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Objective To explore the role of microRNA-145-5p(miR-145-5p)in the regulation of inflammatory response and oxidative stress process in cellular model of atherosclerosis.Methods Human monocytic leukemia THP-1 cells-derived foam cells were constructed in vitro.Then,the relative expression of miR-145-5p in the model and control groups of cells were detected by qRT-PCR.TargetScan database was used to predict the targeting relationship between miR-145-5p and Toll-like receptor 4(TLR4).The 293T cells were divided into wild-type+mimic group,wild-type+mimic negative control(NC)group,and mutant+mimic group,mutant+mimic NC group,and dual luciferase assay was employed to verify the targeting relationship of miR-145-5p and TLR4.Foam cells were cultured in vitro and divided into miR-145-5p mimic group,mimic NC group,miR-145-5p inhibitor group,and inhibitor NC group according to the corresponding treat-ments.The expression of TLR4 at mRNA and protein levels was detected by qRT-PCR and Wes-tern blotting.The contents of TNF-α,IL-1β and IL-6 were detected by ELISA.Biochemical reagent kits were applied for generation of reactive oxygen species(ROS),content of MDA and activity of SOD.Results The expression of miR-145-5p was significantly reduced in the model group than the control group(0.29±0.01 vs 1.00±0.08,t=11.180,P<0.01).Dual luciferase assay showed that luciferase activity was significantly lower in the miR-145-5p mimic group than the mimic NC group(t=8.612,P<0.01).Compared with the mimic NC group,the mimic group had obviously lower mRNA and protein levels of TLR4 and contents of TNF-α,IL-1β,lL-6,ROS and MDA,and higher miR-145-5p expression level and SOD activity(P<0.05,P<0.01).The treatment of inhibi-tor resulted in increased TLR4 mRNA and protein levels and TNF-α,IL-1β,IL-6,ROS and MDA contents,and decreased miR-145-5p expression and SOD activity when compared with the above levels in the inhibitor NC group(P<0.05,P<0.01).Conclusion MiR-145-5p inhibits inflamma-tion and oxidative stress in cellular model of atherosclerosis by targeting TLR4.
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Objective To explore the effect of chemotherapeutic drugs doxorubicin or cisplatin on lipid metabolism of macrophages and its regulatory mechanism.Methods Macrophage RAW264.7 was treated with doxorubicin or cisplatin,and intracellular lipid level was detected by oil red O and ELISA;RNA sequence screen-ing and Western blot were used to confirm the changes of gene expression after chemotherapeutic drug treatment;The effects of silencing ROBO3 on cellular lipid metabolism were explored,and changes in key target genes of lipid metabolism were detected by Q-PCR and western blot.Results Adriamycin or cisplatin induced disturbances in macrophage cholesterol metabolism and exacerbated macrophage foaminess.Further studies showed that the expression of the axon guidance factor receptor,ROBO3,increased and then decreased during the chemotherapeutic drug-induced macrophage foaming process.Further intervention with ROBO3 exacerbates oxldl-induced cholesterol accumulation and foam formation in macrophages.Mechanistically,ROBO3 deficiency promotes the expression of cholesterol synthesis-related gene DHCR24 and inhibits the expression of cholesterol elimination-related gene ABCG1,resulting in cholesterol accumulation in macrophages.Conclusion This study found that ROBO3 plays an important regulatory role in the disruption of cholesterol metabolism and its foaming process in macrophages induced by chemotherapeutic drugs,which may provide new targets and ideas for the prevention and treatment of chemotherapy-related atherosclerosis.
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Atherosclerosis is a chronic disease caused by thickening of the lining of the arteries, narrowing of the lumen or hardening of occlusion, and the incidence is increasing year by year. Studies have found that the inflammatory response is involved in different pathological processes in atherosclerosis. As an important inflammatory factor, interferon-γ participates in the occurrence and development of atherosclerosis by promoting endothelial cell damage, inducing foam cell formation, and promoting plaque formation and rupture. However, studies have shown that interferon-γ can also act on lipid receptors to inhibit the formation of foam cells, inhibit the proliferation of smooth muscle cells and protect against atherosclerosis.This article will review the effect of interfron-γ of atherosclerosis in occurence and development.
