左归丸母代干预对妊娠糖尿病模型大鼠子代胎鼠胰腺发育调控因子PDX-1、HNF-6的影响

Effects of Zuogui Pills(左归丸)Maternal Intervention on Regulatory Fators of Pancreatic Development in the Offspring of Gestational Diabetes Mellitus Model Rats

目的探讨左归丸母代干预对妊娠糖尿病(GDM)模型大鼠子代胎鼠的影响及机制。方法通过雌雄同笼制备孕鼠90只,随机分为正常组,模型组,门冬胰岛素组,左归丸低、中、高剂量组,每组15只。除正常组外,其余孕鼠通过腹腔注射链脲佐菌素的方法复制GDM孕鼠模型。造模成功后左归丸低、中、高剂量组给予左归丸灌胃剂量分别为2.5、5.0、10.0g/(kg·d),正常组和模型组分别给予10ml/(kg·d)生理盐水灌胃,门冬胰岛素组给予门冬胰岛素注射液20U/(kg·d)皮下注射,每日1次,共给药14天。Western-blot法检测胎鼠胰腺胰十二指肠盒因子1(PDX-1)蛋白表达,Real-time PCR法检测胎鼠胰腺肝细胞核因子6(HNF-6)mRNA表达,BSP法检测胎鼠胰腺HNF-6基因甲基化表达。结果与正常组比较,模型组PDX-1蛋白、HNF-6 mRNA及HNF-6甲基化率表达降低(P<0.05);与模型组比较,门冬胰岛素组及左归丸各剂量组PDX-1蛋白表达均升高,左归丸各剂量组HNF-6 mRNA及HNF-6甲基化率表达升高,门冬胰岛素组HNF-6 mRNA表达下降(P<0.05);与门冬胰岛素组比较,左归丸中剂量组PDX-1蛋白,左归丸各剂量组HNF-6 mRNA及HNF-6甲基化率表达升高(P<0.05);左归丸中剂量组PDX-1蛋白、HNF-6 mRNA及HNF-6甲基化率表达高于左归丸高、低剂量组(P<0.05);左归丸高剂量组HNF-6 mRNA及HNF-6甲基化率表达高于左归丸低剂量组(P<0.05)。HNF-6 mRNA表达与其甲基化率呈显著正相关(P<0.05,r=0.779)。结论左归丸可能通过上调胎鼠胰腺发育调控因子HNF-6基甲基化水平,以提升其mRNA表达,从而上调PDX-1蛋白表达,调节胎鼠胰腺的发育,减缓子代相关疾病的发生。

Objective To explore the effect of maternal intervention of Zuogui Pills(左归丸)on the offspring of rats with a model of gestational diabetes mellitus(GDM)and the mechanism.Methods Female rats were caged with male rats for pregnancy.Ninety pregnant rats were obtained and randomly divided into normal group,model group,insulin aspart group,and Zuogui Pills low,medium and high dose groups,with 15 rats in each group.The intraperitoneal injection of streptozotocin was used to establish a GDM model in pregnant rats of all groups except for normal group.After modeling,Zuogui Pills was administered by gavage in low,medium and high dose groups at a dosage of 2.5,5.0 and 10.0 g/(kg·d),respectively.In normal group and model group,10 ml/(kg·d)normal saline was given by gavage,while in the insulin aspart group,the subcutaneous injection of 20 U/(kg·d)insulin aspart was administered.All the interventions were given once daily,for 14 days.Western blotting was used to detect the protein expression of pancreatic and duodenal homeobox-1(PDX-1)of the fetal rats.Real-time PCR method and bisulfite sequencing PCR(BSP)method were used to detect mRNA expression and gene methylation expression of hepatocyte nuclear factor-6(HNF-6),respectively.Results Compared to normal group,the model group had lower expression levels of PDX-1 protein,HNF-6 mRNA and its methylation(P<0.05).Compared to model group,the PDX-1 protein expression significantly increased in insulin aspart group and all Zuogui Pills groups;the HNF-6 mRNA expression and its methylation significantly increased in Zuogui Pills groups,while decreased in insulin aspart group(P<0.05).Compared to insulin aspart group,Zuogui Pills medium dose group had higher expression of PDX-1 protein,and all Zuogui Pills groups had higher HNF-6 mRNA and methylation expression(P<0.05).Compared among Zuogui Pills groups,the expression of PDX-1 protein,HNF-6 mRNA and its methylation in the medium dose group were the highest(P<0.05).The expression of HNF-6 mRNA and methylation in the high dose group were significantly higher than in low dose group(P<0.05).The expression of HNF-6 mRNA was positively correlated with its methylation(P<0.05,r=0.779).Conclusion Zuogui Pills can up-regulate the methylation level of HNF-6 to enhance its mRNA expression and further up-regulating the protein expression of PDX-1,thereby regulating the pancreas development of fetal rats and reducing the incidence of related diseases.