Inhibitory Effects And Molecular Mechanisms Of Scutellarin On In-Stent Restenosis After Percutaneous Coronary Intervention

Background:Percutaneous coronary intervention（PCI）is the most widely used myocardial reperfusion therapy worldwide,which can effectively increase the survival rate and improve the quality of life of patients with coronary artery disease and myocardial infarction.However,after PCI,the new intima evolves into unstable atherosclerotic（AS）plaques due to the gradual thickening of the intima within the stent,leading to in-stent restenosis（ISR）,which may even rupture and progress to acute adverse cardiovascular events,seriously affecting patient prognosis.There is a lack of stent-coating drugs in clinical use,only paclitaxel and rapamycin（sirolimus）and their derivatives are available,which have problems such as delayed endothelial healing and increased thrombosis in the stent,which seriously affect the therapeutic outcome.Therefore,it is essential and urgent to find anti-ISR stent-coating drugs with proven efficacy and fewer adverse effects.Our previous study has found that Scutellarin,an active ingredient from the Yunnan specialty medicinal plant Calendula officinalis,could protect vascular endothelial cells,and had significant antithrombotic and strong antiplatelet aggregation effects in animal thrombosis models,suggesting that Scutellarin has potential anti-ISR effects and deserves further research and development.Atherosclerosis（AS）is an important pathological basis for PCI.To clarify the therapeutic effect of Scutellarin on ISR after PCI,it is necessary to focus on the efficacy of Scutellarin on AS.An APOE^-/-mouse experimental atherosclerosis model was replicated to verify the effect of Scutellarin on AS at the overall level in this study.Next,a small porcine coronary artery stent model for AS was constructed to validate the effect of Scutellarin on the prevention and treatment of ISR after PCI at an overall level,and the effect of Scutellarin-coated stents on the inhibition of ISR after PCI was evaluated.Further,we replicated the H₂O₂injured VSMCs model from the perspective of vascular smooth muscle cells（VSMCs）,which are closely related to ISR,and observed the effects of Scutellarin on the proliferation,migration,apoptosis,phenotypic transformation,and oxidative stress of VSMCs.To clarify the potential mechanism of action of Scutellarin against ISR,transcriptomic sequencing and molecular docking techniques were applied to explore the signaling pathways and key targets of Scutellarin inhibition of ISR after PCI.The interactive regulation of PI3K/AKT/m TOR and IKKs/NF-κB pathways by Scutellarin was verified at the molecular level,with the aim of systematically elucidating the mechanism of action of Scutellarin in the treatment of ISR at the overall,cellular,and molecular levels.Objective:To study the inhibitory effect of Scutellarin on instent restenosis after PCI and its molecular mechanismsMethods:1 Therapeutic effect of Scutellarin on atherosclerosis in Apo E^-/-miceReplication of an APOE^-/-mouse model of experimental atherosclerosis1.1 Histopathological observation:pathological examination of plaque tissues using bulk oil red O staining,HE stains,oil red O staining and Masson staining were performed to evaluate the effects of Scutellarin on the degree of plaque tissue lesions,lipid deposition and collagen fiber proliferation.1.2 Transmission electron microscopy:changes in the microstructure of the aorta of Apo E^-/-mice after treatment with different doses of Scutellarin.1.3 The effect of enzyme linked immunosorbent assay（ELISA）on TNF-αand IL-1βin mice serum.1.4 Immunofluorescence was used to detect the effects of different doses of Scutellarin on the expression of OPN,SM22αandα-SMA,which are phenotypic markers of smooth muscle cells in vascular tissue.1.5 Western blot detection of the effects of Scutellarin on the expression of SM22α,α-SMA and OPN,phenotype transformation markers in smooth muscle cells of mouse aortic vascular tissue.1.6 Western Blot detection the effect of Scutellarin on the expression of apoptosis-related genes Bax,Bcl-2,and Cleaved-caspase-3 in mouse aortic vascular tissue.1.7 The serum superoxide dismutase（SOD）activity,malondialdehyde（MDA）,nitric