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Aim To explore the effect of tanshinone II A (Tan II A) on reverse cholesterol transport in atherosclerosis model mice and RAW264. 7 cells and the underlying mechanism. Methods Thirty-two male LDLR -/- mice were randomly divided into four groups. These mice were fed with normal diet or high fat diet for 12 weeks. The control group and model group were given normal saline. Tan II A group and atorvastatin group were given Tan II A solution and atorvastatin solution for 12 weeks. RAW264. 7 cells were induced with oxidized low-density lipoprotein (ox-LDL) 100 mg • L-
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Aim To examine the effect of peptide P3 on lipid accumulation in RAW264.7 cells and the underlying molecular mechanism. Methods MTT method was used to screen the concentration of peptide P3 and oxidized low density lipoprotein(ox-LDL),and RAW.264.7 cells were induced to form foam cells by ox-LDL with 80 mg·L
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Aim To investigate the effeets of Dracocephalum Moldavica total flavonoids (TFDM) on the foaming lipoprotein (ox-LDL), and to further elucidate the mechanism of anti-atherosclerosis (AS) of TFDM. Methods RAW264. 7 maerophages were cultured in vitro and induced to become foam cells by ox-LDL stimulation, and inflammation of mouse monocyte macrophage leukemia cells (RAW264. 7) induced by oxi-dized low density TFDM(25, 50, 100 mg • L"
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Resumo Fundamento A formação de células espumosas ocorre devido ao aumento em lipoproteína plasmática de baixa densidade (LDL) e desregulação da inflamação, sendo importante para o desenvolvimento da aterosclerose. Objetivo Avaliar o perfil do fator de necrose tumoral alfa (TNF-α) e da interleucina-6 (IL-6) no método de formação da célula espumosa existente, otimizando esse protocolo. Métodos A LDL foi isolada, oxidada e marcada com sonda de isotiocianato de fluoresceína (FITC). As células espumosas foram geradas de célula derivada de monócitos humanos THP-1 e incubadas na ausência (controle) ou presença de FITC-ox-LDL (10, 50, 100, 150 ou 200 μg/mL), por 12, 24, 48 ou 72 horas. A FITC-ox-LDL na célula foi quantificada por microscopia. O ensaio de imunoabsorção enzimática foi avaliado para quantificar a IL-6 e o TNF-α, com um p <0,05 considerado significativo. Resultados Todas as concentrações de FITC-ox-LDL testadas apresentaram fluorescência mais alta em comparação com o controle, demonstrando maior acúmulo de lipoproteínas nas células. Quanto mais alta a concentração de FITC-ox-LDL, maior a produção de TNF-α e IL-6. A produção de IL-6 pelas células espumosas foi detectada até o valor de 150 µg/mL da LDL máxima de estímulo. Concentrações acima de 50 μg/mL de LDL estimularam maior liberação de TNF-α comparado ao controle. Conclusões Nosso modelo contribui para o entendimento da liberação de IL-6 e TNF-α em resposta a várias concentrações de ox-LDL usando o método otimizado para a formação de células espumosas.
Abstract Background The formation of foam cells occurs due to the increase in low-density plasma lipoprotein (LDL) and dysregulation of inflammation, which is important for the development of atherosclerosis. Objective To evaluate the profile of tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) in the existing foam cell formation method, optimizing this protocol. Methods The LDL was isolated, oxidized, and labeled with a Fluorescein isothiocyanate (FITC) probe. Foam cells were generated from THP-1 human monocyte-derived cells and incubated in the absence (control) or presence of FITC-ox-LDL (10, 50, 100, 150, or 200 μg/mL), for 12, 24, 48, or 72 hours. The accumulated FITC-ox-LDL in the cell was quantified by microscopy. The enzyme-linked immunosorbent assay was evaluated to quantify the IL-6 and TNF-α, with p < 0.05 considered significant. Results All the FITC-ox-LDL concentrations tested showed a higher fluorescence when compared to the control, showing a greater accumulation of lipoprotein in cells. The higher the concentration of FITC-ox-LDL, the greater the production of TNF-α and IL-6. The production of IL-6 by foam cells was detected up to the value of 150 µg/mL of the maximum stimulus for LDL. Concentrations above 50 μg/mL LDL stimulated greater release of TNF-α compared to control. Conclusions Our model contributes to the understanding of the release of IL-6 and TNF-α in response to different concentrations of ox-LDL, using an optimized method for the formation of foam cells.
