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Effects of NF-ΚB on ABCA1 Expression and Cholesterol Efflux in THP-1 Derived-foam Cells Induced by AngII

Author: YueBing
Tutor: ChenZhiJian
School: Huazhong University of Science and Technology
Course: Internal Medicine
Keywords: Atherosclerosis Angiotensin II Foam cells NF - kappa B Angiotensin II ABCA1
CLC: R543.5
Type: Master's thesis
Year: 2007
Downloads: 148
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Abstract

The first part of NF-kappa B in THP-1 macrophage-derived foam cells cholesterol and cholesterol efflux in the role of AngII mediated the purpose of THP-1 monocytic cell line induced to differentiate into foam cells, THP-1 macrophage cells derived foam cells for the study, angiotensin II (Ang II) the role of cholesterol efflux in THP-1 macrophage-derived foam cells cholesterol and intracellular to explore nuclear factor-kappa B (NF-kappaB). Method with PMA human THP-1 monocytic cell line induced differentiation into macrophages incubated 48 hours to allow macrophages into foam cells, 50mg / L of oxidized low density lipoprotein (ox-LDL), were randomly divided into Group 3 (1) control group: adding sterile PBS; (2) Ang II group: to join Ang II (10-5 mol / L), (3) the TPCK pre-incubation group: TPCK (10 micromol / L) pre-incubation for 1 hour and then Ang II (10-5 mol / L); Add to apoA-I (10 mg / L) stationary culture for 24 hours. Enzymatic by a fluorescence spectrophotometer (HITACHI 650-60 type), the excitation wavelength at 325nm, 415nm for fluorescence emission wavelength detection of extracellular and intracellular cholesterol content. For measuring cellular cholesterol efflux (a liquid scintillation counting method), 0.37 x 106 Bq / ml [3H] cholesterol and 50 mg / L ox-LDL incubated macrophages 48 hours after the packet, then the above method. Results Ang Ⅱ caused by THP-1-derived macrophages cholesterol levels significantly increased, with the control group compared to the increased 140.5% (p lt; 0.05); TPCK pre-incubation significantly reduce the increase in AngII-induced foam cell cholesterol content, compared to the Ang Ⅱ group decreased by 24.1% (p lt; 0.05), the intracellular cholesterol than the control group. Ang Ⅱ group foam cells cholesterol efflux rate (9.31 ± 1.4)% compared with the control group decreased by 46.4% (p lt; 0.05), the TPCK pre-incubation cholesterol efflux rate compared with Ang Ⅱ group was significantly increased 41.1% (p lt; 0.05) Conclusion Ang Ⅱ significantly increase the cholesterol content in THP-1-derived macrophage cells derived foam cells, inhibition of cholesterol outflow in the foam cells, promote the formation of foam cells, accelerate artery atherosclerosis hardening; TPCK pre-hatching inhibition of NF-kappa B activation can significantly with inhibition of Ang Ⅱ caused by intracellular cholesterol levels increase to reduce AngII outflow inhibition of macrophage cholesterol, reduce the degree of foam cells. The second part of the NF-kappa B in AngII-mediated effect of THP-1 macrophage-derived foam cells ABCA1 expression purpose THP-1 monocytic cell line induced to differentiate into foam cells, THP-1 macrophages source foam cells for the study, to explore nuclear factor-kappa B (NF-kappaB) in the role of ABCA1 expression and regulation of angiotensin II (angiotensin II, Ang II) THP-1 macrophage-derived foam cells. Method with PMA human THP-1 monocytic cell line induced differentiation into macrophages incubated 48 hours to allow macrophages into foam cells, 50mg / L of oxidized low density lipoprotein (ox-LDL), were randomly divided into 3 (1) bubble control group: adding sterile PBS, (2) Ang II group: to join Ang II (10-5 mol / L), (3) the TPCK pre-incubation group: TPCK (10 micromol / L) pre-incubation for 1 hour Add to Ang II (10-5 mol / L); join apoA-(I10mg / L) was allowed to stand for 48h. Reverse transcription - polymerase chain reaction (RT-PCR) and Western blot were used to detect changes in ABCA1 mRNA expression of ABCA1 protein. 0,30 min, 1h, 2h and 4h, the use of the the Sandwich ELISA law detection of NF-kappa B activation level. Results of mRNA and protein expression indicated gray value the AngII group than in the control group, respectively, a decrease of 41% and 30.4% (p lt; 0.05); of TPCK pre-hatch group gray value compared with AngII group increased 30% and 19%, respectively (p lt; 0.05), but ABCA1mRNA, protein expression was significantly lower than the control group. The AngII cells NF-kappa B (P65) activated the nuclear translocation reached a peak in 30 to 60 minutes, 2 to 4h still maintain a high level. TPCK pre-incubation activation of NF-kappaB nuclear translocation no significant peak is maintained at a lower level. Conclusion AngII significantly inhibited ABCA1mRNA protein TPCK significantly reduce pre-incubation AngII ABCA1mRNA, inhibition of protein expression. AngII instantly significant activation of NF-kappaB and TPCK pre hatch significantly inhibited AngII activation of NF-kappaB.

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