二甲双胍对高糖环境下的大鼠胎盘病理变化影响的机制研究

Mechanism study on effect of metformin on pathological changes of rat placenta in high glucose environment

目的探究二甲双胍对高糖环境下的大鼠胎盘病理变化影响。方法将12只造模成功的妊娠期糖尿病雌性大鼠随机分为糖尿病组(n=6)和二甲双胍组(n=6)。二甲双胍组采用二甲双胍灌胃给药,剂量为300 mg/kg/d,每3 d称重调整药物剂量;糖尿病组给予等体积的饮用水,持续至第16.5天,处死取材;观察分析胎盘组织HE染色的结果;通过RNA-Seq测序筛选出差异表达基因,并对差异基因进行GO和KEGG富集分析。结果二甲双胍组大鼠血糖显著低于糖尿病组,二甲双胍组的孕鼠体重、胎盘重、胎盘系数及活胎数均显著高于糖尿病组(P<0.05);糖尿病组胎盘纵切面面积显著变大、胎盘各层厚度显著增加(P<0.05)。转录组测序共筛选出318个差异表达基因,与糖尿病组相比,二甲双胍组上调基因142个、下调基因176个;选取5个基因进行实时定量PCR验证,检测结果与RNA测序结果一致。GO分析显示差异表达基因主要富集在生物进程、细胞组分、分子功能等;KEGG富集结果提示TGF-β信号通路、Hippo信号通路、Relaxin信号通路等差异基因数量较多;通过GO和KEGG联合分析,筛选出Tgfb2、Notch1、Wnt11、Fbn1、Col1a2为二甲双胍治疗的新的效应基因。结论二甲双胍的干预治疗可有效缓解妊娠期糖尿病大鼠胎盘的病理改变;转录组测序提示许多基因存在差异性表达,但其具体机制亟待进一步探究。

Objective:To explore the effect of metformin on the pathological changes of placenta in high glucose environment.Methods:Twelve female rats were successfully induced gestational diabetes,and then randomly divided into diabetes group(n=6)and metformin group(n=6).The rats in metformin group was administrated intragastric with 300 mg/kg/day metformin,and the dose was adjusted by weighing every three days.The rats in diabetic group was given equal volume of drinking water until 16.5 th day.The rats were executed for sampling.The results of HE staining of placental tissue were observed and analyzed.The differentially expressed genes were screened by RNA-Seq sequencing,and GO and KEGG enrichment analysis was performed on the differentially expressed genes.Results:The body weight,placental weight,placental coefficient and number of live births in metformin group were significantly increased compared with the diabetes group(P<0.05).The longitudinal section area of placenta and the thickness of placenta layers were significantly increased in the gestational diabetes group(P<0.05).A total of 318 differentially expressed genes were screened by transcriptome sequencing(P<0.05).Compared with the diabetes group,there were 142 up-regulated genes and 176 down-regulated genes in metformin group.Five genes were selected for real-time quantitative PCR validation.The detection results were consistent with the RNA sequencing results.GO analysis showed that differentially expressed genes were mainly enriched in biological processes,cellular components,molecular functions.KEGG enrichment results indicated that there were more differentially expressed genes in TGF-βsignal pathways,Hippo signaling pathways,Relaxin signaling pathways,etc.Through the combined analysis of GO and KEGG,Tgfb2,Notch1,Wnt11,Fbn1 and Col1a2 were selected as new effector genes for metformin treatment.Conclusions:Pretreatment of metformin can effectively relieve the pathological changes of placenta in rats with gestational diabetes.The results of transcriptome sequencing show that many genes exhibit differential expression,and their specific mechanism needs to be further explored.