue gene expression of VEGF was lower in PE group(P<0.05).Placenta weight had positive correlation with VEGF mRNA expression in placenta(P<0.05).4 VDR expression was increased in preeclampsia placentaIn placenta of PE group,VDR showed a trend for higher mRNA expression with no significant difference.High protein expression was verified in PE group placenta by western blot and immunohistochemistry staining.5 Correlation between VDR mRNA with umbilical cord serum vitamin D,placenta weight,newborn birth weight and newborn birth lengthInterestingly,umbilical cord serum 25(OH)D concentrations had negative correlation with VDR mRNA expression in placenta(r=-0.405,P<0.05).Placenta weight,newborn birth weight,newborn birth length had adverse correlation with VDR mRNA expression in placenta(r=-0.523,P<0.05;r=-0.419,P<0.05,r=-0.427,P<0.05,respectively),suggesting that placenta development and fetus growth may have relationship with vitamin D.Summary:In preeclampsia pregnancy,serum levels of 25(OH)D in maternal and umbilical cord blood were significantly lower compared with normal pregnancy.Low level of 25(OH)D in preeclampsia pregnancy may have relationship with placenta development and fetus growth.Part II Effect of 1,25(OH)2D on hypertensive response to Reduced Uterine Perfusion Pressure in pregnant ratsObjective:We surveyed the effect of 1,25(OH)2D injection in RUPP rats on maternal and fetal outcome.Methods:Reduction in uteroplacental perfusion(RUPP)in rat model was conducted in Three-month-old female Sprague-Dawley rats.Animals were divided into 3 groups:normal pregnant(NP group,n=12),pregnant with RUPP(RUPP group,n=14),and RUPP plus 1,25(OH)2D(RUPP+VD group,n=15).On day 1,7 and 14,RUPP+VD group received 120 ng/100 g body wt./wk of 1,25(OH)2D.At day 19 of gestation we measured proteinuria,cardiac function with echocardiography,arterial pressure and offspring number.A variety of rat serum biochemical levels were determined containing renal function,serum lipid,serum glucose and serum calcium,phosphorus and alkaline phosphatase.Rat placenta,heart and kidney slices were fixed for ultrastructural cytology.VDR,CYP24A1 gene expressions were studied in rat placenta,heart and kidney.Results:1 1,25(OH)2D alleviated symptoms of preeclampsia of RUPP ratCompared to rats of NP group,the RUPP procedure significantly increased the blood pressure(P<0.001),proteinuria P<0.01),fetal mortality(P<0.001),and significantly decreased sum of placental weights(P<0.05)and sum of fetal weights P<0.05)in rats of RUPP group,which agreed with previous research.By contrast,there was a decrease in mean blood pressure(P<0.05),proteinuria(P<0.01)and fetal mortality P<0.05),and an increase in sum of placenta weights(P<0.05)and sum of fetal weights(P<0.05)in rats of RUPP +VD group compared to rats of RUPP group.However,the average placental and fetal weight had no significant differences between the RUPP and RUPP+VD group since the total number of pups in RUPP group was significantly reduced.Additionally,maternal hyperglycemia in RUPP group was relieved in RUPP +VD group(P<0.05).The free calcium was remarkably elevated in maternal serum of RUPP+VD group rats compared to NP group(P<0.001)and RUPP group(P<0.05).All together,it indicated that 1,25(OH)2D should take a role in alleviating symptoms of preeclampsia.2 1,25(OH)2D alleviated the damage of heart,kidney and placenta in RUPP rat2.1 1,25(OH)2D reduced myocardial cell mitochondrial damageAlthough HE stain images of rat heart showed no obvious damage in histological structure among three groups,transmission electron microscopy images showed myocardial cell swelling and mitochondrial crest disappeared in heart of RUPP group compared to NP group,and the assay of echocardiography shown that left ventricular volume(diastole)and left ventricular internal diameter(systole)were significantly decreased,and the ratio of left ventricular anterior wall(diastole)to weight,and ratio of left ventricular posterior wall(systole)to weight were significantly increased(P<0.05)in RUPP group compared to NP group.These results suggested that the impaired ultrastructure occurred in rats of RUPP group,and mitochondrial damage was reduced in heart of RUPP+VD group compared to RUPP group.By PCR assay,vitamin D receptor(VDR)expression in the heart had no remarkable differences among three groups.However,as a VDR-target gene,expression of CYP24A1 gene in the heart was significantly increased by 1,25(OH)2D treatment(P<0.05)compared to NP group and RUPP group.It indicated that 1,25(OH)2D should play a role in alleviating mitochondrial impairment of heart of RUPP rat probably via its genomic response.2.2 1,25(OH)2D alleviated glomerular structure damage in kidney of RUPP ratIn the RUPP group,maternal kidney tissues had glomerular atrophy compared to the NP group.The glomerular atrophy was lessened in the RUPP+VD group compared to the RUPP group.Using transmission electron microscopy,the glomerular filtration membrane in the RUPP group was damaged,with foot processes fusion of the podocyte and basement membrane thickening compared to NP group.In support of the light microscopy results,the glomerular filtration membrane in the RUPP+VD group was less damaged.Using PCR,VDR expression in the kidney did not differ among the three groups.However,renal expression of VDR-target gene,CYP24A1 was increased in the RUPP+VD