| Intra-uterine growth retardation(IUGR)is a serious problem in animal husbandry production,which refers to the growth and development damage of mammalian embryo/fetus or its organs during pregnancy.IUGR fetus often occurs catch-up growth,which could result in type 2 diabetes mellitus,cardiovascular disease,increased fat mass,and obesity.Placental insufficiency induced by oxidative stress during the pregnancy is the major cause of IUGR.In addition,IUGR could also cause damage to the intestinal function and microbiota.Curcumin has a variety of biological activities such as anti-inflammatory,antineoplastic,and antioxidant.However,the effects of dietary supplementation of curcumin on placental function and intestinal function of IUGR remains unclear.Therefore,this study was conducted to investigate the effects of IUGR on porcine placenta.Then,we used the IUGR mice model of protein restriction to study the effects and potential mechanisms of curcumin supplementation in pregnancy diet on maternal placental function and intestinal function of mice offspring with IUGR.The study is divided into five parts.The research contents and results are as follows:1.A comparative study of normal porcine placenta and intrauterine growth retarded porcine placentaThis experiment was conducted to study the effects of IUGR on proliferation,apoptosis and antioxidant gene and protein expression of porcine placenta.In this experiment,the placentas of normal birth weight and IUGR piglets were selected from newly delivered sows,and they were divided into the control group and IUGR group.The results showed as follows:(1)Stronger PCNA immunostaining was observed in the control group,while weaker PCNA staining was observed in the IUGR group;(2)The apoptosis rate of the IUGR group was significantly higher than that of the control group(P<0.05);(3)The results of qRT-PCR showed that compared with the control group,the mRNA expression of NRF2,NQO1,SOD1,SOD2,GCLC and GCLM in placenta of the IUGR group was significantly decreased(P<0.05).While the expression of HO-1 mRNA showed no significant difference between the control group and the IUGR group(P>0.05);(4)The protein expression levels of NRF2 and HO-1 in the control group and the IUGR group were not significantly different(P>0.05).The above results showed that IUGR could be caused by decreased proliferation,increased apoptosis and reduced antioxidant gene expression of porcine placental cells.2.Protective effect and mechanism of curcumin on oxidative damage of human trophoblast HTR8/SVneo cellsThe aim of the present study was to investigate effect of curcumin pretreatment on the oxidative damage of HTR8/SVneo cells induced by H2O2 and its molecular mechanism.Four groups were established in this experiment:(1)The control group;(2)H2O2 group;(3)H2O2+2.5μM curcumin group;(4)H2O2+5μM curcumin group.The results showed that:(1)H2O2 significantly reduced the cell viability,and the 5μM curcumin pretreatment group significantly increased the cell viability(P<0.05);(2)The activities of CAT and GSH-Px were significantly increased and the accumulation of ROS was significantly decreased in curcumin pretreatment group(P<0.05);(3)Flow cytometry showed that 5μM curcumin pretreatment significantly reduced the apoptosis rate of HTR8/SVneo cells(P<0.05);(4)The results of qRT-PCR showed that H2O2 stimulation alone significantly increased the mRNA levels of NRF2,HO-1,GCLC,GCLM and NQO1(P<0.05),whereas pre-treatment of 5μM curcumin further up-regulated the expression of NRF2,GCLM and NQO1(P<0.05).H2O2 treatment alone increased the transcription level of Bax,and pre-treatment with 5μM curcumin increased the expression of BCL-2(P<0.05).In addition,pre-treatment with 5μM curcumin increased the gene expression of SLC2A3(P<0.05);(5)H2O2 stimulation upregulated the protein expression of NRF2 and NQO1(P<0.05).Pre-treatment with 2.5 or 5μM curcumin up-regulated the protein expression of nuclear NRF2,total-NRF2,HO-1 and NQO1(P<0.05);(6)In addition,both H2O2 and curcumin stimulated NRF2 nuclear translocation in HTR8/SVneo cells.However,we observed that pre-treatment with curcumin induced more obvious NRF2 nuclear translocation compared with the H2O2 treatment group;(7)Transfected HTR8/SVneo cells with a NRF2 siRNA for 24 h,after the transfection with si-NRF2 in curcumin and H2O2 treatment group,we observed markedly decreased mRNA and protein expression level of NRF2,HO-1 and NQO1(P<0.05).The cell viability and the activation of CAT and GSH-Px were significantly decreased with knockdown of NRF2(P<0.05).The above results showed that curcumin could protect HTR8/SVneo cells from H2O2induced oxidative stress by activating NRF2 signaling pathway.3.Effects of dietary supplementation of curcumin on placental function and fetal growth in mice with intra-uterine growth retardationThe objective of this study was to investigate the effects of dietary supplementation of curcumin during pregnancy on placental function and fetal growth in a mouse model of IUGR fed the protein restriction diet.32 pregnant mice were divided into 4 groups:(1)normal protein(19%protein)diet(NP);(2)protein restriction(8%protein)diet(LP);(3)protein restriction diet+100 mg/kg curcumin(LPL);(4)protein restriction diet+400 mg/kg curcumin(LPH).At GD 19.5,6 pregnant mice per group(a total of 24 mice)were randomly selected and anesthetized with isoflurane and then underwent a cesarean section.The fetal weight,placental weight,and placental diameter were measured.The results showed that:(1)The fetal weight of the LP group significantly decreased compared with the NP group(P<0.05).The fetal weight of the LPL group significantly increased compared with the LP group(P<0.05),while it was lower than the NP and LPH groups.The placental weight of the LP group remarkably decreased compared with other 3 groups(P<0.05).The fetal/placental weight ratio significantly decreased in the LP group(P<0.05);(2)Compared with the NP group,the LP group showed enhanced maternal serum corticosterone level(P<0.05).Maternal serum progesterone levels were reduced in all 3 groups fed the protein restriction diet(P<0.05);(3)The placental GSH-Px activity was significantly