Effect Of Sodium Ferulate On Gene Expression In Human Umbilical Vein Endothelial Cells Treated With Ox-LDL

Atherosclerosis (AS) is one of serious cardiovascular disease that is a danger to health, which increases the the morbidity and mortality rates day by day. Therefore, it is essential to select effective drugs for prevention and treatment to atherosclerosis, explore the mechanism of the drugs, and find the sensitive target goal. Microarray provides a platform for examining the complex mechanisms, gene interactions and gene regulations involved in the process of atherosclerosis. Sodium ferulate(SF), is a Na salt of ferulic acid, which is an effective component of Chinese herb medicines angelica and szechwan lovage rhizome. It is reported that sodium ferulate has beneficial pharmacological effects such as inhibition of platelet aggregation and thrombus formation, prevention of oxidation, decrease of free radicals production and acceleration of their clearance, decrease of serum lipids and inhibition of oxidation of LDL both in animals and humans. Therefore, we established a model in vitro using human umbilical vein endothelial cells that are treated with Ox-LDL as a damage factor and intervened with SF as a protective fator in order to find the key genes in the process of preventing AS with SF, which provides a new theoretical and experimental basis for effective clinical application of SF.PartⅠEffect of SF on cultured HUVEC by Ox-LDLObjective:To investigate the effect of sodium ferulate on the proliferation, migration and apoptosis induced by Ox-LDL in vascular endothelial cell.Methods:Human umbilical vein endothelial cells (HUVEC) were treated with different concentrations of of Ox-LDL (25mg/L,50mg/L,75mg/L) for 6h,12h,24h and 48h and the cell proliferation, migration and apoptosis were assessed by MTT, scratch test, flow cytometry and confocal microscopy respectively. After finding out the best concentration point of apoptosis induced by Ox-LDL, HUVEC were exposed to different concentration of SF (5μM, 10μM,20μM) for 1h, and then were culcured with Ox-LDL (50 mg/L) for 24h in the presence of SF to explore the effects of SF on the proliferation, migration and apoptosis induced by Ox-LDL.Results:Compared with normal control group, Ox-LDL could inhibit proliferation, migration and increase apoptosis of HUVEC in a dose-and time-dependent way. SF could promote the proliferation and migration, decrease the apoptosis of HUVEC Ox-LDL-treated HUVEC in a dose-and time-dependent way.Conclusion:Ox-LDL could inhibits the proliferation, migration and induce apoptosis of HUVEC. SF could increase the proliferation, migration and reduce the apoptosis of HUVEC induced by Ox-LDL.PartⅡEffect of SF on Gene expression profile in Ox-LDL-stimulated HUVECObjective:Screen differentially expressed genes in Ox-LDL-stimulated HUVEC in response to SF using analysis of gene expression profileMethods:A human genome70-mer oligonucleotide microarray, representing 21,329 well-characterized Homo sapiens genes(22K Human Genome Array), was obtained from CapitalBio Corporation. The arrays were scanned using a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. To determine the significant differentially expressed genes, Significance Analysis of Microarrays (SAM, version 3.02) were performed. Cluster 3.0 and Treeview software were used to carry out average linkage clustering for differentially expressed genes. The functions of these differentially expressed genes were retrieved using MAS software.Results:A total of 55 genes were differentially expressed in ox-LDL treatment group compared to control. Two of the genes were down-regulated while 53 of them were up-regulated by ox-LDL. Compared to ox-LDL alone group, a total of 42 genes were down-regulated by SF administration. There are 20 down-regulated genes in protection group compared to control according to SAM software analysis.Among the 53 ox-LDL induced up-regulated genes, the 32 genes significantly down-regulated to a similar level of control by sodium ferulate administration were identified through the analysis of gene profiles. Among the 32 genes, inflammation-related chemokines(CXCL1, CXCL2, CXCL3, CCL20, CXCL2), inflammatory cytokines (IL-1β, IL-6) and growth factors(AREG, EREG) were expressed with significantly differences. These factors play essential roles in more than one signal transduction pathway. This shows that SF plays the role of anti-inflammatory to protect injured endothelial cell.Conclusion:SF could inhibit the over expressed genes of inflammation-related chemokines(CXCL1, CXCL2, CXCL3, CCL20, CXCL2, IL-8), inflammatory cytokines (IL-1β, IL-6) and growth factors(AREG, EREG) in HUVEC induced by Ox-LDL.PartⅢEffect of SF on inflammatory factors expression and possible molecular mechanism in ox-LDL-induced HUVECObjective:To investigate the effect of SF on inflammatory factors expression and explore its’possible molecular mechanism in Ox-LDL-induced HUVEC.Methods:Inflammatory genes expression, inflammatory factors production and NF-κBp65 protein were assessed by quantitative real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot respectively.Results:Ox-LDL significantly induced all inflammatory factor expressions in mRNA and protein level. After SF were added to the ox-LDL-treated cells, all inflammatory factors mRNA expressions and productions were markedly decreased as compared with ox-LDL alone group. Ox-LDL induced NF-κB activation and translocation from cytoplasm to nucleus. Ox-LDL incubation induced an increased expression of NF-κB p65 protein (the active subunit of the NF-κB complex) in the nuclear extracts of HUVEC. When SF were added to the ox-LDL-treated cells, the expression of NF-κB p65 protein in the nuclear extracts was markedly decreased as compared with ox-LDL alone group in a dose-dependent way.Conclusion:SF plays a protective effect on endothelial cells via inhibition of inflammatory factors expressions. The activation of NF-κB may be the potential mechanism involved.