oxide（NO）,reduced glutathione（GSH）glutathione peroxidase（GSH-PX）and H₂O₂were measured in each group of mice using the kits.1.8 The amount of ROS in the aortic tissue homogenates of each group of mice was determined using dihydroethidium staining（DHE）fluorescent probe.2 Study on the inhibition of restenosis after PCI with Scutellarin-coated stents2.1 In this study,an AS minipig coated stent model was constructed,and different doses of Scutellarin were coated on the stent surface,with rapamycin-coated stents as positive control group.After general anesthesia,Quantitative coronary angiography（QCA）and optical coherence tomography（OCT）were performed on the day of the experiment and on day 28,and the parameters related to ISR were observed and recorded.2.2 Animals were sacrificed by execution 28 days after stent implantation in AS minipigs,and HE stains was performed on specimens from the stented segment vessels to assess the injury score,inflammation score and vascular endothelialization score of the stented segment vessels under light microscopy,to evaluate the safety and efficacy of Scutellarin-coated stents against ISR,and to explore the possibility of Scutellarin as a coronary stent coating drug.3 Effects of Scutellarin on VSMCs proliferation,migration,apoptosis,and oxidative stressVSMCs（Rat thoracic aortic smooth muscle cells,A7r5 line）were cultured in vitro to replicate the H₂O₂ injured model.3.1 The effect of Scutellarin on the proliferation of VSMCs was examined using the CCK-8 assay.3.2 Wound healing assay to detect the effect of Scutellarin on VSMCs migration.3.3 Flow cytometry to detect the effect of Scutellarin on the cell cycle of VSMCs.3.4 Immunofluorescence assay to detect the effects of Scutellarin on VSMCs phenotype conversion markers SM22α,α-SMA,OPN protein.3.5 Western blot was used to detect the effects of Scutellarin on the expression of SM22α,α-SMA and OPN proteins in VSMCs,respectively.3.6 Fluorescent probes to detect the fluorescence intensity of ROS in individual cells and mitochondria.3.7 Detection of total activities of SOD,Mn SOD,MDA and NADPH oxidase activities in VSMCs with the kits.4 Transcriptomic sequencing and molecular docking technologies to predict potential targets and pathways of action of Scutellarin against ISR4.1 Transcriptomic sequencing of APOE^-/-mouse aortic vascular tissuesIllumina PE150 sequencing was performed based on the output of the library data and the merging of effective concentration requirements.The clean reads were compared to the transcriptome data and the RNA-Seq sequencing data were compared and analyzed using Hisat2 software.4.2 Molecular docking was performed using Discovery Studio software,through data mining of Scutellarin anti-atherosclerosis targets and pathways to provide an experimental basis for subsequent experimental studies.5.Studies on the regulation of PI3K/AKT/m TOR and IKKs/NF-κB signaling pathways by Scutellarin5.1 Western blot assay to detect the expression of PI3K/AKT/m TOR pathway related proteins and their altered protein after phosphorylation.5.2 The effects of Scutellarin on the regulation of PI3K/AKT/m TOR pathway were observed after treatment with AKT1 inhibitor MK2206（20μmol/L）.The effects of Scutellarin on phenotypic transition and apoptosis of VSMCs were examined.5.3 The effect of Scutellarin on PI3K/AKT/IKKs/NF-κB signaling pathway was observed after pretreatment with NF-κB inhibitor BAY11-7082（2μmol/L）;the effect of Scutellarin on VSMCs phenotypic transition was examined.Results:1 Scutellarin treats AS through anti-inflammatory,antioxidant,apoptosis-inducing,and phenotype-transforming modulation of VSMCs.1.1 Histopathological results:the results of bulk oil-red O staining,HE stains,Oil-red O staining and Masson staining suggested that Scutellarin could significantly reduce lipid deposition in aortic plaque tissue,reduce inflammatory response,inhibit collagen proliferation in plaque tissue,and reduce the degree of lesion and vascular damage in aortic vascular plaque tissue in APOE^-/-mouse AS model.1.2 The ELISA results showed that Scutellarin significantly reduced the levels of serum TNF-αand IL-1β.1.3 Transmission electron microscopy results:the endothelium of the blood vessels in the normal