higher in the LPL group compared with the LP group(P<0.05).The levels of placental MDA content,a marker of lipid peroxidation markedly increased in the LP group(P<0.05),while treatment with curcumin reduced the concentration of MDA to the control level in the LPL and LPH groups;(4)H&E staining showed that the LP group had a strikingly reduced blood sinusoids area(P<0.05).And the LPH group showed a significantly increased area compared with the LP group(P<0.05).TUNEL assay showed that the protein restriction diet enhanced the placental apoptosis in the LP group.While dietary supplementation of curcumin alleviated the apoptosis in placenta(P<0.05);(5)The LP group showed significantly decreased mRNA expression of Nrf2 compared with the other 3 groups(P<0.05).The LP group exhibited significantly decreased Ho-1 expression compared with the NP group(P<0.05).Igf1 mRNA expression significantly decreased in the LP and LPL groups(P<0.05).VEGF mRNA expression significantly elevated in the LPL group compared with the LP group(P<0.05).Snat2 mRNA expression markedly decreased in all 3 groups fed the protein restriction diet(P<0.05);(6)Fetal liver qRT-PCR results showed that compared with NP group,the mRNA expression levels of Sod1,Sod2,Cat,GSH-Px,Nrf2,Ho-1,Gclc and Nqo1 in LP group were significantly decreased(P<0.05).However,dietary supplementation of curcumin significantly increased the mRNA expressions of Sod1,Sod2,Cat,Gclc and Nqo1(P<0.05);(7)Dietary supplementation of curcumin could significantly increased NRF2 and HO-1 protein expression levels in fetal liver(P<0.05).The above results showed that dietary supplementation of curcumin could improve maternal placental function and fetal growth in mice with IUGR.4.Effects of dietary supplementation of curcumin on intestinal function in mice with intra-uterine growth retardationThe present study investigated whether dietary supplementation of curcumin during pregnancy and lactation on intestinal function of of IUGR mice induced by protein restriction diet.In total,36 C57BL/6 mice(24 females and 12 males,6-8 weeks old)were randomly divided into three groups based on the diet before and throughout pregnancy and lactation:(1)normal protein(19%);(2)protein restriction(8%);(3)protein restriction(8%)+600 mg/kg curcumin.Offspring administered a control diet after weaning until postnatal day 35.Six male and six female mice were randomly selected from each group to slaughter and collect blood and jejunum for index measurement.The results showed that:(1)compared to the NP group,the LP group showed a significant increase in the body weight and serum glucose levels of male mice on PND 35(P<0.05).In addition,the total cholesterol levels were significantly decreased in the LPC group compared to that in the LP group(P<0.05);(2)Compared with the NP group,the LP group showed a significant decrease in the SOD activity and T-AOC content(P<0.05);(3)HE staining results showed that compared with the LP group,the LPC group showed significantly decreased in the crypt depth of jejunum and the increased the ratio of villus height to crypt depth.AB-PAS staining showed that compared with NP group,the loss of goblet cells caused by LP diet could be improved after dietary supplementation of curcumin(P<0.05);(4)Immunostaining results showed that the proportion of PCNA positive cells in jejunal villi of LP group was significantly lower than that of NP group(P<0.05),while the apoptosis rate was significantly increased(P<0.05);(5)In addition,compared with the NP group,the LPC group showed a significant increase in the mRNA expression of Nrf2,Sod2 and Ocln compared with the LP group(P<0.05).The above results showed that dietary supplementation of curcumin during pregnancy and lactation lessens intestinal damage in male mice with IUGR.5.Effects of dietary supplementation of curcumin on intestinal microflora diversity and species composition in mice with intra-uterine growth retardationThe experimental design was the same as experiment 4.After slaughter,cecal contents were collected for 16S rRNA sequencing.The results showed that:(1)In the mice cecum,the dominant microflora at the phylum level were Firmicutes and Bacteroidota,and those at the genus level were Lactobacillus、Desulfovibrio and Lachnospiraceae_NK4A136_group;(2)At phylum level,compared with NP group,the ratio of Firmicutes/Bacteroidota in LP group was significantly decreased(P<0.05).However,there was no significant difference between the NP and LPC groups(P>0.05).At the genus level,compared with the NP group,the relative abundance of Lactobacillus in LP group was significantly reduced(P<0.05).The relative abundance of Desulfovibrio in the LP group has increased significantly(P<0.05),while dietary supplementation of curcumin made the relative abundance of Desulfovibrio in the LPC group close to that in the NP group;(3)PCoA analysis results showed that the samples of NP group and LP group showed an obvious separation trend,indicating that the gut microbiota structure of the two groups was significantly different.The LPC group was distributed between the NP group and the LP group,and was closer to the NP group;(4)Moreover,Lactobacillus was positively correlated with the SOD activity,and it was negatively correlated with Il-1β expression(P<0.05).Desulfovibrio was negatively correlated with the SOD activity and the j ejunal expression of Sod1,Bcl-2,Card11 and Zo-1(P<0.05).The above results showed that dietary supplementation of curcumin during pregnancy and lactation could regulate the cecal microflora of IUGR mice,and can improve the species composition of gut microflora.In summary,in the IUGR group,the expression of PCNA was significantly decreased,the apoptosis rate was increased,and the expression of antioxidant genes was significantly decreased in the porcine placenta.Dietary supplementation of curcumin improved placental function and fetal growth in mice with IUGR induced by a protein restriction diet.Dietary supplementation of curcumin also alleviated the intestinal injury and regulated the composition of intestinal microbiota in male mice offspring with IUGR. |
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