Abstract:Aim To investigate the effect and mechanism of paeonol on the secretion of exosomes in THP-1 cells induced by lipopolysaccharide (LPS). Methods THP-1 cell culture supernatant exosomes were extracted by ultracentrifugation; the morphology of exosomes was observed by transmission electron microscope (TEM), and the marker proteins Alix, TSG101, CD9 and CD63 were detected by Western blot, and the dynamic light scattering (DLS) was used to detect exosome particle size. CCK-8 was used to detect the effect of different concentrations (0,0, 1,0.1,0.01 and 0.001 mg/L) of LPS on the survival rate of THP-1 cells to determine the optimal concentration; CCK-8 was used to detect the survival rate of THP-1 cells in different concentrations of paeonol (0,0, 0,0 and 15 μmol/L) medium for different time (2,4 and 48 h), to determine the optimal concentration and time; the amount of exosome protein was detected by BCA; the levels of N-SMase2 and p38 MAPK proteins in the cells were detected by Western blot. Results The results showed that the microvesicle structure obtained by ultracentrifugation was exosome. When the dose of LPS was 1 mg/L, it was suitable; when paeonol was administered at 60 μmol/L, the cell survival rate was higher at 24 h. The exosome secretion of the LPS group was significantly increased compared with the blank group, and the N-SMase2 protein level was increased, and the effect was similar to that of DNR. The exosome secretion of the paeonol group was significantly reduced compared with the LPS group, and the expression of N-SMase2 protein was decreased, and the effect was similar to that of GW4869. LPS promoted phosphorylation of p38 MAPK, and paeonol and SB203580 inhibited p38 MAPK phosphorylation.Paeonol inhibited the secretion of exosome of THP-1 cells after LPS stimulation. Conclusion Paeonol inhibits the secretion of THP-1 cell exosome, which is related to the inhibition of p38 MAPK/N-SMase2 pathway.

Abstract:Aim To investigate the effects of licochalcone A(Lico A)on the expression of inflammatory cytokines in macrophages of human acute monocytic leukemia cell line (THP-1) induced by lipopolysaccharide (LPS) in vitro. Methods 100 μg /L phorbol ester (PMA) was used to induce THP-1 cells for 48 hours, after the differentiation of THP-1 cells, which became macrophage, and cells were divided into control group, LPS group and LPS combined with concentration (20, 0,5 mg/L) of Lico A. The levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were detected by ELISA. The levels of Toll like receptor-4(TLR-4) and nuclear factor kappa B (NF-κB) mRNAs were tested by real-time PCR. The levels of TLR-4, NF-κB,Iü kappa B kinase alpha (IKKα), phosphorylated IKB-alpha (p-IKB-α), cyclooxygenase-2 (COX-2) and isoform of nitric oxide synthase (iNOS) proteins were examined by Western blot. Results The expression levels of TNF-α, IL-1 and IL-6 were up-regulated in the v macrophages after stimulated by LPS, Lico A could reduce the elevated expression levels of TNF-α, IL-1 and IL-6 induced by LPS. The expression of TLR-4 significantly increased after stimulated by LPS and NF-κB was activated. Lico A could reverse the above changes and prevent the activation of NF-κB. Conclusion Lico A could inhibit LPS-induced inflammatory response in THP-1 macrophages via TLR-4 /NF-κB pathway.

Abstract:AimTo determine the effect of oxLig-1, synthesized by an chemical method, on the monocyte chemoattractant protein-1 (MCP-1) expression in human THP-1 monocytes.MethodsoxLig-1 was identified by mass spectrometry and nuclear magnetic resonance (NMR) analysis.Human THP-1 monocytes were treated with different concentration (15, 30 and 60 mg/L) of oxLig-1, the apoptosis of the cell was detected by flow cytometry using Annexin-V and PI.The mRNA and protein levels of MCP-1 were measured by RT-PCR and ELISA methods, respectively.ResultsThe structure of synthetic oxLig-1 identified by LC-MS and NMR analysis showed the same as the natural structure of oxLig-1.There were no differences in THP -1 cell apoptosis, as evidenced by FCM at a high concentration of oxLig-1 (60 mg/L).The mRNA and protein expressions of MCP-1 were increased in a dose-dependent manner of oxLig-1, and the maximum value was observed at the concentration of 40 mg/L.ConclusionThe oxLig-1 could upregulate the expression level of MCP-1 in human THP-1 cells.

