| Objective:To study the effects and influence mechanism of maternal intervention of tonifying kidney recipes on fetal pancreas development of GDM model rats by animal experiment in vivo;To study the effect of serum containing tonifying kidney agent on the differentiation of embryonic stem cells into insulin-secreting cells by cell induction experiment in vitro,so as to provide a new scientific basis for the treatment and mechanism of clinical GDM.Methods:The first part:GDM rat model was established by intraperitoneal injection of streptozotocin（STZ,N group was injected with normal saline）in pregnant rats which were prepared by keeping male and female rats together in the cage in a ratio of 2:1.The rats with successful model reproduction were divided into model group（M）,insulin aspartate group（MD）,Liuwei Dihuang pill group（LW）,Zuogui pill group（ZG）,Yougui pill group（YG）,Shenqi pill group（SQ）.Insulin asparagus was injected subcutaneously at a dose of 20U·kg-1,and the other groups were gavaged intragastorically at a dose of 5g·kg-1respectively.N and M groups were gavaged with the same dose of normal saline once a day for 2 weeks.During the experiment,the general conditions of the rats were observed daily,and the random blood glucose,body weight,food intake and other indexes were detected regularly;After the experiment,collected the samples and detected the serum insulin level of the rats,weighed the placenta,observed the placenta pathology.And detected the placenta IGF2 expression to determine the placenta development status;Detected the m RNA and protein expressions of PDX-1,Ngn3,HNF-3β,NKX6.1 and Insuin in fetal pancreas.Detected the protein expressions of DNMT1,DNMT3A and USP7 in fetal pancreas;Detected the expression of PDX-1 and Ngn3 methylation in fetal mouse pancreas.The second part:Using kidney-tonifying agent containing serum to intervene embryonic stem cells to induce differentiation into insulin-secreting cells,the experiment was divided into normal group（N）,Zuogui pill induced group（ZG）,Liuwei Dihuang pill induced group（LW）,Yougui pill induced group（YG）and Shenqi pill induced group（SQ）,and observed the cell growth morphology and induction every day.Detected the m RNA and protein expression of SOX17,FOXA2,Ngn3,Nkx6.1 and Insulin after the different induction periods;Detected the methylation expression of PDX-1 and Ngn3;Identified the cell induction by dithizone staining;Determined the expression of Insulin and C-peptide in cell culture medium.Results:The first part:（1）After the preparation of the model,the GDM model rats showed obvious symptoms of polydipsia,polyphagia and polyuria.The pathological changes of pancreatic tissue were vacuolar changes,pancreatic cell decrease,inflammatory cell infiltration and obvious bleeding points.There was no change in the weight of the placenta,and the placental tissue showed some pathological changes including telangiectasia in the decidua area,lysis and necrosis of trophoblast cells,vacuolar changes in cytoplasm.The random blood glucose and food intake of rats in M group were significantly higher than those in N group（P<0.05）,and the body weight was significantly decreased（P<0.05）.The m RNA and protein expression of placental IGF2,pancreatic PDX-1,fetal mouse pancreatic PDX-1,HNF-3β,Ngn3,NKX6.1and Insulin in M group were significantly lower than those in N group（P<0.05）.The expressions of DNMT1,DNMT3A and USP7 methyltransferase in fetal pancreas were significantly decreased（P<0.05）.The methylation rates of PDX-1 and Ngn3 in fetal mice of M group were significantly decreased（P<0.05）,and the methylation rates were positively correlated with gene expression（P<0.05）.（2）After the intervention of kidney tonifying agent,the state of GDM rats,pathological damage of pancreas and placenta were improved to varying degrees.Compared with M group,blood glucose and body weight of rats in MD and kidney tonifying agent groups were significantly decreased（P<0.05）,however,compared with MD group,blood glucose and body weight of rats in kidney tonifying agent group were significantly increased（P<0.05）.Compared with M group,the m RNA and protein expressions of placental IGF2,pancreatic PDX-1,fetal mouse PDX-1,HNF-3β,Ngn3,NKX6.1 and Insulin in MD and ZG groups were significantly increased（P<0.05）,the m RNA expressions of PDX-1,Ngn3,NKX6.1 in LW group were no different from those in M group,the m RNA of HNF-3βwas increased（P<0.05）,and m RNA of insulin was decreased（P<0.05）.The m RNA of Ngn3、Nkx6.1 in YG group were significantly increased than that in M group（P<0.05）,and the expression of Insulin was decreased（P<0.05）,the expression of PDX-1 and HNF-3βwere at the same level than those in M group.The m RNA level of PDX-1、Ngn3、HNF-3βin SQ group were no different from those in