group of mice was intact,and the endothelial gap was small.In the model group,endothelial cells were seen to be shed,or even almost completely disappeared,and a large amount of plasma amorphous components were attached to the endothelium.After the intervention of Scutellarin,the degree of endothelial damage was significantly reduced,and the endothelial structure tended to be intact,with occasional mitochondrial edema and lipid vesicle deposition.1.4 Immunofluorescence results showed that Scutellarin affected the phenotypic conversion of VSMCs in mouse vascular tissues,increased VSMCs of contractile phenotype and decreased VSMCs of synthetic phenotype,and up-regulated the protein expression ofα-SMA,SM22-αand down-regulated the expression of OPN protein in mouse vascular tissues.1.5 Western blot showed that Scutellarin upregulated the expression ofα-SMA and SM22-αin VSMCs and downregulated the expression of OPN in VSMCs.1.6 The results of the oxidative stress assay showed that Scutellarin significantly increased serum SOD,GSH-PX and NO levels,and decreased serum MDA and H₂O₂levels in the APOE^-/-mouse AS model.1.7 The results of DHE staining showed Scutellarin reduced the amount of ROS in the aortic tissue homogenates of APOE^-/-mice.2 Scutellarin-coated stents significantly inhibit ISR after PCI in minipigs2.1 The QCA results suggested that the medium and high dose coated stent groups of Scutellarin could effectively inhibit coronary artery intimal hyperplasia in the AS minipig stent model,and the degree of stenosis was significantly lower than that in BMS group（P<0.05）.2.2 The OCT results suggested that compared with the BMS group,the de novo intimal area and the degree of stenosis were reduced in both the medium-and high-dose scutellarin coated stent groups（P<0.05）,and the residual lumen area in both the high-dose Scutellarin-coated stent group and the positive control group were significantly larger than the other experimental groups（P<0.05）.It was also observed that different degrees of neointimal hyperplasia of the coronaryand lumen reduction were seen in the BMS group and the low-and medium dose groups of Scutellarin.2.3 HE stains showed that at 28 days after stent implantation,Scutellarin-coated stents significantly reduced coronary artery neointima thickness,neointima area and restenosis in a dose-dependent manner compared to the BMS group,with a difference（P<0.05）.Compared to the BMS group,Scutellarin reduced the inflammation score around the stent（P<0.05）.3 Scutellarin inhibits the proliferation and migration of VSMCs by regulating the phenotypic transition of VSMCs through antioxidants3.1 The results of CCK-8 method showed that Scutellarin significantly inhibited the abnormal proliferation of VSMCs.3.2 The results of the scratch assay suggest that Scutellarin significantly inhibits the migration of VSMCs.3.3 The results of flow cytometry showed that Scutellarin significantly blocked VSMCs in G0/G1 phase.3.4 The results of oxidative stress assay showed that Scutellarin significantly increased the activity of SOD,NO and NADPH oxidase and decreased the MDA level in VSMCs.3.5 The results of the cytofluorimetric probe showed that Scutellarin significantly increased SOD and Mn-SOD activities in VSMCs.4 Transcriptomic results4.1 Transcriptomic sequencing analysis of mouse vascular tissues predicts potential molecular targets and pathways of Scutellarin on anti-atherosclerosis.The sequencing results showed that 1895 differentially expressed genes were up-regulated and 2174 were down-regulated in the model group compared with the normal control group,and 531 differentially expressed genes were up-regulated and574 genes were down-regulated in the Scutellarin group compared with the model group.When the differential genes of the three groups were intersected,a total of 81differential genes were overlapped.GO biological enrichment analysis revealed that they were mainly involved in the positive regulation of cell migration,positive regulation of wound healing,cellular response to transforming growth factorβstimulation,response to transforming growth factor,and positive regulation of wound healing.KEGG results showed that the top ranked signaling pathways were cancer pathway,platelet