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Abstract Introduction: Drug-eluting stents (DES) coated with rapamycin or paclitaxel as antiproliferative substances significantly reduced the incidence of clinical restenosis and had fewer side effects after percutaneous coronary intervention. However, DES coated with rapamycin or paclitaxel still cause restenosis due to abnormal tissue growth which remained a therapeutic problem, particularly in certain subgroups, possibly due to drug concentrations. This study examined the impact of different concentrations of rapamycin and paclitaxel on cytokine, cell viability and proliferation in human aortic smooth muscle cells (HASMC)-derived foam cells. Methods: The foam cell model was established in vitro by incubating HASMC with 20 µg/mL oxidized low-density lipoprotein (ox-LDL) for 48 hours. Subsequently, foam cells were treated with different concentrations (0.01 µg/mL, 0.1 µg/mL, 0.5 µg/mL, 1 µg/mL, 5 µg/mL and 10 µg/mL) of rapamycin or paclitaxel for 48 hours, to measure cytokine, cell viability and proliferation by ELISA and MTT, respectively. Finally, viability and proliferation were measured by MTT after the foam cells were treated with 1 µg/mL rapamycin or paclitaxel combined with cytokine antibody for 48 hours. Results: After incubation of HASMC with ox-LDL, the ratios of cholesterol ester and total cholesterol increased significantly (55.29%) (P<0.01). Lipid staining with Oil Red O showed many lipid vacuoles and red dye particles in the cells. Meanwhile, cell viability and proliferation significantly increased compared with the control. This indicated that HASMC had been transformed into foam cells (P<0.01) while rapamycin or paclitaxel concentrations ≥0.1 µg/mL can significantly decrease the foam cell proliferation (P<0.05 or P<0.01), and 1 µg/mL of rapamycin or paclitaxel appeared the most effective concentration. As for cytokines, rapamycin or paclitaxel concentrations ≥1 ug/mL could significantly increase the level of inflammatory cytokines IL-6 (P<0.05 or P<0.01), which was enhanced with the increase of drug concentration. However, rapamycin or paclitaxel concentrations ≥1 µg/mL could significantly reduce the levels of anti-inflammatory cytokines IL-35 and transforming growth factor beta (TGF-β) (P<0.05 or P<0.01), which decreased with the increase of drug concentration. In addition, rapamycin or paclitaxel combined with anti-IL-1β, anti-IL-6, anti- TNF-α or anti-IL-35 had no significant effect on foam cell proliferation compared to the drug alone. However, rapamycin or paclitaxel combined with anti-IL-10 or anti-TGF-β can significantly enhance foam cell proliferation (P<0.01). In addition, there was no difference in the effects of the same concentrations of rapamycin and paclitaxel on foam cells. Conclusion: Although rapamycin or paclitaxel can reduce foam cell proliferation, too high or too low concentrations could decrease effectiveness. In particular, a high dose can induce foam cells to increase inflammatory cytokines secretion, reduce anti-inflammatory cytokines secretion, and thus affect the inhibiting proliferation. For rapamycin- and paclitaxel-eluting stents, this conclusion may explain the clinical observation of in-stent restenosis after percutaneous coronary intervention. DES coated with an appropriate concentration of rapamycin or paclitaxel may, at least to some extent, contribute significantly to reducing incidence of late in-stent restenosis.
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Verruciform xanthoma (VX) is a rare benign lesion of unknown etiology, with a rough or papillary aspect, painless, sessile, well-defined, most lesions do not exceed 2 cm in their largest diameter, the degree of keratinization of the surface influences color, varying white to red, affecting mainly the gingiva and alveolar mucosa, and can also be seen in skin and genital. Herein, we present a report a clinical case of oral verruciform xanthoma in the buccal mucosa associated with the lichen planus lesion, as well as the morphological and immunohistochemical characteristics of the lesion. The clinical diagnostic hypothesis of oral lichen planus of the white reticular lesions on the buccal mucosa and on the tongue was confirmed by histopathology before a subepithelial connective tissue exhibiting intense inflammatory infiltrate in a predominantly lymphocytic band. In contrast, the hypothesis of the verrucous lesion in the left buccal mucosa was leukoplakia, with histopathological evidence showing exophytic and digitiform proliferations with parakeratin plugs between the papillary projections. Subepithelial connective tissue was characterized by macrophages with foamy cytoplasm (xanthoma cells). An immunohistochemical examination was performed, showing positivity for CD68, a macrophage marker, in addition to testing by Schiff's periodic acid (PAS) with diastasis, which was detected the presence of lipids inside these macrophages. The patient is free of recurrences of verruciform xanthoma and is being monitored due to the presence of lesions of oral lichen planus.