M group,the expression of Nkx6.1 was increased（P<0.05）,the m RNA level of Insulin was decreased（P<0.05）.Protein detection showed that the protein expression of each index in LW and SQ group was no different from that in M group,and the protein expressions of HNF-3βand Ngn3 in YG group were not significantly different from those in M group,while the protein expressions of PDX-1 and NKX6.1 were significantly increased（P<0.05）,and the protein expression of Insulin was significantly decreased（P<0.05）.There were no differences in the m RNA and protein expressions of placental IGF2,pancreatic PDX-1,fetal mouse PDX-1,HNF-3β,Ngn3,NKX6.1 and Insulin between LW,SQ and M group.Compared with the M group,the protein expressions of DNMT1,DNMT3A and USP7 in MD and renal tonification agent groups were significantly increased（P<0.05）;Compared with the MD group,the expressions of DNMT1,DNMT3A and USP7 in ZG group were significantly increased（P<0.05）,and the other three groups were significantly decreased（P<0.05）.Compared with the M group,the methylation rates of PDX-1 and Ngn3 in MD,ZG and LW groups were significantly increased（P<0.05）,while the methylation rates of YG and SQ groups were significantly decreased（P<0.05）.Compared with the MD group,the methylation rates of PDX-1 in ZG group were significantly increased（P<0.05）,and there was no difference in the other groups.The second part:（1）Endoderm period:Compared with N group,there was no difference in SOX17 immunohistochemical optical density between ZG and LW groups,but it was significantly decreased in YG and SQ groups（P<0.05）.Foxa2 in ZG group was significantly increased（P<0.05）.Compared with N group,SOX17 protein expression in ZG group was significantly increased by Western-blot detection（P<0.05）,while it was significantly decreased in other groups（P<0.05）.Foxa2 in kidney tonifying agent group was significantly lower than that in N group（P<0.05）,and that in other kidney tonifying agent groups was significantly lower than that in ZG group（P<0.05）.Compared with N group,the m RNA expression of SOX17 and FOXa2 had no difference,and the other groups were significantly decreased（P<0.05）.（2）The islet precursor cell phase:Compared with N group,the m RNA expression and gene methylation rate of PDX-1 in ZG group were significantly increased（P<0.05）,while the protein expression was significantly decreased（P<0.05）.The m RNA,gene methylation rate and protein expression of PDX-1 in the other three groups were significantly decreased compared with ZG group（P<0.05）.（3）Endocrine precursor cell stage:Compared with N group,there were no differences in Ngn3 m RNA,gene methylation rate and protein expression in ZG group,while those in other groups were significantly decreased compared with ZG group（P<0.05）.（4）Cell phase of insulin secretion:Compared with N group,the expression of m RNA and protein of NKX6.1,Insulin in ZG group were significantly increased（P<0.05）,and the expression of Insulin m RNA in LW group was significantly increased（P<0.05）.The others were significantly lower than those in ZG group.Conclusion:（1）Kidney-tonifying recipes can improve the placental function of GDM rats by regulating the intrauterine high glucose environment and placental development,and affect the expression of HNF-3β,PDX-1,Ngn3,NKX6.1and Insulin in the epigenetic form of PDX-1 signaling pathway in the pancreas development of offspring,and further affect the pancreas development of offspring.Zuogui pill can promote the development of pancreas of offspring by increasing the methylation rate of PDX-1 and Ngn3,while Yougui pill,Liuwei Dihuang pill and Shenqi pill can not promote the development of pancreas of offspring by the opposite regulation.（2）Kidney-tonifying agents affected the expression of HNF-3β,PDX-1,Ngn3,NKX6.1 and Insulin at different stages of inducing mouse embryonic stem cells to differentiate into insulin-secreting cells through epigenetics,in order to influence the formation rate of insulin-secreting cells.Zuogui pill can promote the formation of insulin-secreting cells by up-regulating the methylation rate of PDX-1 and Ngn3 to up-regulate the expression of markers at various stages,while Yougui pill,Liuwei Dihuang pill and Shenqi pill can not promote the formation of insulin-secreting cells by the opposite effect.Markers in the stages of mouse embryonic stem cell induction toward insulin-secreting cells are exactly key factors in the PDX-1 signaling pathway.（3）In this study,the effect mechanism of kidney tonifying agent on pancreatic development was explained at both animal and cell levels,which reflected the importance of TCM"tonifying kidney and filling essence",and provided experimental basis for the mechanism research and clinical treatment of GDM. |
PDF Full Text Request