Mast cells change cellular cholesterol content and inhibit cholesterol efflux from THP-1 macrophage-derived foam cells

AIM:Mast cells (MC) are present in the arterial intima,the site of atherogenesis. The present studies explore the effect of MC on cholesterol content,distribution and efflux in THP-1 macrophage-derived foam cells (THP-1FCs）. METHODS:THP-1FCs were incubated with high-density lipoproteins 3 (HDL₃) in the absence or presence of mast cell granules (MCGs) harvested from compound 48/80-stimulated rat peritoneal MC. The intracellular cholesterol level,cholesterol effluxing capacity,ATP-binding cassette transporter A1(ABCA1) mRNA and HDL₃ treated with MCGs were detected to characterize the role of MC on intracellular cholesterol. RESULTS:MCGs had high levels of cellular total cholesterol(TC),free cholesterol(FC) but not esterifed cholesterol(EC) compared to control group where the TC concentrations ranged from 527.3 mg/g to 917.9 mg/g cellular protein with EC accounting for 7.6% of the cholesterol. Cholesterol efflux was 14% less in MCGs group compared to control group. ABCA1 mRNA expression in MCG-treated THP-1FCs remained unchanged in 20 hours. In contrast,treatment of HDL₃ with MCGs resulted in rapid degradation of the main HDL₃ apoliproteins,apoA-Ⅰ. SDS-PAGE revealed that a minor polypeptide band with about 26 kD molecular mass appeared below the apoA-Ⅰband. Densitometric analysis of the gel demonstrated that ≈ 28% of apoA-Ⅰhad been degraded by the MCGs. CONCLUSION:These results indicate that MC decreases cholesterol efflux,increases cellular accumulation in TC and FC by depleting HDL₃ and apoA-Ⅰ,but not by inhibiting ABCA1 mRNA expression.     

Effects of anthocyanin on cholesterol efflux from mouse peritoneal macrophage foam cells

AIM: To explore the effect of anthocyanin on cholesterol efflux and elucidate its possible molecular mechanism. METHODS: Mouse Peritoneal macrophages were loaded with 50 mg/L AcLDL to induce macrophage-derived foam cells. Cholesterol efflux from macrophage-derived foam cells induced by anthocyanin was determined by enzymatic methods. PPAR-γ mRNA and protein expression in macrophage-derived foam cells was assayed by quantitative PCR and western blotting. RESULTS: Anthocyanins had the capacity of promoting cholesterol efflux from mouse peritoneal macrophage-derived foam cells and increased PPAR-γ mRNA and protein expression. CONCLUSION: Anthocyain-induced cholesterol efflux may be related to its enhancing PPAR-γ mRNA and protein expression.     

Effect of astragalus polysaccharides on cholesterol efflux in THP-1 macrophage-derived foam cells

AIM: To study the effect of astragalus polysaccharides (Aps) on cholesterol efflux in THP-1 macrophage-derived foam cells. METHODS: After exposed to Aps at different doses, cholesterol efflux and ABCA1 protein levels in cultured THP-1 macrophage-derived foam cells were determined by a γ counter and flow cytometry, respectively. RESULTS: Aps increased cholesterol efflux in THP-1 macrophage-derived foam cells with dose dependent pattern and resulted in an increase in the expression of ABCA1 protein in THP-1 macrophage-derived foam cells. CONCLUSION: The increase in cholesterol efflux by Aps might be related to the up-regulation of ABCA1.     

Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

AIM: To study the protective effect of puerarin on the atherosclerosis of RAW264.7-derived foam cells. METHODS: The model of foam cells was established by incubating the RAW264.7 cells with ox-LDL. The cholesterol uptake was evaluated by a DiI-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264.7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3Ⅱ, P62, CD36, ABCA1, LAL and p-AMPK were determined by Western blot. RESULTS: Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3Ⅱ, ABCA1 and LAL in puerarin group were higher than that in ox-LDL group, while the protein levels of P62 and CD36 were obviously decreased, and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3Ⅱ, ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION: Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264.7 macrophages, which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation, and reduce the uptake of lipids by CD36 negative regulation.     

