Study On The Mechanism Of Bushen Antai Granule Regulating PI3K/Akt Signaling Pathway To Improve Maternal Fetal Vascular Recasting

Objective Based on the early stage of the Bushen Antai Granules in the treatment of recurrent abortion and experimental observation,to further explore the Bushen Antai Granules infusion of regulating mechanism of recurrent miscarriage,this experiment by studying the Bushen Antai Granules drug-containing serum on the outer placental microvascular endothelial cell(PMVEC)of the body,PI3K/Akt signal pathway as the core,to observe the effect of bushen Antai Granule on the expression of VEGF,VEGFR-2,PI3 K,Akt m RNA and protein and P-Akt protein in signal pathway,and to observe the effect of Bushen Antai Granule on PMVEC multiplication,locomotion and tube formation,to reveal the connotation of bushen Antai Granule in preventing and treating recurrent miscarriage from cellular and molecular mechanism.It provides a scientific basis for the treatment of recurrent miscarriage and also helps to promote its modern research and development.MethodsCollect healthy pregnant women at six to eight weeks in the first affiliated hospital of anhui medical university department of gynaecology do abortion villus tissue,cultivate PMVEC.The method of gastric perfusion was used in SPF male SD rats,the experimental group was given the equivalent dose of 11.7 g/kg of bushen Antai Granule to prepare the medicated serum,the Control Group,the serum of SD male rats was prepared by intragastric administration of deionized water.Design three pairs of VEGFR-2 si RNA,using si RNA liposome mediated transfection PMVEC,RT-PCR method selection activity;The PMVEC after stable passage was randomly assignment to normal serum group,si RNAnc normal serum group,drug serum group,si RNA normal serum group,and si RNA drug serum group.Detection of PMVEC proliferation by the MTT method,the experimental determination of PMVEC Transwell migration,Tube Formation detection of PMVEC tube Formation;The effect of Bushen Antai Granules on expressions of VEGF,VEGF-R2,PI3 K and AKT m RNA in PMVEC were checked by RT-PCR.Western blot was used to check the effect of Bushen Antai Granules on the expression of VEGF,VEGFR-2,PI3 K,Akt,p-Akt in PMVEC.The effect of Bushen Antai Granules on expressions of VEGF,VEGF-R2,PI3 K and Akt in p MVEC were checked by immunofluorescence technique.Results1.PMVEC proliferation in each group was detected by MTT method The MTT results of PMVEC in each group were shown at different time points: Made comparable with averagel serum group,there was no obvious difference in PMVEC multiplication in Normal Serum Group of SIRNANC(P>0.05),while the effect of drug serum group on PMVEC multiplication was significantly increased(P<0.05),and the PMVEC multiplication of si RNA normal serum group was significantly decreased(P<0.05);Compared with the si RNA averagel sera group,the PMVEC multiplication of si RNA drug sera group was up-regulated(P<0.05 at 24 h,P<0.01 at 48h).2.Transwell assay was used to determine PMVEC migration in each group Made comparable with the averagel serum group,there was no obvious difference in PMVEC locomotion in si RNAnc normal serum group(P>0.05),while the PMVEC migration in drug serum group was increased(P<0.05),and the si RNA normal serum group had significant inhibitory effect on cell migration(P<0.01);Compared with the si RNA averagel serum group,the effect of si RNA drug serum on cell migration was significantly increased(P<0.01).3.PMVEC Formation in each group was detected by Tube Formation Made comparable with the averagel serum group,there was no obvious difference in the number of PMVEC tubes in si RNAnc normal serum group(P>0.05),while the number of components of drug serum increased(P<0.05),and the si RNA normal serum group had significant inhibitory effect on PMVEC tube formation(P<0.01);Compared with the si RNA normal serum group,the effect of si RNA drug serum on PMVEC tube formation was obviously increased(P<0.01).4.The effect of Bushen Antai Granules on the contents of VEGF,PI3 K,VEGFR-2and AKT m RNA in PMVEC of each group were checked by RT-PCR When the expression level of β-actin m RNA was consistent in each group,there was no significant difference in the expression of VEGF,VEGFR-2,PI3 K and AKTm RNA in si RNAnc normal serum group compared with the normal serum group(P>0.05),while the m RNA expression of VEGF,VEGFR-2,PI3 K and AKT increased in drug serum group(P<0.05),and the m RNA expressions of VEGF,VEGFR-2,PI3 K and AKT in si RNA normal serum group were significantly decreased(P<0.01);Compared with the si RNA normal serum group,the m RNA expressions of VEGF,VEGFR-2,PI3 K and AKT were increased in si RNA drug serum group(P<0.05).5.The Western blot was used to check the effect of Bushen Antai Granules on the expressions of AKT,VEGF,PI3 K,VEGFR-2 and p-Akt protein in PMVEC in each groupWhen the expression level of β-actin in each group was consistent,there were no significant differences in VEGF,VEGFR-2,PI3 K,AKT,p-AKT in si RNAnc normal serum group when compared with the normal serum group(P>0.05),while the expression of VEGF,VEGFR-2,PI3 K,AKT,p-AKT protein increased in drug serum group(P<0.05),and the protein expressions of VEGF,VEGFR-2,PI3 K,AKT and p-AKT in si RNA normal serum group were obviously down-regulated(P<0.01);Compared with the si RNA normal serum group,VEGF,VEGFR-2,PI3 K,AKT,p-AKT were obviously up-regulated in si RNA drug serum group(P<0.01).6.The effect of Bushen Antai Granules on the expressions of AKT,VEGF,PI3 K and VEGFR-2 in PMVEC of each group were detected by immunofluorescence assay Compared with the normal serum group,there were no significant differences in VEGF,VEGFR-2,PI3 K and Akt proteins in si RNAnc normal serum group(P>0.05),while the content of VEGF,VEGFR-2,PI3 K and Akt protein increased in drug serum group(P<0.05),and the protein content of VEGF,VEGFR-2,PI3 K and Akt decreased in si RNA normal serum group(P<0.05);VEGF,VEGFR-2,PI3 K and Akt proteins increased in si RNA-treated serum group compared with si RNA-treated normal serum group(P<0.05).ConclusionBushen Antai granule can increase the expression of VEGF and VEGFR2 content in RSA patients PMVEC,improve PI3K/Akt signaling pathway PI3 K,Aktm RNA and protein content and the expression of P-Akt protein content,to activate the PI3K/Akt signal pathway,and then phosphorylate series downstream regulatory protein,adjust the biological function of endothelial cells,promote the placental microvascular endothelial cell proliferation,migration and tube cavity formation,improve the placenta angiogenesis and promote the formation of placental vascular network,guarantee between maternal-fetal blood supply,the supply of nutrients and normal gas exchange function is normal,d Hold the purpose of pregnancy.