Signal Transduction Mechanisms Of Chlamydia Pneumoniae-induced Disruption Of Cholesterol Homeostasis In THP-1-Derived Macrophages

PartⅠEffects of Chlamydia pneumoniae infection on the Cholesterol Metabolism In THP-1-derived macrophagesObjective To observe the effects of Chlamydia pneumoniae (C.pn) infection on the cholesterol metabolism in THP-1-derived macrophages.Methods C.pn strain AR-39 was propagated in HEp-2 cells by centrifugation-driven infection. The human THP-1 monocytic leukemia cell line differentiated to macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and then were randomly allocated into five groups:①control group;②-⑤different concentrations(1×105,4×105,5×105 and 1×106 IFU) of C.pn infection group; each group was incubated with 50μg/ml LDL for 48 h. The infection of C.pn on THP-1-derived macrophages was figured by transmission electron microscope. Lipid droplets in cytoplasm and the number of foam cells were observed by oil red O staining. The contents of intracellular total cholesterol (TC) and cholesteryl esters (CE) were measured by enzymic chromatometry. The mRNA and proteinexpressions of scavenger receptor A1 (SR-A1), acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), ATP binding cassette transporter Al (ABCA1) and G1 (ABCG1) from each group were determined by RT-PCR and Western-blot, respectively.Results Compared with control group, C.pn infection increased the contents of intracellular lipid droplets, the number of foam cells, TC and CE in a concentration-dependent manner. Moreover, C.pn infection not only up-regulated the mRNA and protein expressions of SR-A1 and ACAT1, but also down-regulated the mRNA and protein expressions of ABCA1 and ABCG1 in a concentration-dependent manner in LDL-treated THP-1 macrophages (all p<0.05). Higher concentrations of C.pn infection (5×105 and 1×106 IFU) group obviously cause the effects as mentioned above (p<0.05).Conclusion C.pn infection may destroy the homeostasis of intracellular cholesterol metabolism by increasing SR-A1 and ACAT1 expressions, and inhibiting ABCA1 and ABCG1 expressions in LDL-treated THP-1-derived macrophages, which leads to foam cell formation.Part II The role of peroxisome proliferator-activated receptor y pathway in Chlamydia pneumoniae-induced imbalance of cholesterol metabolism homeostasisObjective To investigate the effects of peroxisome proliferator-activated receptor y (PPAR y) on the expressions of SR-A1, ACAT1, ABCA1 and ABCG1 regulated by Chlamydia pneumoniae (C.pn) infection, and to discuss the pathways of C.pn-induced imbalance of cholesterol metabolism homeostasis.Methods THP-1 monocytes were induced into macrophages by 160 nmol/L PMA for 48 h. and then were randomly allocated into seven groups:①control group,50μg/ml low density lipoprotein (LDL) for 48 h;②C.pn infection group,50μg/ml LDL plus 1×106 IFU C.pn infection for 48 h;③Rosiglitazone (a specific PPARγligand) plus C.pn infection group, pretreatment with different concentrations of rosiglitazone (1,10,20μmol/L) for 2 h,50μg/ml LDL plus 1×106 IFU C.pn infection for 48 h;④Rosiglitazone group,20μmol/L rosiglitazone for 48 h. Lipid droplets in cytoplasm and the number of foam cells were observed by oil red O staining. The contents of intracellular cholesteryl esters were detected by enzymic chromatometry. The mRNA and protein expressions of PPARγ, SR-A1, ACAT1, ABCA1 and ABCG1 were determined by RT-PCR and Western-blot, respectively.Results C.pn infection suppressed the expression of PPARγat mRNA and protein levels in concentration-dependent manner in LDL-treated THP-1 macrophages (all p <0.05). From morphological and biochemical criteria, higher concentrations of rosiglitazone (10 and 20μmol/L) markedly inhibited C.pn-induced imbalance of cholesterol metabolism homeostasis and foam cell formation. Compared with C.pn infection group, not only the up-regulation of SR-A1 and ACAT1 but also the down-regulation of ABCA1 and ABCG1 at mRNA and protein levels by C.pn infection were concentration-dependently inhibited by rosiglitazone (all p<0.05).Conclusion C.pn infection induces imbalance of cholesterol metabolism homeostasis and foam cell formation in THP-1 derived macrophages by up-regulating SR-A1 and ACAT1 expressions and down-regulating ABCA1 and ABCG1 expressions partly via PPAR y-dependent pathway. PartⅢThe role of c-Jun NH2 terminal kinase pathway in Chlamydia pneumoniae-induced imbalance of cholesterol metabolism homeostasisObjective To investigate the effects of c-Jun NH2 terminal kinase (JNK) on the expressions of SR-A1, ACAT1, ABCA1 and ABCG1 regulated by Chlamydia pneumoniae (C.pn) infection, and to discuss the pathways of C.pn-induced imbalance of cholesterol metabolism homeostasis.Methods THP-1 monocytes were induced into macrophages by 160 nmol/L PMA for 48 h. and then were randomly allocated into seven groups:①control group,50μg/ml low density lipoprotein (LDL) for 48 h;②different concentrations of C.pn infection group,50μg/ml LDL plus 1×105,4×105,5×105 and 1×106 IFU C.pn infection for 48 h;③SP600125 (a specific JNK inhibitor) plus C.pn infection group, pretreatment with different concentrations of (1,10,20μmol/L) for 2 h,50μg/ml LDL plus 1×106 IFU C.pn infection for 48 h;④SP600125 group,20μmol /L SP600125 for 48 h. Lipid droplets in cytoplasm and the number of foam cells were observed by oil red O staining. The contents of intracellular cholesteryl esters were detected by enzymic chromatometry. The mRNA and protein expressions of PPARγ, SR-A1, ACAT1, ABCA1 and ABCG1 were determined by RT-PCR and Western-blot, respectively.Results From morphological and biochemical criteria, higher concentrations of SP600125 (10 and 20μmol/L) markedly inhibited C.pn-induced imbalance of cholesterol metabolism homeostasis and foam cell formation. Compared with C.pn infection group, not only the down-regulation of PPAR y, but also the regulation of the taget genes of PPAR y (SR-A1, ACAT1, ABCA1 and ABCG1) at mRNA and protein levels by C.pn infection were concentration-dependently inhibited by SP600125(all p<0.05). Conclusion C.pn infection induces imbalance of cholesterol metabolism homeostasis and foam cell formation in THP-1 derived macrophages by up-regulating SR-A1 and ACAT1 expressions and down-regulating ABCA1 and ABCG1 expressions partly via JNK-PPARγdependent pathway.