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Humains , Femelle , Sujet âgé , Xanthomatose/complications , Lichen plan buccal/complications , Immunohistochimie , Xanthomatose/anatomopathologie , Lichen plan buccal/anatomopathologie , Muqueuse de la bouche/anatomopathologieRésumé
BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptorligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.
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Phagocytose , Protéines du système du complément , Adipocytes , Techniques in vitro , Opsonines , Techniques de coculture , Cellules spumeuses , Macrophages , Microscopie de fluorescenceRésumé
ObjectiveRecent studies revealed that the transcription factor EB (TFEB) plays an important role in regulating autophagy, reducing intracellular lipids, and inhibiting atherosclerosis. This study aims to explore the effects of atorvastatin (ATV) on autophagy and cholesterol levels of foam cells by activating TFEB.MethodsHuman mononuclear cell line THP-1 was cultured in vitro and induced to differentiate into macrophages using phorbol ester. Oxidized low-density lipoprotein (oxLDL) was added to macrophages, which were induced for 48 hours to establish a foam cell model. The experiment was divided into four groups: blank group, model group (oxLDL group), oxLDL+ Chloroquine (CQ) group, oxLDL+ ATV group, and oxLDL+CQ+ ATV group. Cells in each group were treated with drugs for 48 h. The toxicity of ATV and chloroquine on the cells was detected by the CCK8 method. Oil red O staining was used to test the level of lipid droplets. Oxidase method was used to detect levels of intracellular free cholesterol (FC), total cholesterol (TC) and others related to. Cholesterol efflux fluorescence analysis was used to determine the cholesterol efflux rate of the cells. Expression of I, P62, TFEB, LAMP 1 protein was determined by Western blot.ResultsThe results of the CCK8 method showed that the cell survival rate decreased significantly with the increase of ATV and CQ concentrations (P<0.01). Compared to the blank group, the levels of lipid droplets, FC, TC, and CE/TC in the model group significantly increased (P<0.05), and the cholesterol outflow rate significantly decreased (P<0.05). Compared to the model group, the intracellular lipid droplets, FC, TC, and CE/TC levels in the oxLDL+CQ group elevated significantly (P<0.05), while the cholesterol outflow rate decreased significantly (P<0.05). The intracellular lipid droplets, FC, TC, and CE/TC contents in the oxLDL+ATV group decreased significantly (P<0.05), and the cholesterol outflow rate increased significantly (P<0.05). Compared to the oxLDL+CQ group, the intracellular lipid droplets, FC, TC, and CE/TC content in the oxLDL+CQ+ATV group decreased significantly (P<0.05), while the cholesterol outflow rate increased significantly (P<0.05). Western blotting results showed that protein expression levels of LC3II/I, P62 and TFEB were decreased in the model histone in comparison to the blank group (P<0.05). Compared to the expression levels of LC3II/I, P62, TFEB and LAMP1 ((1.006±0.052), (0.183±0.013), (0.333±0.020), and (0.957±0.026)) in the model group, the expression levels of oxLDL+CQ histamine ((1.594±0.017), (0.257±0.006), (0.477±0.024), and (0.957±0.026)) were significantly higher (P<0.05).The protein expression levels of LC3II/I, TFEB and LAMP1 in oxLDL+ATV group ((1.146±0.060), (0.540±0.031), and (1.027±0.054)) were increased, while the protein expression levels of P62 (0.115±0.009) were decreased (P<0.05). Compared to LC3II/I and P62 in oxLDL+CQ group, the expression level of oxLDL+CQ+ATV histone was decreased ((1.419±0.036) and (0.165±0.006)), and the difference was statistically significant (P< 0.05).ConclusionATV can increase the cholesterol outflow rate of macrophages, reduce the level of cholesterol and lipid droplets in macrophages, and reduce the formation of foam cells. The mechanisms behind this are still unknown, which may be related to the activation of TFEB by ATV and influencing the process of autophagy.
