Celastrol Inhibits Restenosis Causing By Balloon Injury Through Mediating AKT

Background:Coronary atherosclerotic heart disease(CHD)is a major disease threatening the health of Chinese people today.The mortality rate of CHD is increasing year by year and the onset age of CHD tends to be younger.Percutaneous coronary intervention(PCI)is the main treatment for CHD.The incidence of restenosis,a major complication of PCI,was about 30%to 50%in the last century.After the invention of drug-eluting stents,the incident of restenosis decreased to 10%.To improve patients'long-term survival,it's important to further reduced the incidence of restenosis.Celastrol is a bio-active component extracted from traditional Chinese medicinal plants from Celastraceae family such as Thunder God Vine and Celastrus orbiculatus.Celastrol has been shown to be beneficial in various chronic disease.It's reported that celastrol has powerful anti-inflammatory,anti-oxidant and anti-angiogenic effects.In cardiovascular diseases,the role of celastrol in inhibiting myocardial fibrosis and reducing the size of infarction area after myocardial infarction has been confirmed.Multiple researches described that celastrol could regulate several protein expression and modulate various cell signaling pathway such as ROS/JNK,JAK/STAT and AKT/mTOR.Specially,AKT is vital to the proliferation of VSMC.In this case,we speculate that celastrol could modulate the proliferation of VSMC through modulating AKT.Purpose:1.To investigate whether celastrol could inhibit the proliferation of VSMC to prevent restenosis after vascular balloon injury;2.To verify the inhibition capability of celastrol on VSMC proliferation in vitro;3.To explore the possible signaling pathway that celastrol modulate the proliferation of VSMC.Methods:This research is divided into two aspects:cell experiments and animal experiments.We used balloon catheter to injure carotid arteries in Sprague-Dayley rat to establish the VSMC proliferation model in vivo.And PDGF was used to stimulate VSMC over-proliferate in vitro to verify the effect of celastrol.To further certify the effect of celastrol,we provided the results of EdU,CCK-8,Western Blot,HE staining and immunohistochemistry.Quantity One was used to analyse Western Blot band.ImagineJ was used to observe and analyse HE sections and immunohistochemistry.Data was analyzed by SPSS 20.0 and statistical graph was made by GraphPad Prism 8.0.The other graph was made by Adobe PS 2019 and Adobe AI 2019.Results:The ratio of intima was significantly higher in the model group than the sham group as well as the ratio of vascular lumen was significantly lower.After 14 days of intraperitoneal injection of celastrol lmg/kg/d to postoperative rat,the ratio of intima was decrease and the ratio of vascular lumen was increase compared to the model group.We extracted tissue protein to proceed Western Blot and discovered that the PCNA protein in the treatment group was down-regulated and the ?-SMA was up-regulated compared to the model group(P<0.05).These changes were also observed in the immunohistochemistry result.The celastrol treatment had no effect on rats growth and development.We set up three different concentrations of celastrol in the experiment in vitro:500 nmol/L,750 nmol/L and 1000 nmol/L.We compared the results of EdU and CCK-8 test between each two groups and found out that celastrol could siginificantly inhibit the proliferation of VSMC stimulated by PDGF(P<0.05).The results of Western Blot analysis showed that celastrol down-regulated the expression of proliferative-related protein PCNA(P<0.05),and increased the expression of phenotypic-related protein ?-SMA(P<0.05).These results indicated that celastrol can inhibit the proliferation,migration and phenotypic transformation of VSMC.Further more,we explored the possible signaling pathway of celastrol.Western Blot experiment results were compared between each two groups and found that celastrol can inhibit the AKT protein phosphorylation in VSMC stimulated by PDGF(P<0.05).Next,we stimulated VSMC with AKT activator Sc79 and celastrol,and found that the AKT phosphorylation was increase and the PCNA expression was up-regulated(P<0.05).The results of CCK-8 and scratch test also showed that the inhibition of celastrol to the proliferation and migration abilities of VSMC was reversed.Moreover,adding AKT inhibitor MK2206 to celastrol aggravate the inhibition of proliferation and migration of VSMC(P<0.05).Conclusions:This experiment proved that celastrol could inhibit proliferation,migration and phenotypic transformation of VSMC to prevent restenosis after balloon injury by modulating AKT for the first time.