To investigate the expression changes of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) on Chlamydia pneumoniae (C.pn) induced foam cell formation.Human monocytic cell line (THP-1) was induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and were randomly allocated into four groups: negative control group (50 microg/ml LDL for 48 h); positive control group (50 microg/ml ox-LDL for 48 h); C.pn infection group (50 microg/ml LDL plus 1 x 10(5), 4 x 10(5), 5 x 10(5) and 1 x 10(6) IFU C.pn for 48 h or 1 x 10(6) IFU C.pn for 0, 24, 48 and 72 h); ACAT inhibitor 58-035 plus C.pn infection group (1, 5, 10 microg/ml ACAT inhibitor 58-035 pretreatment for 1 h, 50 microg/ml LDL and 1 x 10(6) IFU C.pn for 48 h). The mRNA and protein expressions of ACAT1 were determined by RT-PCR and Western blot, respectively. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesteryl esters were detected by enzyme-fluorescence.The mRNA and protein expressions of ACAT1 were significantly up-regulated in positive control cells compared those in negative control cells and further upregulated by C.pn infection in a time-dependent and concentration-dependent manner (all P < 0.05). There were significantly increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol in positive control cells as compared with negative control cells and these were further aggravated by C.pn (at the concentrations of 5 x 10(5) and 1 x 10(6) IFU for 48 h) and C.pn infection induced increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol could be significantly attenuated by ACAT inhibitor 58-035 (all P < 0.05).Chlamydia pneumoniae induces THP-1-derived foam cell formation by up-regulating the expression of ACAT1.
Objective To investigate the mechanisms of Chlamydia pneumoniae (C. pn)-induced foam cell formation, the expression of ATP binding cassette transporter AI ( ABCA1 ) and perexisome prolif-erator-activated receptor γ (PPARγ) were examined. Methods THP-1 monneytes were induced into mac-rophages after the addition of 160 nmol/L phorbol myristate acetate (PMA) for 72 h. THP-1-dorived macro-phages when co-cuhured 50 mg/L low density lipoprotein (LDL) were designated randomly in four groups: control (uninfected) group, C. pn infection group, rosiglitazone + C. pn infection group and rosiglitazone group. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellnlur choles-terol ester were detected by enzyme-flnoreseence. The expression of ABCA1, PPARγ, mRNA and protein were determined by RT-PCR and Western blot, respectively. Results C. pn down-regulated the expression of ABCA1, PPARγ at mRNA and protein levels in a concentration-dependent manner in THP-1-derived mac-rophages when co-incubated with LDL. Resiglitazone not only concentration-dependently alleviated the down-regulation of ABCA1 expression by C. pn infection (P<0.05), but also markedly suppressed the accumula- tion of lipid droplets and cholesteryl ester by C. pn at higher concentrations ( 10 and 20 μaol/L). Condu-sion C. pn induces foam cell formation by down-regulating the expression of ABCA1 via PPART pathway, which may provide a new evidence for the development and progression of atherosclerosis initiated by C. pn infection.
Key words:
Chlamydia pneumon/ae; ATP binding cassette transporter AI ; Peroxisome prnlifera-tor-activated receptor γ; Foam cell formation; Atherosclerosis
BACKGROUND Anti-melanoma differentiation-associated protein 5-positive dermatomyositis (MDA5⁺ DM) is characterized by a life-threatening complication of rapidly progressive interstitial lung disease (RP-ILD). Early prediction of RP-ILD can enhance diagnostic accuracy and therapeutic efficacy. This study was conducted to develop a nomogram model for predicting RP-ILD in patients with MDA5⁺ DM. MATERIAL AND METHODS We retrospectively analyzed 53 patients with MDA5⁺ DM, of whom 21 patients were diagnosed with RP-ILD between January 2018 and January 2021. Univariate analysis (t test, Mann-Whitney U test, chi-squared test, or Fisher's exact test) and receiver operating characteristic (ROC) analysis were used to select candidate variables. Multivariate logistic regression analysis was conducted to construct a prediction model, which was subsequently transformed into a nomogram. ROC analysis, calibration curve and decision curve analysis were performed to evaluate the model's performance. The bootstrapping method (resampling=500) was used for internal validation. RESULTS We successfully established a nomogram, called the CRAFT model, to predict RP-ILD in MDA5⁺ DM patients. The model included 4 variables, namely C-reactive protein-to-albumin ratio, red blood cell distribution width-coefficient of variation, fever status, and CD3⁺ T cells. The model presented high predictive power and a good performance in calibration curve and decision curve analysis. In addition, the model had a good predictive ability in internal validation. CONCLUSIONS The CRAFT model could help to predict RP-ILD in patients with MDA5⁺ DM.
It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-alpha could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-alpha in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.
In the presence of low density lipoprotein (LDL), Chlamydia pneumoniae induces macrophage-derived foam cell formation, a typical pathological feature of early atherosclerosis. However, its mechanism has not been fully understood. Peroxisome proliferator-activated receptors (PPARs) are key regulators of macrophage lipid metabolism. This study therefore investigated the role that PPAR alpha and PPAR gamma may play a role in C. pneumoniae-induced foam cell formation. Oil Red O staining and Lipid mass quantification showed that LDL-treated THP-1 macrophages infected with high doses of C. pneumoniae (5x10(5) and 1x10(6)IFU) resulted in the large accumulation of lipid droplets and markedly increased the ratio of intracellular cholesteryl ester (CE) to total cholesterol (TC) (>50%). The results of RT-PCR and Western blot indicated that C. pneumoniae infection dose-dependently suppressed the expression of PPAR alpha and PPAR gamma at mRNA and protein levels in LDL-treated THP-1 macrophages. PPAR alpha (fenofibrate) and PPAR gamma (rosiglitazone) agonists, inhibited the accumulation of intracellular CE by C. pneumoniae in a dose-dependent manner. Furthermore, C. pneumoniae-induced foam cell formation was significantly suppressed by higher doses of fenofibrate (20 and 50microM) and rosiglitazone (10 and 20microM). These results first reveal that C. pneumoniae induces foam cell formation via PPAR alpha and PPAR gamma-dependent pathway, which may contribute to its pro-atherogenic properties.
To investigate whether nuclear factor（NF-kappa-B） activation participate in the regulation and control of ABCA1 gene expression in THP-1 derived-macrophage foam cells. First, THP-1 cells were be transformed into foam cells. Then, foam cells were treated with TNF-alpha（ 10 ng/ml） , or with TPCK（ 10 Mu-mol/L） at 60 min before TNF-alpha stimulation or at different time points after TNF-alpha stimulation. Gene expression of ABCA1 was analyzed using RT-PCR and its protein was detected by Western blot. Sandwich ELISA method was employed to evaluate the nuclear translocation of NF-kappa-B subunit p65. Stimulation of foam cells by TNF-alpha led to a rapid increase of NF-kappa-B p65 nuclear translocation and the peak of NF-kappa-B p65 was 30-60 rain. TNF-alpha could down regulate beth ABCA1 mRNA expression and ABCA1 protein level. Addition of TPCK to the cells at 60 rain before TNF-alpha attenuated the increase of nuclear factor（NF-kappa-B） translocated into the nucleus and the decrease of ABCA1 gene expression induced by TNF-alpha. Contrastively, there was no difference in the level of ABCA1 gene expression between cells incubated with TNF-alpha and cells added TPCK 4 h after stimulation with TNF-alpha. TNF-alpha could swiftly result in NF-kappa-B activation. Activation of NF-kappa-B signal transduction may finally inhibit ABCA1 gene expression, which may play a critical role in cellular cholesterol efflux.
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