ABSTRACT
Background@#Macrophage accumulation in the vascular wall is a hallmark of atherosclerosis. Studies showed that shifting of oxidized lipids-induced inflammatory macrophages towards an anti-inflammatory phenotype by promoting oxidative metabolism attenuated atherosclerosis progression. Therefore, this study aimed to investigate whether metformin, which has ameliorated atherosclerosis in animal models and clinical trials, modulated oxidized low-density lipoprotein (Ox-LDL) induced inflammatory status in macrophages by regulating cellular oxidative metabolism.@*Methods@#Murine raw264.7 macrophages were incubated with Ox-LDL (50 μg/mL) in the presence or absence of metformin (15 μmol/L) for 24 h. Real-time polymerase chain reaction was used to quantify the transcription of classically activated (M1) proinflammatory and alternatively activated (M2) anti-inflammatory markers and mitochondrial DNA copy numbers. Cellular reactive oxygen species (ROS) production and mitochondrial membrane potential were detected by immunofluorescence. Cellular adenosine triphosphate (ATP) synthesis, glucose uptake, and lactic acid production were measured by commercial kit and normalized to cellular lysates. Western blotting analysis was performed to detect the expression of mitochondrial fusion/fission related proteins, enzymes mediating lipid metabolism and signaling pathway of glucose transport. Differences between groups were analyzed using one-way analysis of variance.@*Results@#Metformin improved Ox-LDL-impaired anti-inflammatory phenotype in raw264.7 macrophages as shown by up-regulated transcription of anti-inflammatory markers including interleukin 10 (0.76 ± 0.04 vs. 0.94 ± 0.01, P = 0.003) and Resistin-like molecule alpha (0.67 ± 0.08 vs. 1.78 ± 0.34, P = 0.030). Conversely, Ox-LDL-diminished phosphorylation of Akt was upregulated by metformin treatment (0.47 ± 0.05 vs. 1.02 ± 0.08, P = 0.040), associated with an improvement of mitochondrial function, characterized by decreased ROS generation (2.50 ± 0.07 vs. 2.15 ± 0.04, P = 0.040), increased lipid oxidation, and elevated cellular ATP production (0.026 ± 0.001 vs. 0.035 ± 0.003, P = 0.020). Moreover, metformin-mediated Akt activation increased Akt substrate of 160 kDa (AS160) phosphorylation (0.51 ± 0.04 vs. 1.03 ± 0.03, P = 0.0041), promoted membrane translocation of glucose transporter 1, and increased glucose influx into the cells (4.78 ± 0.04 vs. 5.47 ± 0.01, P < 0.001).@*Conclusion@#This study suggested that targeting macrophage metabolism with new or existing drugs had therapeutic potential for the prevention and treatment of diabetes-accelerated atherosclerosis.
ABSTRACT
BACKGROUND@#Macrophage accumulation in the vascular wall is a hallmark of atherosclerosis. Studies showed that shifting of oxidized lipids-induced inflammatory macrophages towards an anti-inflammatory phenotype by promoting oxidative metabolism attenuated atherosclerosis progression. Therefore, this study aimed to investigate whether metformin, which has ameliorated atherosclerosis in animal models and clinical trials, modulated oxidized low-density lipoprotein (Ox-LDL) induced inflammatory status in macrophages by regulating cellular oxidative metabolism.@*METHODS@#Murine raw264.7 macrophages were incubated with Ox-LDL (50 μg/mL) in the presence or absence of metformin (15 μmol/L) for 24 h. Real-time polymerase chain reaction was used to quantify the transcription of classically activated (M1) pro-inflammatory and alternatively activated (M2) anti-inflammatory markers and mitochondrial DNA copy numbers. Cellular reactive oxygen species (ROS) production and mitochondrial membrane potential were detected by immunofluorescence. Cellular adenosine triphosphate (ATP) synthesis, glucose uptake, and lactic acid production were measured by commercial kit and normalized to cellular lysates. Western blotting analysis was performed to detect the expression of mitochondrial fusion/fission related proteins, enzymes mediating lipid metabolism and signaling pathway of glucose transport. Differences between groups were analyzed using one-way analysis of variance.@*RESULTS@#Metformin improved Ox-LDL-impaired anti-inflammatory phenotype in raw264.7 macrophages as shown by up-regulated transcription of anti-inflammatory markers including interleukin 10 (0.76 ± 0.04 vs. 0.94 ± 0.01, P = 0.003) and Resistin-like molecule alpha (0.67 ± 0.08 vs. 1.78 ± 0.34, P = 0.030). Conversely, Ox-LDL-diminished phosphorylation of Akt was up-regulated by metformin treatment (0.47 ± 0.05 vs. 1.02 ± 0.08, P = 0.040), associated with an improvement of mitochondrial function, characterized by decreased ROS generation (2.50 ± 0.07 vs. 2.15 ± 0.04, P = 0.040), increased lipid oxidation, and elevated cellular ATP production (0.026 ± 0.001 vs. 0.035 ± 0.003, P = 0.020). Moreover, metformin-mediated Akt activation increased Akt substrate of 160 kDa (AS160) phosphorylation (0.51 ± 0.04 vs. 1.03 ± 0.03, P = 0.0041), promoted membrane translocation of glucose transporter 1, and increased glucose influx into the cells (4.78 ± 0.04 vs. 5.47 ± 0.01, P < 0.001).@*CONCLUSION@#This study suggested that targeting macrophage metabolism with new or existing drugs had therapeutic potential for the prevention and treatment of diabetes-accelerated atherosclerosis.