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Objective@#To investigate the expression and clinical significance of circDLGAP4 from peripheral blood in coronary heart disease (CAD). @*Methods@#The relative expression level of circDLGAP4 in peripheral blood leukocytes (PBLs) from 142 CAD patients and 169 healthy controls were detected by real-time PCR. Logistic regression, Spearman correlation and multivariate regression analysis were used to investigate the correlation of circDLGAP4 with CAD. THP-1 macrophages were treated with oxidized low density lipoprotein (ox-LDL) to construct an atherosclerotic foam cell model. The levels of circDLGAP4 mRNA were detected at different time points. @*Results@#The mRNA expression of circDLGAP4 in PBLs of CAD patients was significantly decreased compared with controls (P=0.019). With increased unit (2 -ΔCt ) of circDLGAP4 expression, the risk of CAD occurrence reduced by 41.6% (adjusted OR=0.584, 95% CI: 0.394-0.866, P=0.007). The expression of circDLGAP4 was negatively correlated with T2DM history (β=-0.182,P=0.030). The level of circDLGAP4 in ox-LDL-treated THP-1 macrophages was decreased in a time-dependent manner. @*Conclusion@#The expression of circDLGAP4 was significantly decreased in PBLs of CAD patients and THP-1 macrophages-derived foam cells, and might be a protective factor in the pathophysiology of CAD.
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Objective: The effect of Xiexintang on Toll like receptor 9 (TLR9) signaling pathway in macrophage derived foam cells was studied by in vitro cell experiments. Method: The fifty SPF male SD rats were randomly divided into low, medium and high dose groups (1.4,4.2,12.6 g·kg-1·d -1) and normal groups. Except 20 rats in the normal group, 10 rats in each group were given equal volume of pure water gavage in the blank group. After the last Administration for 7 days, serum was separated,and the serum containing drugs in the low, medium and high dose groups of Xiexintang was prepared. Oxidized low density lipoprotein (ox-LDL) was used to intervene the differentiation of RAW264.7 macrophages into foam cells. The cell foam was identified by oil red O staining. After observing the effect of drug containing serum on the proliferation of macrophage derived foam cells by methye thiazolye telrazlium(MTT) method,the serum containing 20%concentration of each drug was selected to act on the foam cell model. The expression of interleukin(IL) -1β and interferon (INF) -γ was determined by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (Real-time PCR). TLR9, myeloid differentiation factor 88(MyD88)and nuclear factor(NF) -κB were detected by Western blot. Result: Oil red O staining showed that the red particles were obvious after ox-LDL intervention. The foam cell model was successfully prepared. MTT results showed that there was no significant difference in cell proliferation between the high dose group of Xiexintang in the 10%~30%concentration range and the normal group serum. Follow up selection of the serum containing 20%concentration of each dose intervened the foam cells induced by ox-LDL. Compared with the normal group,the model group after ox-LDL intervention induced the high expression of TLR9,MyD88,NF-κB p65,IL-1β,INF-γ (PPκB p65,IL-1β,INF-γ(PPConclusion: Xiexintang containing serum can inhibit ox-LDL-induced RAW264.7 macrophage foaming,and its mechanism may involve regulation of TLR9/MyD88/NF-κB p65 signaling pathway and inhibition of IL-1β and INF-γ overexpression. This may be one of its mechanisms of against atherosclerosis.