activation pathway,leukocyte transendothelial migration pathway,PI3K/AKT signaling pathway,TNF-αsignaling pathway,etc.These pathways are all involved in the regulation of AS by Scutellarin,and the PI3K/AKT/m TOR signaling pathway is significantly enriched,and its specific molecular mechanism of action deserves further study and verification.4.2 Molecular docking results showed that Scutellarin had strong binding to AKT,SM22α,α-SMA,osteopontin（OPN）,NF-κB,with binding capacities-7.14,-8.61,-7.93,-7.72 kal/mol,suggesting that these molecules could be used as targets for Scutellarin anti-atherosclerosis studies.5 Scutellarin inhibits the phenotypic switch of VSMCs against AS by regulating PI3K/AKT/m TOR and IKKs/NF-κB pathways5.1 Detection of PI3K/AKT/m TOR pathway expression in vascular tissues of APOE^-/-mouse AS model:western blot showed that p-PI3K,p-AKT,and p-m TOR expression were significantly higher in the model group compared with the normal group（P<0.01）.Compared with the model group,the expression of p-PI3K,p-AKT and p-m TOR was significantly lower in the Scutellarin（14 and 28 mg/kg）group（P<0.05）.5.2 PI3K/AKT/m TOR pathway expression assay in H₂O₂ injured VSMCs model:Western blot results showed that the expression of p-PI3K,p-AKT,and p-m TOR was significantly higher in the model group compared with the Normal control group（P<0.05）.The expression of p-PI3K,p-AKT,p-m TOR was significantly lower in the Scutellarin（50 and 100μmol/L）group compared with the model group（P<0.05）.5.3 Results of MK2206 treatment experiments:compared with the Normal control group,the expression of p-PI3K,p-AKT,p-m TOR,OPN,and Bcl-2 was significantly higher and the expression of SM22α,α-SMA,Bax,Cleaved Caspase-3 was significantly lower in the Model group（P<0.05）.Compared with the model group,the expression of p-PI3K,p-AKT,p-m TOR,OPN,and Bcl-2 was significantly decreased in the model+MK2206 group,and the expression of SM22α,α-SMA,Bax,Cleaved Caspase-3 was significantly increased in the model+Scutellarin（100μmol/L）group（P<0.05）.Compared with the Normal group,SOD cell activity was significantly lower and MDA activity was higher in the model group（P<0.05）.SOD activity was significantly higher and MDA activity was lower in the model+MK2206 and model+Scutellarin（100μmol/L）groups compared to the model group（P<0.05）.SOD activity was increased,and MDA activity was decreased in the model+MK2206+Scutellarin（100μmol/L）group compared with the model+Scutellarin（100μmol/L）group（P<0.05）.5.4 Results of inhibitor BAY11-7082 pretreatment experimentsCompared with the Normal control group,the expression of p-PI3K,p-AKT,IKKα/β,p-NF-κBp65,OPN was significantly higher and the expression of SM22α,α-SMA,IKBαwas significantly lower in the Model group（P<0.05）.Compared with the Model group,the Model group+BAY group,the Model group+Scutellarin（100μmol/L）group,p-PI3K,p-AKT,IKKα/β,p-NF-κBp65,OPN expression was significantly decreased and SM22α,α-SMA,IKBαexpression was significantly increased（P<0.05）.Compared with model+Scutellarin（100μmol/L）group,model+BAY+Scutellarin（100μmol/L）group p-PI3K,p-AKT,IKKα/β,p-NF-κBp65,and OPN expression were lower and SM22α,α-SMA,and IKBαexpression were significantly higher in the Model+BAY+Scutellarin（100μmol/L）group（P<0.05）.SOD activity was significantly lower and MDA activity was higher in the model group compared with the Normal group（P<0.05）.SOD activity was significantly higher and MDA activity was lower in the model+BAY group,compared with the model+Scutellarin（100μmol/L）group（P<0.05）.SOD activity was significantly higher and MDA activity was lower in the model+BAY+Scutellarin（100μmol/L）group compared with the model+Scutellarin（100μmol/L）group（P<0.05）.Conclusion:1 Scutellarin has a significant therapeutic effect on experimental AS.2 Scutellarin-coated stents inhibit ISR after PCI by suppressing coronary artery neointimal hyperplasia and inflammatory responses.3 Scutellarin inhibits the proliferation and migration of VSMCs and promotes apoptosis by regulating the phenotypic transition of VSMCs;Scutellarin also significantly inhibits the oxidative stress response of VSMCs.4 Scutellarin interactively regulated PI3Κ/AΚT/m TOR andΚΚs/NF-κB signaling pathways by targeting AKT1 and NF-κBp65 sites,respectively.