ABSTRACT
<p><b>OBJECTIVE</b>To establish a novel severe aplastic anemia (SAA) mouse model by interferon-γ (IFN-γ) plus busulphan.</p><p><b>METHODS</b>Thirty clean-class BALB/c female mice were intraperitoneally injected with IFN-γ and intragastrically administrated with busulphan (group I), meanwhile busulphan alone group (n = 30, group II) and normal control group (n = 30, group III). Multi-parameters were compared among the three groups.</p><p><b>RESULTS</b>In group I at day 10 after treatment, the incidence of SAA was 100% and mortality 20% respectively; the WBC, HGB, PLT, absolute reticulocyte count (Ret) and tibial nucleated cell count (TNCC) were (0.8 ± 0.3) × 10(9)/L, (45 ± 20) g/L, (10 ± 8) × 10(9)/L, (15.2 ± 10.2) × 10(9)/L, (12 ± 7) × 10(6)/tibia, respectively, which were significantly different from the other two groups (all P < 0.05). The bone marrow smears and patho-histological examinations showed marked reductions of marrow cell proliferation, and increases of the percentages of non-hematopoietic cells and cellular adipose. The depression was severe and irreversible. In group II, the blood cells count, TNCC and marrow proliferation recovered gradually with erythroid hyperplasia and hematopoietic dysplasia.</p><p><b>CONCLUSIONS</b>IFN-γ plus busulphan can establish a SAA mouse model in a relatively short period, which is more resemble with human SAA.</p>
Subject(s)
Animals , Female , Mice , Anemia, Aplastic , Busulfan , Disease Models, Animal , Interferon-gamma , Mice, Inbred BALB CABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of activated AKT on murine myeloid precursor cells (32D cells), and the effects of IFN-γ on 32D cells and its mechanisms.</p><p><b>METHODS</b>Plasmid transduction was used to enhance the expression of AKT on 32D cells. After the transfected cells treated with IFN-γ for 24 hours, proliferation rate was tested by WST-1, apoptosis by flow cytometry, expression of phosphorylated Erk1/2, Stat3 and phosphorylated Stat3 was determined by Western blot.</p><p><b>RESULTS</b>(1) IFN-γ at low concentration (100 U/ml) enhanced the growth and proliferation of 32D cells, while at high concentration (1000 U/ml) suppressed them. (2) Compared with control groups, low concentration IFN-γ increased (1124 ± 13) Stat3 phosphorylation in 32D-cell, while it high concentration IFN-γ decreased (601 ± 13). 32D cells transfected with activated Akt grew rapidly (0.287 ± 0.010) and had a low apoptotic rate [(9.57 ± 0.17)% (P < 0.05)]. (3) The expression of p-Erk1/2 in transfected 32D-cell was significantly reduced (P < 0.05). (4) Apoptosis rate of IFN-γ treated group was significantly decreased in transfected 32D cells (P < 0.05).</p><p><b>CONCLUSIONS</b>IFN-γ has dual effects on 32D cells, namely, at low concentration enhanced the growth and proliferation of 32D cells, while at high concentration suppressed them. Its mechanisims is possibly through Stat3 pathway. Activated Akt can significantly promote the growth and proliferation of 32D cell and significantly inhibit apoptosis and IFN-γ can regulate cell proliferation and apoptosis through AKT. AKT activation can inhibit the Erk signal pathway, which may be affected by inhibition the modificaton of Raf1.</p>