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Abstract@#Introduction: Atherosclerosis is a chronic inflammatory disease initiated by the accumulation of macrophage derived foam cells in the intima layer of artery. In mice model of atherosclerosis, murine norovirus-4 has been shown to accelerate atherogenesis. In cells, lipid biometabolism is regulated by peroxisome proliferator activated receptor γ (PPARγ). Since PPARγ is predominantly expressed in macrophages and mice macrophages are MNV-1 proliferation-permissive host, we hypothesised that PPARγ ligands may regulate atherogenesis. Methods: MNV-1 was generated via RNA-based recovery system and used to infect the RAW 264.7 cells, then subjected to oxidized low-density lipoprotein (oxLDL)-loaded and treated with ciglitazone or 15-deoxy-Delta(12,14)-PGJ(2)(15d-PGJ2). Foam cell formation was evaluated and the MNV-1 infection in all treatments was confirmed using virus titration (50% tissue culture infective dose; TCID50) and polymerase chain reaction (PCR). Results: Increment of lipid droplets was observed in all oxLDL treatment involving MNV-1 infection, ciglitazone or 15d-PGJ2 in the cytosol of RAW 264.7 cells over time compared to non-oxLDL treated cells. From the cholesterol ester (CE) content analysis amongst the oxLDL-loaded cells however, we found MNV-1 did not elicit increment of CE content. Treatment with 15d-PGJ2 resulted in increase of the CE content in oxLDL-treated cells. Interestingly, MNV-1 and ciglitazone had synergistic effect in reducing the CE content in oxLDL-treated cells. Conclusion: oxLDL stimulates foam cells formation in RAW 264.7 cells. However, MNV-1 infection did not contribute to RAW 264.7 cells derived-foam cells formation. On the other hand, 15d-PGJ2 promotes foam cells formation whilst ciglitazone inhibits the formation of foam cells derived from MNV-1-infected macrophages.
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Objective To study whether Nεcarboxymethyl lysine(CML)can form a good molecular docking with the scavenger receptor CD36and induce a stable interaction.Methods The interaction between CML and CD36was studied by co-immunoprecipitation.The binding mode and affinity of CD36to CML were tested using AutoDock 4.2,iBabel and XQuartz-2.7.7software respectively. Results Co-immunoprecipitation showed that anti-CD36antibody magnetic bead could precipitate CD36from the total protein in RAW264.7cells and anti-CML could detect CD36 binding CML.CD36had a good molecular docking with CML,CD36and CML interacted stably with each other.The affinity of CML to 4Q4Bprotein structure of CD4extracellular domain was -29.62kJ/mol.ARG82,ASN71and THR70were the products of amino acid receptor interaction. Further docking analysis showed that CML could form 3interacting hydrogen bonds with 4Q4B,and the docking prediction inhibition constant was 6.92with a root mean square deviation of 2.54.Conclusion A good molecular docking between CML and 4Q4Bprotein structure of CD36extracellular domain can induce a stable interaction between CML and CD36.Hydrogen bonding is the main interaction mode.
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OBJECTIVE: To investigate the role of cyclophilin A in the foaming process of macrophages induced by free silica( SiO_2). METHODS: The human peripheral blood mononuclear cell THP-1 in the logarithmic growth phase was induced and differentiated into human macrophages with phorbol 12-myristate 13-acetate at 100 μg/L for 48 hours. The cells were divided into 4 groups. The cells in the blank control group were not treated. The cells in the oxidized low-density lipoprotein( ox-LDL) control group were treated with a final concentration of 50 mg/L ox-LDL. The cells of 50 mg/L SiO_2 group were treated with 50 mg/L of SiO_2 and ox-LDL. The cells of 100 mg/L SiO_2 group were treated with 100 mg/L of SiO_2 and 50 mg/L of ox-LDL. After treatment of cells for 48 h,cell viability was measured by MTS method. The lipid accumulation of cells was observed by oil red O staining and colorimetric method; the expression of cyclophilin A in cell supernatant was detected by enzyme-linked immunosorbent assay,and the expression of cyclophilin A in the cells was detected by Western blotting. RESULTS: The cell viability was the highest when the concentration of SiO_2 was 100 mg/L compared with the control and other four SiO_2groups( P < 0. 01). The cell foaming change in the 100 mg/L SiO_2 group observed by oil red O staining significantly increased compared with the blank control group. The expression of total cholesterol,free cholesterol increased in the ox-LDL control group,50 mg/L SiO_2 group and 100 mg/L SiO_2group( P <0. 05),and the cholesterol specific gravity increased( P < 0. 05) compared with the blank control group,meanwhile the expression of cyclophilin A in the cell supernatant increased( P < 0. 05),and the expression in the macrophages cells decreased( P < 0. 05). CONCLUSION: Cyclophilin A is involved in the foaming process of macrophages induced by SiO_2.
