Intervention Mechanism Of Yiqi Liangxue Shengji Recipe On Repair Of Endothelial Cell Injury Based On MTORC1 Signaling Pathway

Percutaneous coronary intervention(PCI)is one of the main treatments for coronary heart disease(CHD).Drug-eluting stents(DES),such as rapamycin-eluting stent,can inhibit the proliferation and migration of vascular smooth muscle cells(VSMCs),but also inhibit the repair of endothelial cells.To promote regeneration of injured endothelial cells and restore intimal integrity as soon as possible is one of the key measures to prevent in-stent restenosis(IRS)and thrombosis after PCI.Yiqi liangxue shengji decoction(YLS decoction)is made up of four Chinese herbs including Astragalus membranaceus,Salvia miltiorrhiza,honeysuckle and cortex moutan.The prophase clinical study suggests that YLS decoction can reduce the trend of ISR and combined adverse cardiovascular events after PCI.The modern pharmacodynamic experiments show that the four drugs have the effects of antigenic microorganism,anti-inflammatory,scavenging oxygen free radicals,oxidative stress damage and improving energy metabolism.These effects may be the mechanism of promoting the repair of endothelial injury by YLS decoction.Mammalian target of rapamycin complex-1(MTORC1)signaling pathway is an important way for rapamycin(RAPA)to regulate the proliferation and migration of endothelial cells and VSMCs.When the energy is lacking,the ratio of AMP/ATP is increased and the AMP activated protein kinase(AMPK)activates mTORC1.The mTORC1 signal is transmitted to two independent target proteins downstream,the p70 ribosomal protein S6 kinase(p70S6K)and 4E binding protein 1(4E-BP1),which controls the cell growth in mammalian.To sum up,we propose that the mechanism of YLS decoction to promote the repair of vascular endothelial cells injuryed by hypoxia/reoxygenation(H/R)is regulating the mTORC1 signaling pathway by reducing oxidative stress and improving energy metabolism to promote proliferation of endothelial cells.Methods:Experiment 1:We prepared human umbilical vein endothelial cells(HUVECs)for H/R model.The experiment had five groups:control group,model group,YLS group,AST group.YLS+AST group.We used CCK 8 method to detect cell proliferation rate,transmission electron microscope to observe cell ultra structure changes,scratch test to test cell mobility and fluorescence probe method to test reactive oxygen species(ROS).The content of malondialdehyde(MDA)was detected by using chromatoptometry and the content of superoxide dismutase(SOD)was detected by using WST-1 method.We detected concentration of ATP and AMP by mass spectrometry.Protein expression level was detected by western blot.All the data was taken statistical analysis.Experiment 2:We prepared HUVECs for H/R model.During the reoxygenation,RAPA was used.The experiment had six groups:control group,model group,H/R+RAPA group,YLS+RAPA group,AST+RAPA group,YLS+AST+RAPA group.We used CCK-8 method to detect cell proliferation rate,scratch test to test cell mobility.Protein expression level was detected by western blot.All the data was taken statistical analysis.Results:Experiment 1:Mechanism of YLS decoction on proliferation of endothelial cells injured by H/R①When the concentration of the serum was 10%,compared with model group,YLS group,AST group and YLS+AST group increased proliferation rate(P<0.05 or P<0.01),YLS+AST group is better than borh YLS group and AST group(P<0.05);②Compared with model group,the cell migration distance of the YLS+AST group was increased(P<0.05);③Compared with model group under the transmission electron microscope,YLS group,AST group and YLS+AST group had better recovery.The nucleus boundary is more clear and the structure of mitochondria is more complete;④Compared with model group,the content of ROS and MDA decreased and the content of SOD increased after the intervention of drug intervention(P<0.05 or P<0.01);⑤ATP/AMP in YLS group and YLS+AST group was significantly higher than the model group(P<0.05 or P<0.01),compared with AST group,YLS+AST group is higher(P<0.01).⑥The p-AMPK/AMPK of YLS and YLS+AST group was significantly lower than that of the model group(P<0.05).The p-mTOR/mTOR,p-p70S6K/p70S6K and p-4E-BP1/4E-BP1 in YLS group,AST group and YLS+ AST group were significantly higher than those in model group(P<0.05).Experiment 2:Intervention of YLS combined with RAPA on proliferation of endothelial cells injured by H/R:①Compared with RAPA group,the proliferation rate of endothelial cells in group YLS+AST+RAPA was significantly higher than that in RAPA group;②Compared with RAPA group,there was no significant impact on cell migration distance in YLS+RAPA group,AST+RAPA group and YLS+AST+RAPA group(P>0.05).③Compared with RAPA group,p-AMPK/AMPK is higher inYLS group,AST group and YLS+AST+RAPA group(P<0.05).No sigignificant differences in p-mTOR/mTOR,p-p70S6K/p70S6K and p-4E-BP1/4E-BP1 between RAPA group and intervention groups.Conclusion:① Yiqi Liangxue Shengji decoction can improve the energy metabolism of endothelial cells injured by H/R,reduce oxidative stress,inhibit the activation of AMPK,regulate the mTORCl signaling pathway,and play the role of protecting endothelial cells proliferation;② The combined treatment of Yiqi Shengxue Shengji decoction and atorvastatin can reduce the inhibitory effect of RAPA on the proliferation of injured endothelial cells.The mechanism is related to the improvement of the energy metabolism of endothelial cells and the inhibition of AMPK activation.