自噬相关基因在小鼠月经样模型子宫内膜崩解过程中的动态表达

Dynamic expression of autophagy related genes during endometrial disintegration in a mouse menstrual-like model

目的探究小鼠子宫内膜崩解过程中自噬相关基因表达的动态变化。方法建立小鼠月经样模型,孕酮撤退后0 h、8 h、16 h和24 h分别处死小鼠取其双侧子宫组织,通过苏木素伊红(HE)染色观察小鼠子宫组织的形态特征变化,采用蛋白质印迹(WB)检测小鼠子宫内膜崩解过程中LC3B和p62蛋白的表达动态变化,实时定量PCR(RT-qPCR)检测小鼠子宫内膜崩解过程中Lc3b、p62、Beclin1、Atg3、Atg5和Atg7的mRNA水平变化。结果孕酮撤退后0 h、8 h、16 h和24 h子宫大体颜色逐渐由浅粉色变为深红色,子宫内膜崩解坏死区域逐渐扩大,24 h时蜕膜化的子宫内膜完全崩解坏死。WB结果显示:孕酮撤退后16 h时,p62的蛋白表达水平较0 h和8 h时显著升高(P<0.05),24 h时p62的蛋白表达水平较其他时间点显著升高(P<0.01);LC3B-Ⅰ蛋白在孕酮撤退后0 h到8 h无显著变化(P>0.05),8 h到24 h其表达量显著降低(P<0.05);孕酮撤退后子宫内膜崩解过程中LC3B-Ⅱ的蛋白表达水平无显著变化(P>0.05)。RT-qPCR结果显示:p62 mRNA水平在孕酮撤退后0 h到16 h无显著变化(P>0.05),而24 h时p62 mRNA水平较8 h和16 h显著降低(P<0.05);Lc3b mRNA水平在孕酮撤退后16 h时较0 h和8 h显著上调(P<0.01),而24 h时Lc3b mRNA水平显著低于撤退后16 h(P<0.01);Beclin1 mRNA水平在孕酮撤退后8 h、16 h和24 h的表达较0 h均显著下降(P<0.05),且撤退24 h后Beclin1 mRNA水平相较8 h和16 h显著降低(P<0.01);Atg3、Atg5和Atg7的mRNA水平孕在酮撤退后0 h到16 h无显著变化(P>0.05),而24 h时的mRNA水平显著低于其他时间点(P<0.05)。结论孕酮撤退后,子宫内膜崩解过程中自噬水平下降,提示自噬参与负调控子宫内膜崩解过程。

Objective:To investigate the expression changes of autophagy genes and its effects during the endometrial disintegration in the mouse menstrual-like model.Methods:A mouse menstrual-like model was established.Mice were executed and bilateral uterine tissues were taken at 0 h,8 h,16 h and 24 h after progesterone withdrawal.The morphology of the endometrium was observed by hematoxylin-eosin(HE)staining.The protein of LC3B and p62 were detected by Western blotting(WB),and real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the dynamic expression of Lc3b,p62,Beclin1,Atg3,Atg5,and Atg7 at the mRNA level during the process of endometrial disintegration in mice.Results:At 0 h,8 h,16 h and 24 h after progesterone withdrawal,the color of the uterus gradually changed from light pink to dark red,and the area of endometrial disintegration and necrosis was gradually enlarged,and the decidualized endometrium was completely disintegrated and necrotic at 24 h.The WB results showed that at 16 hours after progesterone withdrawal,the protein expression level of p62 was significantly increased compared to 0 and 8 hours(P<0.05),and at 24 hours,the protein expression level of p62 was significantly increased compared to other time points(P<0.01).The expression level of LC3B-Ⅰprotein showed no significant change from 0 h to 8 h after progesterone withdrawal(P>0.05),but decreased significantly from 8 h to 24 h(P<0.05).There was no significant change in the protein expression level of LC3B-Ⅱduring the process of endometrial disintegration after progesterone withdrawal(P>0.05).The RT-qPCR results showed that there was no significant change in the level of p62 mRNA from 0 h to 16 h after progesterone withdrawal(P>0.05),while at 24 h,the level of p62 mRNA was significantly reduced compared to 8 h and 16 h(P<0.05).The level of Lc3b mRNA was significantly upregulated at 16 hours after progesterone withdrawal compared to 0 and 8 hours(P<0.01),while at 24 hours,the level of Lc3b mRNA was significantly decreased than that at 16 hours after progesterone withdrawal(P<0.01).The expression of Beclin1 mRNA levels significantly decreased at 8,16,and 24 hours after progesterone withdrawal compared to 0 hours(P<0.05),and the mRNA levels of Beclin1 significantly decreased at 24 hours after progesterone withdrawal compared to 8 and 16 hours(P<0.01).After progesterone withdrawal from 0 h to 16 h,Atg3,Atg5 and Atg7 were not significantly changed at the mRNA level,whereas they were all down-regulated at the mRNA level than those at other time points(P<0.05).Conclusions:After progesterone withdrawal,autophagy levels decreased during endometrial disintegration,suggesting that autophagy is involved in negatively regulating the endometrial disintegration process.