Abstract:Aim To investigate the effect of adrenaline on the expression of ATP binding cassette transporter A1 (ABCA1) mRNA and scavenger receptor AI(SR-AI) mRNA in THP-1 macrophages and to identify its possible mechanism mediated byβ-adrenoceptor.Methods THP-1 derived macrophages were preincubated for an hour with aβ1-adrenoceptor antagonist metoprolol(10 nmol/L to 100μmol/L) and aβ2-adrenoceptor antagonist butoxamine(2.5 nmol/L to 25μmol/L),respectively,before treatment with 1μmol/L adrenaline for 24 h.Then ABCA1 and SR-AI mRNA expression were determined by RT-PCR.Results RT-PCR analysis showed that compared with control(without adrenaline),1μmol/L adrenaline significantly decreased ABCA1 mRNA expression and significantly increased SR-AI mRNA expression (P<0.05).These stimulatory effects of adrenaline on ABCA1 mRNA and SR-AI mRNA expression in THP-1 macrophages were reversed by theβ1-adrenoceptor antagonist metoprolol or theβ2-adrenoceptor antagonist butoxamine in a concentration -dependent manner.Furthermore,the effect of theβ2-adrenoceptor antagonist butoxamine reached a platform at the concentration of 2.5μmol/L.Conclusion Adrenaline up-regulates SR-AI mRNA expression and down-regulates ABCA1 mRNA expression,probably being mediated viaβ1-adrenoceptor orβ2-adrenoceptor activation.

Abstract:Aim To investigate the effect of 7-difluoromethyl-genistein(FMGEN) on oxidative stress-induced cell adhesion between vascular endothelial cells and mononuclear cells and the underlying mechanism.Methods Fluorescent light spectrophotometer was used to detect the cell adhesion between vascular endothelial cells and mononuclear cells.The concentrations of E-selectin and intercellular adhesion molecule(ICAM-1) in the cell culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA).The activation of P38 mitogen-activated protein kinase (P38-MAPK) was analyzed by Western blotting.Results Exposure of vascular endothelial cells to H_2O_2 for 24 h increased the adhesion between vascular endothelial cells and mononuclear cells and the release of E-selectin and ICAM-1 in vascular endothelial cells;however,these effects of H_2O_2 were inhibited by FMGEN in a concentration-dependent manner. Treatment of vascular endothelial cells with H_2O_2 for 24 h resulted in the significant activation of P38-MAPK and the activation of P38 induced by H_2O_2 was inhibited by FMGEN.SB203580,a specific inhibitor of P38-MAPK blocked the adhesion between vascular endothelial cells and mononuclear cells and the release of E-selectin and ICAM-1 in vascular endothelial cells induced by H_2O_2.Conclusion FMGEN antagonizes oxidative stress-induced cell adhesion between vascular endothelial cells and mononuclear cells,which is associated with inhibition of the release of E-selectin and ICAM-1 via down-regulating the activation of P38-MAPK.

Abstract:Aim Ubiquitin-proteasome pathway is an important pathway of protein degradation in cells, it is involved in many physiological processes including cell cycle regulation, DNA repair and cell apoptosis. To study the effect of ubiquitin-pro teasome pathway on apoptosis in THP-1 cells and the possible mechanism of apoptosis, THP-1 cells were treated and the related indexes were detected. Methods THP-1 cells were treated with MG132 (5 μmol/L) for 24 h. Ubiquitin, ubiquitin-activat ing enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin-protein ligase (E3) and 26S proteasome genes expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). Apolipoprotein B (ApoB) concentration in THP-1 cells and cell apoptosis rate were detected by flow cytometric analysis (FCA). Results RT-PCR showed that E1, E2, E3 genes expression were decreased in treatment group and the concentration of ApoB and apoptosis rate were increased obviusly. Conclusions Protesome inhibitr MG132 could inhibit the expression of E1, E2 and E3, UPP activity was inhibited. Degradation of ApoB decreased and so cell apoptosis rate increased. The apoptosis of macrophages in the early stage of atherosclerosis could delay the atherogenesis. This experiment is to detect the novel mechanism of macrophages from the regulation of ubiquitin-proteasome pathway, it could be a new target to prevent the atherogenesis.