| Background and objective:Restenosis after percutaneous transluminal angioplasty is a self-repair response of the vessel wall in response to mechanical injury.Intimal hyperplasia is one of the lead-ing causes of restenosis,in which vascular smooth muscle cells play an essential role in intimal hyperplasia.Inositol-tetraphosphate 1-kinase（ITPK1）regulates the synthesis of inositol tetraphosphate and the downstream products inositol penta-and inositol hexa-phosphate,which plays an important role in necroptosis and regulate plasma membrane Ca2+-Cl-channels.Rocaglamide（Roc-A）has the ability to inhibit NF-?B activity and inhibits NF-?B by decreasing phosphorylation of e IF4E to inhibit transla-tion.This study aimed to discuss:（1）the expression of ITPK1 before and after Platelet growth factor（PDGF-BB）injury,and the effect and mechanism of ITPK1 expression on PDGF-induced smooth muscle cell proliferation.（2）The mechanism of Roc-A inhi-bition of proliferation and migration of VSMCs and its regulation of ITPK1 expression.Methods:This study was conducted in three parts.1.Screening for differentially expressed genesDifferentially expressed genes（DEGs）between the two groups were analyzed based on the data set GSE46560 of blood samples from restenosis subjects and control subjects,with P<0.05 and|log FC|>1 as screening conditions.Enrichment analysis of GO and KEGG signalling pathways was performed using DAVID to obtain key gene modules by constructing protein-protein interaction（PPI）networks.2.Effect of ITPK1 on PDGF-induced proliferation and migration of VSMCsRat thoracic aortic smooth muscle cells（A7r5）were cultured in vitro,and the cells were stimulated with 25ng/ml of Platelet growth factor（PDGF-BB）to create an envi-ronment of injury.The relationship between ITPK1 and smooth muscle cell prolifera-tion and migration was examined using the CCK8 and scratch assays;the fluorescence of reactive oxygen species generation was observed by the DCFH-DA fluorescent probe;and the expression of ITPK1,PCNA,NF-κB,m TOR and other related proteins were detected by Western blot.3.Mechanism of Roc-A inhibition of proliferation and migration of VSMCs and its regulation of ITPK1 expressionRat thoracic aortic smooth muscle cells were cultured in vitro.PDGF-BB was used to stimulate the cells to establish an injury model and screen the optimal drug action concentration.The control group,PDGF-BB group and PDGF-BB+Roc-A（10,25 and50 n M）were set up for the experiment.The effects of different concentrations of Roc-A on the proliferation and migration of smooth muscle cells were examined by CCK-8,flow cytometry（Ed U and cell cycle assays）and Transwell assays;the accumulation of reactive oxygen species was examined by the fluorescent probe DCFH-DA;and the expression of proteins related to smooth muscle cell dedifferentiation,proliferation,mi-gration,autophagy,and signaling pathways was examined by protein immunoblotting.Results:1.The ITPK1 gene was obtained by screening,and ITPK1 expression was signif-icantly reduced in PDGF-BB-treated cells.2.ITPK1 inhibited PDGF-induced proliferation and migration of smooth muscle cells.（i）PDGF group injury resulted in increased cell activity（P<0.01）,increased ex-pression of PCNA,CDK2 and cyclin E（P<0.05）,increased S-phase cells（P<0.0001）and reduced scratch wounds（P<0.001）,while ITPK1 overexpression resulted in re-duced cell viability（P<0.001）,decreased expression of PCNA,CDK2 and cyclin E expression（P<0.05）,reduced S-phase cells（P<0.001）,and increased scratch"open-ings"（P<0.01）.（ii）Cellular reactive oxygen species fluorescence was reduced in the overexpression treatment compared to the PDGF group（P<0.001）.（iii）ITPK1 regu-lates the proliferation and migration of VSMCs through the NF-κB/m TOR pathway.3.Roc-A inhibited the proliferative migration of VSMCs.（i）The Roc-A treatment inhibited proliferative migration and expression of associated cyclins in a dose-depend-ent pattern,reducing cells in S-phase（P<0.05）.（ii）Different concentrations of Roc-A attenuated the fluorescence intensity of the probe DCFH-DA and inhibited PDGF-BB-induced autophagy（P<0.01）.（iii）Detection of m TOR and NF-κB phosphorylation lev-els revealed a concentration-dependent inhibition of protein phosphorylation levels by Roc-A（P<0.05）.（iv）Roc-A increased ITPK1 expression（P<0.0001）.Conclusions:ITPK1 has the ability to regulate the proliferation and migration of smooth muscle cells that inhibit proliferation migration and reactive oxygen species accumulation in VSMCs via the NF-κB/m TOR pathway,thereby reducing endothelial proliferation.Roc-A adjusts ITPK1 expression and the m TOR/NF-κB pathway,inhibiting smooth muscle cell proliferation migration and improving endothelial proliferation.This plays a vital role in preventing and treating restenosis after the intervention. |
