Study On The Effect And Mechanism Of Resveratrol Combined With Atorvastatin On Endothelialization After Drug-eluting Stent Implantation

Effects of resveratrol and atorvastatin on re-endothelialization after drug-eluting stent implantation in abdominal aorta of rabbitsObjectives:To make abdominal aorta balloon injury model in rabbit,and then implant a drug-eluting stent(DES)into the injured segment.To observe the effects of resveratrol and atorvastatin on poststent re-endothelialization by optical coherence tomography after DES implantation.Methods:Ninety-four rabbits(male,2.5-3.5kg)were randomized into 4 groups:control group,atorvastatin group(2.0 mg/kg/day,n=22),resveratrol group(50 mg/kg/d,n=24),combination group(atorvastatin 2.0 mg/kg/day + resveratrol 50 mg/kg/d,n=24).The abdominal aorta balloon injury model were made in every rabbit after feeding high fat diet for 3 weeks.Following that the DES(Firebird 2 3.5*13mm)was implanted into the narrow part after abdominal aorta balloon injury in rabbit at the beginning of 8th week.According to the experimental protocol,the animals in each group were given drug by gavage until experimental end point(postoperative day 28).The intimal coverage of the vascular grafts in the DES was checked by optical coherence tomography(OCT).Neointimal thickness(mm),neointimal area(mm2)were analyzed by OCT.And then re-endothelialization of neoendothelial of target vessels was observed under the scanning electron microscope.Results:OCT imaging showed that compared with the combination group,the mean neointmal area was larger than that in resveratrol(RV)group and atorvastatin(Ator)group(1.56±0.09 vs 1.44±0.10,1.56±0.09 vs 1.50±0.10,P<0.05).Similarly,mean neointmal thickness was thicker in combination group compared to resveratrol group and atorvastatin group separately(211.42±24.96 vs 182.03±19.1,211.42±24.96 vs 196.64±24.07,P<0.05),the difference was statistically significant.Scanning electron microscopy results showed that the covered area of neoendothelial was significantly delayed compared with the Ator,RV and Ator + RV group.It had been showed that uncovered stent struts in RV group;while there were higher coverage of endothelial cell on the stent struts surface and struts interval area.Conclusions:Resveratrol or atorvastatin,both can accelerate re-endothelialazation after DES implantation.The effect of combination therapy is superior to the single drug group in the acceleration of re-endothelialization after stent implantation.There are clinical significant for prevention of poststent thrombosis.To investigate the mechanisms on endothelial-like differentation of bone marrow mesenchymal stem cells in rats induced by Resveratrol and Atorvastatin treatmentObjectives:Bone marrow mesenchymal stem cells(BMSCs)was isolated from bone marrow in rat.To observe the therapeutic effects of resveratrol and atorvastatin on BMSCs,and then to find the evidence of PI3K/Akt/eNOS involved in the mechanisms of BMSCs transformation under the inervention of both resveratrol and atorvastatin.The findings will provide theoretical basis for clinical application of resveratrol and atorvastatin combination therapy.Methods:BMSCs were Separated from bone marrow of tibia and femur in SD rats(4-6week old,120g-160g)under sterile conditions.BMSCs shows the property of adherent to plastic,so only the adherent cells were selected to continue cultured.The BMSCs were collected for cellular morphological observation and identification by inverted microscope.The third passage(P3)BMSCs were used in each experiment.We selected P3 BMSCs to identify the Surface marker expression of positive indicator CD90,negative indicator CD45 by flow cytometry.BMSCs were induced differentiation toward adipogenic and osteogenic.According to experimental protocol,P3 BMSCs were incubated with different drugs.Atorvastatin groups were divided into 6 groups:control group(0.1%DMSO),0.001?M,0.01?M,0.1?M,1?M and 10?M;Resveratrol group were divided into 7 groups:control group(0.1%DMSO),0.1 ?M,0.5 ?M,1 ?M,2.5 ?M,5?M,and 10 vM;Rapamycin group:correspondingly divided into 8 groups depending on the concentration of the intervention drug.:Control(0.1%DMSO),0.1 ?M,1 ?M,10 ?M,100 ?M,1 ?M,10?M,100?M.The Cell Counting Kit-8(CCK-8)method was used to detect the proliferation of BMSCs;Western Blot was used to detect the expression of Bax and Bcl-2 protein in BMSCs of different groups.Flow cytometry was used to detect BMSCs apoptosis in different groups.Transwell device was used to observe the migration of BMSCs.Also,western Blot assay was used to detect VE-Cadherin and CD31(PECAM-1)in BMSCs.Similarly,before and after the intervention of each experimental drug,the PI3K inhibitor LY294002(30 ?mol/L)was used to pretreat for 2 hours.The expression of Akt,p-Akt,eNOS,p-eNOS and CXCR4 protein in each experimental group was detected through Western Blot method.Results:The surface marker of BMSCs was tested through flow cytometric assay.The results showed that the expression of CD90 was positive,the positive rate was 83.6%respectively;the expression of CD45 was negative,the positive rate was 0.0%.After adipogenic induction,the lipid droplets stained red color with oil red O appeared in adipogenic medium.After osteogenic induction culture,it showed red mineralized nodulesstained in medium after stained with alizarin red.The results of CCK-8 showed that the cell survival best time was drug intervention 48 hours and the concentration of RV 1?M,Ator 0.1 vM.When BMSCs cultured in rapamycin(RPM)-containing medium for 48 hours,the RV +Ator treated BMSCs showed better survival than RV or Ator treated group(224.26±15.43 vs 135.69±8.62,224.26±15.43 vs 166.66±9.15,P<0.001),the difference was statistically significant.BMSCs apoptosis was also tested by Flow cytometry when cells intervention with RPM.Then we analyzed the bcl-2 and bax to identify the protective effect of atorvastatin using western blot.Wester blot results showed that compared with the Control group,the expression of Bax and Bcl-2 protein were up-regulated in BMSC cells treated with RPM in each experimental group,especially in RV+Ator group,the difference was statistically significant(p<0.05).Transwell test results showed that the migrating cells of Ator and RV+Ator treated group was increased significantly that compared with RV group(20.9 ± 4.5 vs 40.0 ± 8.1,P<0.001,,20.9 ±4.5 vs 45.8±6.1,P<0.001).Wester blot also was used to tested endothelial markers after 12 days of RV and Ator combined intervention to BMSCs.The results showed that compared with Control group,the expression of CD31 and VE-Cadherin increased in the combination group,(p<0.05).Wester blot was used to tested the expression of of Akt,p-Akt,eNOS,p-eNOS and CXCR4 in BMSCs.The results showed that compared with control group,the expressions of eNOS and CXCR4 were up-regulated.The expression of p-Akt and p-eNOS in BMSCs was increased than that in the RV or Ator group.The PI3K inhibitor LY294002 showed the ability to down-regulate p-Akt,p-eNOS and CXCR4 protein expression.AS result the effects of RV and Ator combined intervention could be inhibitedon proliferation and migration of BMSCs by LY294002.Conclusions:Resveratrol and atorvastatin can promote BMSCs proliferation and migration,inhibit cells apoptosis.The RV and Ator combination enhanced the protect effects on BMSCs.The PI3K/Akt/eNOS pathway activation may be involved in the mechanisms of protect effects which induced by RV and Ator combination therapy.