To Explore the Mechanism of Xintongtai Medicated Serum on Inhibiting Apoptosis of Rabbit Aortic Vascular Smooth Muscle Cell by Interfering PI3K/Akt/HIF-1α Signaling Pathway / 世界科学技术-中医药现代化

ABSTRACT

Objective Bioinformatics analysis combined with cell experiment to explore the effect of Xintongtai medicated serum on apoptosis of rabbit aortic vascular smooth muscle cells by down-regulating PI3K/Akt/HIF-1α.Methods ox-LDL was used to induce the apoptosis of VSMC so as to establish the atherosclerotic cell model.The CCK8 method was used to select the optimal concentration of Xintongtai medicated serum.The main chemical components of Xintongtai were collected by TCMSP,the information of active compounds was collected by PubChem,the targets of active compounds were predicted by SwissTargetPrediction,the targets of"atherosclerosis"and"apoptosis"were collected by genecards and disgenet,and the protein-protein interaction(PPI)network was constructed by string platform.David online analysis gene ontology(GO)enrichment analysis function and Kyoto Encyclopedia of genes and genomes(KEGG)enrichment analysis.VSMC was divided into blank serum group,model group,Xintongtai medicated serum group and phosphatidylinositol 3 kinase(PI3K)inhibitor(LY294002)group,and Xintongtai medicated serum+ LY294002 group.The apoptosis of VSMC was detected by TUNEL and flow cytometry,and the apoptosis rate was calculated.The mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 were determined by polymerase chain reaction(PCR).Protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 were determined by Western blot.α-SMA(Contractive VSMC specific marker)Fluorescence quantification of VSMC was determined by cellular immunofluorescence.Results The optimal concentration selected by CCK-8 was 20%middle dose Xintongtai medicated serum.Compared with the blank group,the VSMC early apoptosis rate,late apoptosis rate and total apoptosis rate in model group were increased(P<0.01),the mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 of the model group were up-regulated(P<0.01),the protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 of the model group were up-regulated(P<0.01).Compared with the model group,the VSMC early apoptosis rate,late apoptosis rate and total apoptosis rate in Xintongtai medicated serum group,LY294002 group,and Xintongtai medicated serum+LY294002 group decreased(P<0.01 or P<0.05),and the mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 of Xintongtai medicated serum group,LY294002 group,and Xintongtai medicated serum+LY294002 group were down regulated(P<0.01 or P<0.05),the protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 of Xintongtai medicated serum group,LY294002 group,and Xintongtai medicated serum+LY294002 group were down regulated(P<0.01 or P<0.05),the α-SMA increased significantly(P<0.01 or P<0.05).Compared with the Xintongtai medicated serum group,the VSMC early apoptosis rate,late apoptosis rate and total apoptosis rate were no difference(P>0.05),the mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 were no difference(P>0.05),the protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 were no difference(P>0.05),the α-SMA was no difference(P>0.05).Conclusion Xintongtai medicated serum may down regulate PI3K/Akt/HIF-1α Signal pathway and downstream apoptosis related factors to alleviate the ox-LDL induced VSMC apoptosis,so as to stabilize vulnerable arterial plaque.