Effects Of ADMA On The Expression Of MIF In THP-1 Monocyte-Derived Macrophage Cells And Preliminary Research Of The Mechanisms

Objective:1. To observe the effects of ADM A on differentiation of THP-1 monocyte to macrophage.2. To research the mechanisms of ADMA on THP-1 monocyte-derived macrophage cells by investigating the effects of ADMA on the expression of MIF in THP-1 monocyte-derived macrophage cells and antagonism effect of L-arg and MnTBAP.Methods:After THP-1 monocyte cells were exposed to 30μmol/L ADMA for 48h, Morphologic changes of the cells were observed with microscope. THP-1 monocyte cells were differentiated into macrophage cells after cultured in RPMI 1640 media containing 150nmol/L PMA for 48h. The macrophage cells were identificated by Immunocytochemistry. After ADMA of different concentrations (1μmol/L,5μmol/L, 10μmol/L,20μmol/L,30μmol/L) were incubated with THP-1 monocyte-derived macrophage cells for 24h and 20μmol/L ADMA was incubated with THP-1 monocyte-derived macrophage cells for Oh,6h,12h,24h,48h, the expressions of MIF in mRNA and protein were measured by means of RT-PCR and Enzyme-Linked Immunosorbent assay respectively. After preconditioning for 2h with 1mmol/L L-arg or 100μg/ml MnTBAP, THP-1 monocyte-derived macrophage cells were exposed to 20μmol/L ADMA for 24h. The expressions of MIF in mRNA and protein were measured by means of RT-PCR and Enzyme-Linked Immunosorbent assay respectively.The intra-cellular levels of NO and ROS were measured by fluorescent probe.Results:1. After THP-1 monocyte cells were exposed to 20μmol/L ADMA for 48h, Morphologic changes of the cells were similar with the effect of PMA. Round floating monocyte cells became clostridial adhereing macrophage cells. Inversion rate surpassed 95%.2. Compared with the control group, treatment of THP-1 macrophages with 5μmol/L, 10μmol/L,20μmol/L,30μmol/L ADM A for 24h resulted in an increase in the mRNA and protein levels for MIF in cultured THP-1 monocyte-derived macrophage cells (P<0.05)3. Compared with the control group, treatment of THP-1 macrophage with 20μmol/L ADM A for 12h,24h,48h, resulted in an increase in the mRNA and protein levels for MIF (P<0.05).4. Compared with the control group, the intra-cellular levels of ROS raised up obviously in 20μmol/L ADMA group (P<0.01). and the intra-cellular level of ROS decreased obviously in 20μmol/L ADMA+100μg/ml MnTBAP group(P<0.01). Compared with ADMA group, the intra-cellular level of ROS decreased obviously in 20μmol/L ADMA+100μg/ml MnTBAP group (P<0.01).5. Compared with the control group, the intra-cellular level of NO raised up obviously in 20μmol/L ADMA group (P<0.05). and the intra-cellular level of NO decreased obviously in 20μmol/LADMA+1mmol/L L-arg group (P<0.01). Compared with ADMA group, the intra-cellular level of NO decreased obviously in 20μmol/L ADMA+1mmol/L L-arg group (P<0.01).6. Compared with the control group, treatment of THP-1 macrophages with 20μmol/L ADMA for 24h resulted in an increase in the mRNA and protein levels for MIF in cultured THP-1 monocyte-derived macrophage cells (P<0.01). Compared with the control group, treatment of THP-1 macrophages with 20μmol/L ADMA+100μg/ml MnTBAP and 20μmol/L ADMA+1mmol/L L-arg for 24h resulted in an decrease in the mRNA and protein levels for MIF in cultured THP-1 monocyte-derived macrophage cells (P<0.01). Compared with ADMA group, treatment of THP-1 macrophages with 20μmol/L ADMA+100μg/ml MnTBAP and 20μmol/L ADMA+1mmol/L L-arg for 24h resulted in an decrease in the mRNA and protein levels for MIF in cultured THP-1 monocyte-derived macrophage cells (P<0.01).Conclusion:1. ADMA could promote THP-1 monocyte to differentiate into macrophage cell.2. ADMA could upregulate the expression levels of protein and mRNA for MIF in cultured THP-1 monocyte-derived macrophage cells and in a dose-and time-dependent manner,which might result in the atherosclerosis.3. L-arg and MnTBAP could antagonize up-regulation of the expression levels of protein and mRNA for MIF of ADMA on cultured THP-1 monocyte-derived macrophage cells.4. The mechanisms of up-regulation of the expression levels of protein and mRNA for MIF of ADMA on cultured THP-1 monocyte-derived macrophage cells were concerned with inflammation and oxidative stress.