Effects of oxidized low density lipoprotein and lysophosphatidylcholine on cholesterol efflux from mouse peritoneal macrophage foam cells

AIM:To explore the effects of oxidized low density lipoprotein (OxLDL) and one of its component— lysophosphatidylcholine (LPC) on cholesterol efflux from mouse macrophage foam cells.METHODS:(1) Cholesterol efflux induced by apoAI from mouse peritoneal macrophage foam cells loaded with OxLDL or acylated LDL(AcLDL) was measured. (2) Cholesterol efflux induced by LPC and apoAI from macrophage foam cells separated from normal or apoE gene deficient (E⁰) mouse loaded with AcLDL were measured.RESULTS:(1) When the macrophage foam cells were incubated with apoAI, cholesterol efflux from AcLDL-induced macrophage foam cells increased significantly compared to that of OxLDL-induced macrophage foam cells. (2) LPC promoted cholesterol efflux from macrophage foam cells in relation to both dosage and time. When LPC was incubated with E⁰ mouse macrophage foam cells, the released cholesterol mass was significantly lower than that of normal mouse macrophage foam cells. It was also found that cholesterol efflux induced by apoAI normally occurred in E⁰ mouse macrophage foam cells.CONCLUSIONS:(1) OxLDL accumulated cholesterol in macrophages and impair cholesterol efflux. (2) LPC induced cholesterol efflux from macrophage foam cells, which may occur via apoE pathway.     

Action of ATP binding cassette transporter A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells

AIM:To study the action of ATP binding cassette transporter(ABC) A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells.METHODS:After exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) at different concentration for 24 hours, cholesterol efflux and ABCA1 mRNA level were determined by FJ-2107P type liquid scintillator and reverse trancriptase-polymerase chaim reaction(RT-PCR), respectively.RESULTS:Oxidized LDL promoted cholesterol efflux in THP-1 macrophages and 22(R)-hydroxycholesterol increased cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner and DIDS inhibited cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner. Exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and DIDS at different concentration for 24 hours, resulted in increase and decrease in the expression of ABCA1 mRNA in THP-1 macrophage-derived foam cells in a dose-dependent manner, respectively.CONCLUSION:ABCA1 playes an important role in cholesterol efflux in THP-1 macrophage-derived foam cells.     

Effects of ghrelin on the expression of acyl coenzyme A:cholesterol acyltransferases-1 during formation of foam cells

AIM: To investigate the effects of ghrelin on the expression of acyl coenzyme A:cholesterol acyltransferases-1 (ACAT-1) during the formation of foam cells. METHODS: The human monocytic leukemia cell line THP-1 was used in the study. The differentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate 13-acetate (PMA). Macrophages were incubated with oxidized LDL (ox-LDL) to generate foam cells. Ghrelin of different concentrations were used during the formation of foam cells. The ACAT-1 protein and mRNA levels were detected by Western blotting and RT-PCR. The variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer. RESULTS: Ghrelin reduced the content of cholesterol ester in foam cells obviously. ACAT-1 protein and mRNA levels were also decreased. Ghrelin reduced ACAT-1 protein mass and mRNA level in a dose-dependent manner. CONCLUSION: Ghrelin might retard the formation of atherosclerosis via down-regulating the expression of ACAT-1.     

Effect of ginkgolide B on cholesterol content in PC12 cells under high cholesterol culture

AIM: To explore the effect of ginkgolide B (GB) on high cholesterol-induced dysfunction in PC12 cells. METHODS: The rat brain microvascular endothelial cells and astrocytes were used to establish the blood-brain barrier (BBB) model by in vitro co-culture in the Transwell device with PC12 cells. Cholesterol at the concentration of 40 μmol/L was added into the side of brain microvascular endothelial cells of the Transwell. Various concentrations of GB were applied to deal with the culture system. The morphological changes of the cells were observed under microscope. The safe doses of GB were screened by MTT assay. The level of cholesterol in the PC12 cells was determined by the high-performance liquid chromatography. RESULTS: The morphology of PC12 cells in control group was normal,with round,short spindle or triangle shape,cytoplasm extension,two-pole short processes,good refraction and obvious nucleus.The doses of GB determined by MTT assay were 1 μmol/L for low dose,5 μmol/L for middle dose and 25 μmol/L for high dose. Compared with control group, the cholesterol obviously increased in model group with statistical difference (P<0.05). GB at dose of 25 μmol/L for 16 h exposure had the optimum results to decrease the cholesterol level in PC12 cells. However, no significant difference between GB high dose group and simvastatin group was observed (P>0.05). CONCLUSION: The active monomer of ginkgo leaf, GB, may effectively reduce the cholesterol content in PC12 cells with the condition of BBB in vitro.     

