The Research For The Mechanism Of Danlou Table Promoting The Proliferation And Migration Of Endothelial Cells Based On The PI3K/Akt Signaling Pathway

Objectives : to explore the mechanisms of how Danlou tablets promote proliferation and migration of HAEC cells on the basis of following aspects: the effects of Danlou tablets on proliferation,cell migration,cell cycle profile and expression of cell cycle related factors; the effects of Danlou tablets on secreting VEGF, SDF1 and e NOS by HAEC cells; the effects of Danlou tablets on proteins expression of PI3K-AKT pathway related protein with or without PI3 K inhibition by antagonist.Methods1.GroupsCells were synchronized at G0/G1 phase by no serum for 24 hours and then were divided into 5 groups: serum starvation group,control group, Danlou tablets group,Rosuvastatin gtoup and mixed serum group.Each groups were treated with 10% different drug-containing serum or without serum for 24 hours before the next experiment.2. Drug-containing serum preparationFour groups of rabbits were dosed with saline, Danlou tablets(2.5g/kg/d), Rosuvastatin(5g/kg/d) or Danlou tablets(2.5g/kg/d) combined with Rosuvastatin(5g/kg/d) for 5 days. Blood was collected form aortaventralis and centrifuged at the 1000rmp/min speed for 15 min after 1 hour of the last dose.Then the serum was stored at-70℃.3. Cell proliferation2000 HAEC cells at exponential phase were plated in 96-well plated and were then synchronized by no serum for 24 hours before the cells were treated without or with different drug-containing serums for another 24 hours. Then the cell culture medium was collected and stored at-70℃, and the cell proliferation activity was assessed by Cell Titer-Glo.4. Cell migration200ul of exponential phase cells(1×105/ml) were plated in the upper chamber, and 600 ul of pre-cultured medium was added in the lower chamber for 24 hours in cell culture incubator. Migrating cells were stained and counted under microscope.5. Elisa for VEGF、SDF1 and e NOSDetecting the amount of SDF1,e NOS and VEGF in the supernatant after 24 hours by ELISA.6. Flow cytometry for cell cycle phaseAfter 24 hours’ culture with medium supplemented 10% drug-containing serum, cell cycle profiles were determined by flow cytometry.7. Western blot for detecting the expression of PCNA,Cyclin D1 and CDK4Detect the expression of PCNA, CDK4 and Cyclin D1 of HAEC cells after the culture with medium supplemented 10% drug-containing serum.8.Western blot for detecting the expression of PI3 K,Akt,p-PI3 K and p-AktDetect the expression of PI3 K,Akt,p-PI3 K and p-Akt of HAEC cells after being cultured with medium supplemented 10% drug-containing serum.Results1. Cell proliferationThe results indicated that:the proliferation rate for groups of Danlou group, Rosuvastatin group and mixed group were 42.88%,43.49%,102.52% separately. The mixed group has the highest proliferation rate with statistical significance(P<0.01 or P<0.05).Compared with Rosuvastatin, the proliferation rate of Danlou group have no significant statistical significance(P>0.05).2. Cell migrationThe results indicated that:all four groups migrated better than serum starvation group(P<0.01). Compared with the control group, all three drug-containing serums significantly promoted the migration of HAEC. Compared with Danlou and Rosuvastatin groups, more cells migrated in the mixed serum group with statistical significance(P<0.05). Compared with Danlou Group more cells migrated in Rosuvastatin group without statistical significance(P>0.05).3. Cell cycle phaseThe results indicated that:the proliferation indexes for cell starvation group, control serum group, Danlou group, Rosuvastatin group and Mixed serum group were 17.59%, 41.77%, 51.87%, 50.74% and 58.34% separately. All four groups proliferated faster than serum starvation group with statistical significance(P<0.01). Compared with control serum group, the proliferation indexes of all three groups with drug-containing serum significantly elevated(P<0.01). The mixed group proliferated better than Danlou and Rosuvastatin groups(P<0.05). There is no statistical different between Danlou and Rosuvastatin groups.4. Expression of VEGF、SDF-1、e NOSThe results indicated that:the secretion of VEGF 、 SDF-1and e NOS was significantly increased compared with cell starvation group(P<0.01). The secretion of the three factors above in all three drug-containing groups were higher than control group(P<0.01 or P<0.05), the mixed group is higher than Danlou and Rosuvastatin groups. There is no statistical different between Danlou and Rosuvastatin groups(P>0.05).5. Expression of PCNA、Cyclin D1 and CDK4The results indicated that:(1)PCNA:compared with serum starvation and control groups, expression of PCNA in all three drug-containing groups is significantly higher(P<0.01), but the control group increased without statistical significance compared with serum starvation(P>0.05). There is no statistical significance among three groups with drug-containing serum(P>0.05).(2) Cyclin D1:expression of Cyclin D1 in all three groups with drug-containing serum were significantly higher than both cell starvation and control serum groups(P<0.01). expression of Cyclin D1 in mixes group is higher than Danlou and Rosuvastatin group(P<0.05);no statistical significance was observed between Danlou and Rosuvastatin group(P>0.05).(3)CDK4 :there is no difference among different groups(P>0.05).6. Expression of PI3K、Akt、p-PI3K、p-AktThe results indicated that:(1)PI3K and p-PI3K:there is no difference of PI3 K expression in each group(P>0.05);compared with no serum, phosphorylation-PI3 K in the other groups were significantly increased(P<0.01);Compared with control group, phosphorylation of PI3 K in all three groups with drug-containing serum were increased(P<0.01);the mixed group is significantly higher than both Danlou and Rosuvastatin groups(P<0.05),no statistical difference was observed between Danlou and Rosuvastatin groups(P>0.05).(2)Akt and p-Akt:compared with serum starvation group, expression of AKT in all the other groups increased(P<0.01);while no significant difference was observed among all four groups(P>0.05); phosphorylation-AKT increased significantly in all three drug-containing groups compared with serum starvation(P<0.01),phosphorylation of AKT in control group increased without statistical significance(P>0.05);phosphorylation of AKT in all three groups with drug containing serum increased with statistical significance(P<0.01);compared with both Danlou and Rosuvastatin groups, the mixed group increased significantly(P<0.01). There is no statistical significance in between Danlou and Rosuvastatin groups(P>0.05).Conclusion1. Danlou tablet has effects of promoting HAEC proliferation and migration, improving endothelial cells function. The effects were enhanced in combination with Rosuvastatin.2. The effect of Danlou tablet and combination with Rosuvastatin in promoting HAEC proliferation base on the up-regulation of PCNA and Cyclin D1 and promotion from G0/G1 to S and G2/M phases.3.The effects of Danlou tablet and combination with Rosuvastatin in endothelial proliferation and migration may be related with the activation of PI3K/Akt signaling pathway function and up-regulation of p-PI3 K and p-Akt protein.