P300介导VEGF下调致孕期酒精暴露模型子代小鼠宫内生长受限

Possible mechanism of intrauterine growth restriction in offspring mice induced by alcohol exposure during pregnancy

目的探讨P300介导血管内皮生长因子(VEGF)下调致孕期酒精暴露模型子代小鼠宫内生长受限的可能机制。方法选取SPF级c57小鼠60只。按照雌雄2∶1合笼,次日清晨观察雌鼠阴栓,有阴栓者即为妊娠成功(妊娠成功30只),记妊娠0.5 d,即胎鼠胎龄E 0.5 d。此时开始给予酒精灌胃(5μL/g/d 50%乙醇)建立胎儿酒精综合征(FAS)模型设为实验组,另设对照组(予生理盐水灌胃),实验组及对照组每组各12只[6只雌鼠因灌胃失败未纳入实验]。检测出生体质量;利用化学比色法测定胎盘组织中组蛋白乙酰化酶活性;利用荧光测定法测定胎盘组织中组蛋白去乙酰化酶活性;定量PCR检测组蛋白乙酰化酶p300和PCAF表达,定量PCR和Western blot检测VEGF表达;ChIP-PCR技术检测p300与VEGF启动子结合水平。结果与对照组相比,实验组小鼠出生体质量显著降低(P<0.01),且存在出生后生长发育迟缓;实验组小鼠胎盘中组蛋白乙酰化酶活性较对照组明显下降(P<0.05),而去乙酰化酶活性两组比较差异无统计学意义(P>0.05);实验组小鼠胎盘组织中p300、VEGF表达较对照组明显下降(P<0.05);ChIP-PCR发现,与对照组相比,实验组p300与VEGF启动子结合水平显著降低(P<0.05)。结论孕期酒精暴露通过p300介导组蛋白乙酰化修饰下调胎盘组织中VEGF表达,进而引起子代宫内发育受限。

Objective To explore the possible mechanism of intrauterine growth restriction in offspring mice induced by alcohol exposure during pregnancy.Methods 60 SPF grade c57 mice were selected.According to the male and female cages at a ratio of 2:1,the vaginal plugs of the female mice were observed in the early morning of the next day.Those with vaginal plugs were considered as successful pregnancy,and the pregnancy was recorded as 0.5 days,that is,the gestational age of the fetal mice was E0.5 days.At this time,alcohol gavage(5ul/g/d 50%ethanol)was started to establish a fetal alcohol syndrome(FAS)model,which was set as the experimental group,and another control group(gavage with normal saline)was established.12 mice each[6 female mice were not included in the experiment due to failure of gavage].The birth weight was detected.The activity of histone acetylase in placental tissue was determined by chemical colorimetry.The activity of histone deacetylase in placental tissue was determined by fluorescence assay.The expression of histone acetylase p300 and PCAF was detected by quantitative PCR.The expression of vascular endothelial growth factor(VEGF)was detected by quantitative PCR and Western blot.The binding level of p300 to VEGF promoter was detected by ChIP-PCR.Results Compared with the control group,the birth weight of the mice in the experimental group was significantly reduced(P<0.01),and there was postnatal growth retardation.The intrauterine stillbirth rate in the experimental group was significantly increased(P<0.05).Histone acetylase activity was significantly decreased(P<0.05),while deacetylase activity had no significant difference between the two groups.p300 and VEGF expressions in placental tissue of alcohol-exposed mice were significantly decreased(P<0.05).ChIP-PCR found that the binding level of p300 to the VEGF promoter was significantly reduced compared with the control group(P<0.05).Conclusion Alcohol exposure during pregnancy down-regulates the expression of VEGF in placental tissue through p300-mediated histone acetylation modification,thereby causing restriction of intrauterine growth in offspring.