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Objective To study the role of natural antiox-LDL subclass IgM antibodies in formation of atherclerosis.Methods The macrophages from mice were divded into LDL group,Cuox-LDL group and control group.The macrophages in LDL group and Cuox-LDL group were precultured with LDL and Cuox-LDL.Adhesion experiment was performed in control group.3A6,5G8 and 2H7 were incubated.The macrophages were divided into 3A6 group,5G8 group and 2H7 group.Adhesion experiment was performed by adding the pretreated 125I Cuox-LDL.The binding parameters of macrophages and 125I Cuox-LDL in different groups were recorded.Foam cells were analyzed by cell lipid analysis with oil red O staining.Results The bound 125I Cuox-LDL level was significantly lower in Cuox-LDL group than in control group and LDL group (P<0.01).No significant difference was found in bound 125I Cuox-LDL level between 5G3 group and 2H7 group (P>0.05).The bound 125I Cuox-LDL level was significantly lower in 3A6 group than in control group,5G8 group and 2H7 group (P<0.01).Oil red O staining showed no oil drop in macrophages,but a small number of oil drops in 3A6 group and a large number of oil drops in control group.The cholesterol and TG in macrophages reduced 45% and the TG reduced 170% in 3A6 group,which were significantly lower than those in control group (P<0.01).Conclusion Antiox-LDL IgM subclass antibody 3A6 can effectively prevent the binding of unactivated macrophages to ox-LDL,reduce the occurrence and progression of atherosclerosis.
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OBJECTIVE:To establish the method for the content determination of rapamycin (RAPA) in human monocyte THP-1 derived foam cells,and to study the effects of RAPA targeting preparation(RAPA-NP-Apt)targeting at foam cells. METH-ODS:Foam cells model were established through THP-1 cells were induced by oxidized low density lipoprotein. Foam cells were incubated with 200 ng/mL RAPA or 200,400,800 ng/mL RAPA-NP-Apt for 60 min. The content of RAPA was determined by HPLC. The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-water(90:10,V/V) at flow rate of 1.0 mL/min. The column temperature was set at 40 ℃,and the detection wavelength was 278 nm. The sample size was 20 μL. RESULTS:The concentration of RAPA ranged 50-6400 ng/mL (r=0.99996) with average recovery of 98.72%(RSD=0.62%,n=3). RSDs of inter-day and intra-day were not more than 6.15%(n=6),RSD of stability was lower than 2%(n=6),and RSD of repeatability was 1.64%(n=6). After foam cells were incubated with RAPA or low-concentration,medi-um-concentration and high-concentration of RAPA-NP-Apt,the contents of RAPA were 12,43,98,140 ng/106 cells. CONCLU-SIONS:The method is simple,stable and reproducible. It can be used for content determination of RAPA in foam cells. RA-PA-NP-Apt can improve the effects of RAPA targeting at foam cells.
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Objective To investigate the effects of microRNA-155 (miR-155) on immuno-inflammatory reaction of foam cells by targeting MyD88 and the possible mechanism. Methods RAW264.7 macrophages were cultured in vitro and transfected with miR-155 mimics, miR-155 inhibitor, MyD88 siRNA and their negative control respectively, then ox-LDL stimulation was given to build foam cell model. The expression of MyD88 in foam cells was detected by RT-qPCR and Western blot assay. Moreover, the expression levels of interleukin (IL)-10, TGF-β1 and MCP-1 in supernatant were determined by ELISA. Results After being transfected with miR-155 mimics, the mRNA level of MyD88 remained unchanged compared with that of control group (P>0.05). The protein level of MyD88 decreased significantly (P<0.05), and the expression levels of IL-10, TGF-β1 and MCP-1 in supernatant also decreased significantly (P<0.05). After being transfected with miR-155 inhibitor, the mRNA level of MyD88 remained unchanged compared with that of control group (P>0.05). The expression levels of MyD88 protein and inflammatory cytokines increased significantly (P<0.05). After being transfected with MyD88 siRNA, the expression levels of MyD88 mRNA and protein decreased significantly, and the expression levels of inflammatory cytokines also decreased significantly (P<0.05). Conclusion miR-155 can negatively regulate inflammation by targeting MyD88 through the inhibition of translation.