Effects of angiotensin Ⅱ on ATP binding cassette transporter A1 in THP-1 derived foam cells

AIM:To study the influence of angiotensin Ⅱ (AngⅡ) on ATP-binding cassette transporter A1 in THP-1 derived foam cells. The variance of the expression of ABCA1, the content and the effluent rate of cholesterol were also investigated. METHODS:The regulatory effect of AngⅡ on the expression of ABCA1 mRNA and protein in THP-1 derived form cells were measured by RT-PCR and Western blotting. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer, cholesterol effluent was measured by liquid scintillator. RESULTS:A positive facilitative effect of Ang Ⅱon form cells was observed. Total cholesterol content were increased significantly by Ang Ⅱ treatment (P<0.05). The mRNA and protein of ABCA1 were down-regulated significantly by Ang Ⅱ stimulation (P<0.05). Irbesartan reduced the total cholesterol content significantly (P<0.05). Meanwhile, the increase in the effluent rate of cholesterol and the expression of ABCA1 were observed (P<0.05). CONCLUSION:The effects of Ang Ⅱ on the formation of foam cells and atherosclerosis may be correlated to the activation of AT1 receptor and down-regulation of ABCA1.     

Effect of curcumin on the proliferation and apoptosis in LNCap cells

AIM:To observe the effect of curcumin on the proliferation and apoptosis of prostate cancer cell line LNCap. METHODS:LNCap cells were treated with different doses (10 μmol/L, 20 μmol/L, 30 μmol/L, 40 μmol/L) of curcumin and its effects were analyzed in cell growth and apoptosis by microscope, MTT colorimetric assay and flow cytometry. The expression of prostate specific antigen (PSA) was measured by AXSYMTM system-chemical luciferase methods and expression of androgen receptor (AR) was detected by Western blotting. RESULTS:The results showed that curcumin inhibited the proliferation of LNCap cells. The cell growth was inhibited by curcumin in a dose dependent manner and the optimal dose and time was 40 μmol/L, 24 h. Curcumin induced apoptosis in LNCap cells, the most dramatic dose was 40 μmol/L curcumin, at this dose the apoptosis rate was 9.23%. Curcumin inhibited the expression of PSA in LNCap cells and the most dramatic dose and time was 40 μmol/L, 24 h. The PSA in this group was 20% of the control group. Curcumin inhibited the expression of AR on prostate cancer cells. CONCLUSION:Curcumin decreases proliferation and induces apoptosis in LNCap cells in a dose-dependent manner. Curcumin also inhibites the expression of PSA and AR on LNCap cells.     

Progress on role of exosomes derived from leukemia cells

Exosomes are bilayer-lipid membrane nanovesicle from almost all living cell types which are involved in intercellular substance transporting and signaling communication. Exosomes are 30~120 nm in diameter, can transfer bioactive molecules including DNA, RNA, microRNA, protein as well as lipids derived from parents' cells to recipient cells by body fluids, and specifically influence their physiological or pathological conditions. Leukemia is due to malignant proliferation of hematopoietic stem and progenitor cells. It was reported that leukemia cells derived exosomes play a key role in disease progression, drug resistance, and predict prognosis. This paper will outline the role of exosomes derived from leukemia cells and provide important information to help explore the molecular pathogenesis, biomarker as well as therapeutic target of leukemia.     

Effect of FAK-related non-kinase on apoptosis in hepatic stellate cells

AIM: To evaluate the inhibitory effect of FRNK on the phosphorylation of FAK and apoptosis in hepatic stellate cells (HSCs). METHODS: After stimulated with fibronectin, HSCs was transfected with FRNK plasmid by cationic liposome method. The apoptosis of FRNK-induced HSCs was examined by Annexin-V/propidium iodide double-labeled flow cytometry (FCM), gel electrophoresis and transmission electron microscope. The protein levels of FRNK, FAK and p-FAK (Tyr397) in HSCs were assayed by Western blotting, and RT-PCR was used to detect the expression of mRNA. RESULTS: The expression of FRNK was enhanced and the phosphorylation of FAK was inhibited after FRNK was transiently transfected into HSCs in vitro. The apoptotic rate in HSCs exposed to FRNK plasmid for 48 h was higher than that in the non-FRNK plasmid group ［(25.37±1.92) % vs (9.28±1.05) %, P<0.01］, and accompanied by a significant increase in caspase-3 activity both in the protein and in the mRNA level ［(264.17±12.60 vs 185.82±9.69), P<0.01; (4.19±0.48 vs 1.07±0.27), P<0.01］. CONCLUSION: In HSCs, the expression of FRNK is enhanced and the phosphorylation of FAK is inhibited after FRNK transfection. FRNK induces the HSCs apoptosis.     