group compared to the NP and RUPP groups(P<0.05).It indicated that 1,25(OH)2D should play a role in alleviating kidney impairment of RUPP rat probably via its genomic response.2.3 1,25(OH)2D attenuated trophoblast cell damage in placenta of RUPP ratBy HE stain images,obvious distinctions between three groups were observed more in the segment of villus of rat placenta than in other segments of placenta.In the trophoblast of villus,cytoplasm became decreased and its nuclear was irregular with deeper stained in RUPP group compared to NP group.The cytoplasm in trophoblast was increased and its nuclear was round with lighter stained in RUPP+VD group compared to RUPP group.By transmission electron microscopy images,microvillus was barely on the surface of trophoblast cell and nucleus of trophoblast was seen with increased heterochromatin in RUPP group compared to NP group.In RUPP+VD group,microvillus was few and pyknotic nucleus was ameliorative in irregular shape with slight heterochromatin compared to RUPP group.By PCR assay,VDR expression in placenta of RUPP group was higher than that in NP group and was not different significantly from that in RUPP+VD group.As a VDR-target gene,placenta CYP24A1 expression,however,was not significantly different among the three groups.By immunohistochemistry staining,VDR expression at protein levels was more in villus than that in other segments of placenta in all three groups.VDR protein expression in villus of RUPP group was higher than that in NP group and lower than that in RUPP+VD group.These results indicated that the improvements of placenta structure and function mentioned above are correlated with 1,25(OH)2D treatment and may be mediated by its non-genomic response.3 1,25(OH)2D alleviated hyperglycemia in RUPP ratMaternal hyperglycemia in RUPP group was relieved in RUPP +VD group(P<0.05).In addition,total cholesterol decreased in RUPP and RUPP+VD groups(P<0.01;P<0.05,respectively).Maternal serum triglyceride similar lowered in RUPP group(P<0.05).Summary:we proved that 1,25(OH)2D addition beginning at the early gestation had various effect on blood pressure,fetus and placenta,heart,kidney and metabolism in RUPP rat model.Part III 1,25(OH)2D alleviated placenta trophoblast cell damage of RUPP rat by inhibiting placenta apoptosis and stimulating placenta autophagyObjective:We established 1,25(OH)2D alleviated placenta trophoblast cell damage of RUPP rat by decreasing the levels of oxidative stress,endoplasmic reticulum(ER)stress,inhibiting placenta apoptosis and stimulating placenta autophagy.Methods:The level of oxygen species of rat placenta was assayed with DHE stain.The apoptosis level of rat placenta was assayed with TUNEL stain.We observed markers expression of oxidative stress,endoplasmic reticulum stress,apoptosis and autophagy by western blot and immunohistochemistry staining.Results:1 1,25(OH)2D decreased the levels of oxidative stress in placenta of RUPP ratLevels of oxygen species in placenta were higher in RUPP group than that in NP group,and lower in RUPP+VD group than that in RUPP group.The expression of P47-phox,an oxidative stress marker,at mRNA and protein levels was higher in placenta of RUPP group than that in NP group,and was not significantly different in placenta between RUPP+VD group and NP group.2 1,25(OH)2D decreased endoplasmic reticulum(ER)stress level in placenta of RUPP ratBy immunohistochemical and immunoblot,the protein expression of GRP 78,CHOP and Caspase 12,the ER stress markers,was higher in placenta of RUPP group than that in NP group.Except GRP 78 expression was higher in placenta of RUPP+VD group than that in NP group,the expression of CHOP and Caspase 12 was not significantly different in placenta between RUPP+VD group and NP group.3 1,25(OH)2D inhibited apoptosis in placenta of RUPP ratBy TUNEL image,the number of the apoptosis cells in placenta was RUPP group,RUPP+VD group and NP group in turn.Caspase 3 protein expression decreased in RUPP group and RUPP+VD group compared with NP group in immunohistochemistry staining.In the meantime,1,25(OH)2D treatment markedly down-regulated Cleaved Caspase 3 protein expression compared to RUPP group.4 1,25(OH)2D stimulated autophagy in placenta of RUPP ratBy immunoblot analysis,the expression of Beclin 1 and LC3B-II,autophagy markers,was decreased in placenta of RUPP group compared to NP group,and increased in RUPP+VD group compared to RUPP group.Immunohistochemical staining further shown that Beclin 1 protein expression was higher in placenta of RUPP+VD group than that in RUPP group and NP group.LC3B expression was not different among three groups since the antibody reacted with LC3B-I and LC3B-II at the same time and the total protein had no change.Summary:1,25(OH)2D alleviated placenta trophoblast cell damage of RUPP rat by inhibiting placenta apoptosis and stimulating placenta autophagy.Conclusions:1 Low level of 25(OH)D in preeclampsia pregnancy may have relationship with placenta development and fetus growth.2 we proved that 1,25(OH)2D addition beginning at the early gestation had various effect on blood pressure,fetus and placenta,heart,kidney and metabolism in RUPP rat model.3 1,25(OH)2D alleviated placenta trophoblast cell damage of RUPP rat by inhibiting placenta apoptosis and stimulating placenta autophagy. |
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