吡格列酮对母型-合子型过渡时期小鼠胚胎抗氧化能力的影响

Effects of pioglitazone on early mouse embryos during maternal to zygotic transition

本试验通过H_2O_2建立小鼠胚胎氧化损伤模型,添加PPARγ激动剂吡格列酮对胚胎进行体外培养,DCHFDA测定胚胎内部ROS水平,半定量PCR测定胚胎内部抗氧化基因和NADPH氧化酶基因的表达。结果显示:(1)经过48h处理后,5μmol/L吡格列酮处理组的4-细胞率(P>0.05)和囊胚率(P<0.05)都比对照组的高;(2)DCHFDA检测发现,H_2O_2+PIO处理组的ROS水平比H_2O_2处理组的低(P<0.05);(3)H_2O_2降低抗氧化酶Gpx1、Gpx2的表达(P>0.05),而吡格列酮能上调Gpx1、Gpx2的表达(P<0.05);(4)H_2O_2上调NADPH氧化酶亚型Nox1、Nox3表达,吡格列酮能下调Nox1、Nox3的表达,差异性均不显著(P>0.05),但H_2O_2明显上调NADPH氧化酶亚型Duox2和NOX调节亚基Noxo1的表达水平(P<0.05),吡格列酮均能明显抑制Duox2和Noxo1的表达(P<0.05)。结果表明:吡格列酮能增加抗氧化酶的表达以及抑制NADPH氧化酶的表达,从而有效调控胚胎内部ROS水平,促进胚胎发育。

Peroxisome proliferator-activated receptor gamma （PPARγ） has been involved in NRF2 antioxidant pathway,and regulates the expression of genes associated with antioxidase and NADPH oxidase. To understand functional roles of PPARγ on the development of early embryos and to explore the antioxidant mechanisms of PPARγ in early mouse embryos,the embryo model of oxidative damage was established by H2O2 and cultured in M16 medium supplemented with optimal concentration pioglitazone. ROS level in embryos were measured by DCHFDA,gene expression level was analyzed by semi-quantitative reverse transcription PCR. As a result, we found that：（1）5μmol/L pioglitazone group is higher than control group in 4-cell rate （P〈0.05） and blastocyst rate （P〈0.05）;（2）through DCHFDA fluorescence detection, the ROS level of H2O2 ＋PIO group is significantly lower than H2O2 group （P〈0.05）;（3） H2O2 decreased the antioxidase expression of Gpxl,Gpx2 （P〈0.05）, pioglitazone significantly improved the expression of Gpx1, Gpx2 （P〈0.05） ;（4） H2O2 increased the NADPH oxidase subtype expression of Noxl and Nox3 （P〈0.05）,pioglitazone suppressed the expression of Nox1 and Nox3, the diversity is not remarkable （P〈0.05）. But H2O2 significantly increased the NADPH oxidase subtype expression of Duox2 as well as Nox regulatory subunit Noxol （P〈0. 05）, pioglitazone significantly suppressed the expression of Duox2 and Noxo1（P〈0.05）. The results indicated that pioglitazone effectively regulated the level of ROS inside the embryos through increasing the antioxidase expression and decreasing NADPH oxidase expression,and promoted embryo development.