Regulation of ghrelin on expression of ATP-binding cassette transporter A1/G1 in THP-1 derived foam cells

AIM: To investigate the regulation of ghrelin on the expression of ATP-binding cassette transporter A1 and G1 (ABCA1/ABCG1)during the foam cell formation. METHODS: The human monocytic leukemia cell line (THP-1)was chosen in our study. The differentiation of THP-1 cells into macrophages was induced by using phorbol myristate acetate (PMA). Macrophages were then incubated with oxidized LDL (ox-LDL)to generate foam cells. Ghrelin of different concentrations were treated at different time points during foam cell formation. The ABCA1/ABCG1 protein and mRNA levels were detected by Western blotting and RT-PCR. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer. RESULTS: Ghrelin reduced the content of lipid droplet in foam cells, and increased the efflux of intracellular cholesterol significantly. Ghrelin increased ABCA1 protein mass and mRNA level in dose-dependent manner. The changes of ABCG1 protein and mRNA level were the same as ABCA1. CONCLUSION: Ghrelin interfere atherosclerosis by up-regulating the expression of ABCA1 and ABCG1.     

Mast cells derived from stem cells of umbilical cord blood

Mast cells (MCs) play a key role in the pathogenesis of allergic diseases. Tissue MCs are originated from hematopoietic stem cells in bone marrow. In recent years, it was reported that human mast cells could be differentiated from stem cells of umbilical cord blood. In this review, we summarize the development in this novel area.     

Effect of homocysteine on the apoptosis of cultured human umbilical vein endothelial cells

AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.     

Effect of acyl coenzyme A: cholesteryl acyltransferase 1 antisense oligonucleotides on the formation of foam cells

AIM: To study the effect of acyl coenzyme A: cholesteryl acyltransferase 1 (ACAT1) antisense oligonucleotides on the formation of foam cells (FC). METHODS: THP-1 cells were cultured and differentiated into macrophages (MP) by phorbol myristate acetate (PMA). Over-expressing ACAT1 gene THP-1 cells were constructed. The ACAT1 antisense and missense oligonucleotides conducted by LipofectamineTM 2000 were incubated with above cells. Ac-LDL was added 6 h later and incubated for 24 h. The expression of ACAT1 protein was detected by Western blotting. The ACAT activity was measured by quantifying the incorporation of ［1-14C］ oleoyl CoA into cholesteryl esters. The formation of foam cells was detected by oil red O staining. RESULTS: The ACAT1 antisense oligonucleotides inhibited the activity of ACAT in macrophages and over-expressing ACAT1 gene THP-1 cells. It also inhibited the formation of foam cell in macrophages and over-expressing ACAT1 gene THP-1 cells with lipid loading. The missense oligonucleotides did not show the inhibitory effects. CONCLUSION: The ACAT1 antisense oligonucleotides inhibit the activity of ACAT and the formation of foam cells.     

Effect of fucoxanthin on growth and apoptosis of HSC-T6 cells

AIM: To explore the effect of fucoxanthin (Fu) on the growth and apoptosis of HSC-T6 cells. METHODS: HSC-T6 cells were divided into blank control group, negative control group and drug groups (treated with different concentrations of Fu). The cell viability was detected by CCK-8 assay at 24 h, 48 h and 72 h after Fu treatment. The cell cycle distribution and apoptotic rate were analyzed by flow cytometry. The protein expression of Bcl-2 and Bax were detected by Western blot. RESULTS: Compared with blank control group, the viability of HSC-T6 cells was inhibited by Fu at concentrations of 15~75 μmol/L in a dose- and time-dependent manner (P < 0.01). The cell ratio of G₁ phase was significantly decreased (P < 0.01) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.01) in 60 μmol/L Fu group after 24 h. The cell ratio of G₁ phase was significantly decreased (P < 0.05) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.05) in 15 μmol/L and 30 μmol/L Fu groups in a dose-dependent manner after 48 h. The early cell apoptotic rates and total cell apoptotic rates were significantly increased in the Fu treatment groups in a dose-dependent manner (P < 0.05). The protein expression of Bax was significantly increased in the Fu treatment groups and the protein expression of Bcl-2 was significantly decreased in 30 μmol/L and 60 μmol/L Fu groups (P < 0.05).CONCLUSION: Fu inhibits the growth of HSC-T6 cells possiblely via arresting the cell cycle at S phase and G₂ phase. The apoptosis of HSC-T6 cells induced by Fu might be via down-regulating the protein expression of Bcl-2 and up-regulating the protein expression of Bax.     

Effect of lipopolysaccharide on apoptosis of human umbilical vein endothelial cells

AIM: To explore the mechanism of lipopolysaccharide (LPS) on vascular endothelial cell(VEC) damage. METHODS: By using cytometry techniques, we studied the effects of LPS on apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: LPS was able to induce apoptosis of HUVECs in a time-dose-dependent fashion.CONCLUSION:Apoptosis might play a role in LPS-induced damage of vascular endothelial cells.