| Proline is a cyclic a-amino acids,which plays an important role in protein synthesis,organism metabolism,wound healing,anti-oxidant and immune reactions.It was also found that exogenous addition of proline could not only facilitate the growth and developmet of embryo,placenta and conceptus on sows,but also prevent intrauterine growth retardation(IUGR).In the early stages of neonatal development,it should be regarded as a conditionally essential amino acid.The aim of our study was to investigate the molecular mechanism of proline which affects fetal development and placental cells proliferation in order to provide a theoretical basis for improving the reproductive performance of sows.Firstly in this paper,the effects of 1%proline on the fetal and placental development and related indexes of Huanjiang sows were discussed at 70 days of gestation.Work with these piglets had shown that the average fetal weight of proline group was 122.07±29.43g,which was significantly higher than that of blank control group(96.06±11.62g,P<0.05).Compared with the control group(6.87±0.40),the average number of fetuses in the proline group(7.87 ± 0.35)was higher,but the difference was not significant(P>0.05).Furthermore,proline significantly decreased the GC content(P<0.05)with 20.34±0.24ng/mL of proline group and 21.31±0.31ng/mL of the control group.The relative expression level of GR mRNA in proline group and control group were respectively 0.67 ± 0.02 and 1.10 ± 0.01,which was significant difference(P<0.05).In addition,GR? and GR? protein expression levels in proline group respectively were 1.00 ± 0.02 and 1.21 ± 0.01,while in the control group respectively were 1.02 ± 0.03 and 1.11 ± 0.01,and there was no significant difference between the two groups(P>0.05).Of all seven GR exonl variants expressed in pigs,only to find that GR exon 1-5 and exon 1-9 were expressed in placenta.Moreover,GR mRNA expression of exonl-5 was significantly decreased by proline,wheras GR exon 1-9 did not change(P>0.05).Following by placental chorionic cell line(BeWo)as a model,the molecular mechanism of proline which mediated placental cell proliferation and apoptosis of placental cells in vitro was preliminarily discussed.BeWo cells were cultured in different concentrations of proline(0?g/mL,2.5?g/mL,5?g/mL,7.5?g/mL,10?g/mL,12.5?g/mL,15?g/mL)for 24h,48h and 72h,We found that cells treated with 2.5 ?g/mL and 5 ?g/mL for 48h did significantly promote cell proliferation and inhibit apoptosis(P<0.05).Then preliminary selected this appropriate concentration and time to analyze related genes expression.The QRT-PCR analysis indicated that adding 2.5?g/mL and 5?g/mL concentration of proline significantly increased the mRNA expression level of CDK4,OLR1 and anti apoptotic gene Bcl2 in BeWo cells(P<0.05),but had no significant effect on the Caspase-3 and Bcl-2 expression.Additiontely,GR mRNA and protein expression of proline group were significantly down-regulated(P<0.05),which was consistent with the Spl and CREB mRNA(P<0.05),whereas the Sp1 negative regulatory factor of p53 gene was inverse(P<0.05).What's more,only 2.5 ?g/mL concentration was found that SLC7A5 and mTOR gene expression was significantly higher than the control group(P<0.05)These above results manifested that proline might inhibit GR transcription by lowering exon 1-5 mRNA leading to decline GC content in placenta,which finally improved the phenomenon of fetal IUGR.Meanwhile,proline promote placenta chorionic cells proliferation might through decrease GR gene and protein expression by down-regulating the transcription factors gene of Sp1 and CREB interation with up-regulating the negative regulatory factor p53 gene,which enhanced mTOR and SLC7A5 expression of the signaling pathway. |
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