ABSTRACT
To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms. Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway. Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05). Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.
Subject(s)
Humans , Adenosine Triphosphate , Pharmacology , Endothelial Progenitor Cells , Gene Expression Regulation , Leukocytes, Mononuclear , Cell Biology , Lipopolysaccharide Receptors , Genetics , Lipopolysaccharides , Pharmacology , Myeloid Differentiation Factor 88 , Genetics , NF-kappa B , Metabolism , Signal Transduction , Toll-Like Receptor 4 , GeneticsABSTRACT
Objective:To explore Toll-like receptor 2 (TLR2) and TLR4 polymorphism in Han people from Hunan region and its association with coronary atherosclerotic heart disease.Methods:Sanger sequence and statistical analysis were performed to identify the polymorphism of TLR2 and TLR4 genes in 347 unrelated Hunan Han subjects,including 180 healthy people (control group) and 167 patients with coronary atherosclerotic heart disease (coronary atherosclerotic heart disease group).Results:There was no significant difference in the genotype frequency and allelic frequency for TLR2 SNP2258G＞A and TLR4 SNP896A＞G between the 2 groups (P＞0.05),while there was significant difference in the TLR4 SNP1196C＞T between the 2 groups (P＜0.05).Conclusion:TLR4 SNP 1196C ＞T polymorphism is associated with coronary atherosclerotic heart disease in Chinese Han populationin in Hunan region.
ABSTRACT
Objective To explore the effect of endothelial progenitor cells (EPCs) on the proliferation of co-cultured rat vascular smooth muscle cells(VSMCs). Methods Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch (HES) and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum (FBS), VEGF,bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of α-SM-actin, calponin of VSMCs special antigen. Co-culture system of EPCs and VSMCs was established by transwell permeable support. FBS (20%) was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method,protein quantitation and flow cytometry after co-culture for 6, 12, 24,48 and 72 h. Results After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the control group(P＜0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G_1 phase increased markedly compared with the control group(P＜0.05). The maximal inhibitory effect was observed at 48 h. Conclusion Early EPCs could inhibit the proliferation of VSMCs.