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.     

Allicin attenuates macrophage-derived foam cell apoptosis by inhibiting caspase-12

AIM: To investigate the inhibitory effect of allicin on apoptosis and caspase-12 activation of macrophage-derived foam cells, and to elucidate the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with allicin (12.5, 25 and 50 mg/L) or 4-phenylbutyric acid (PBA, 4 mmol/L) for 1 h and then treated with oxidized low-density lipoprotein (ox-LDL, 100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were examined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of caspase-3 in the cells and lactic dehydrogenase (LDH) in the medium were measured. The protein levels of caspase-12 were determined by Western blot. The intracellular lipid accumulation was measured with oil red O staining and the content of intracellular total cholesterol was determined by enzymatic colorimetry. RESULTS: Similar to the endoplasmic reticulum stress (ERS) inhibitor PBA, allicin inhibited ox-LDL-induced injury of RAW264.7 macrophages in a concentration-dependent manner, as determined by the increased cell viability and the decreased LDH leakage, apoptosis and caspase-3 activity. The decrease in cell viability and increases in LDH leakage and apoptosis induced by TM (an ERS inducer) were also suppressed by allicin. Moreover, similar to PBA, allicin remarkably inhibited ox-LDL- or TM-induced activation of caspase-12. Furthermore, allicin remarkably attenuated ox-LDL-induced lipid accumulation in the RAW264.7 cells and foam cells formation in a concentration-dependent manner. CONCLUSION: Allicin may inhibit macrophage-derived foam cell apoptosis induced by ox-LDL, and the mechanism is partially related to suppressing the activation of caspase-12.     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

Effect of quercetin on ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages

AIM：To investigate the effect of quercetin (QUE) preconditioning on oxidized low-density lipoprotein (ox-LDL)-induced lipid accumulation and peroxidation in mouse RAW264.7 macrophages and the underlying molecular mechanisms. METHODS：RAW264.7 cells were pretreated with different concentrations (20, 40 and 80 μmol/L) of QUE for 30 min and then treated with ox-LDL (100 mg/L) for 24 h. Intracellular lipid droplets were assayed by oil red O staining. Extracellular lactate dehydrogenase (LDH) and malondialdehyde (MDA) and intracellular reactive oxygen species (ROS) were determined to characterize the membrane integrity and the lipid peroxidation, respectively. The mRNA and protein levels of CD36, an important scavenger receptor which mediates ox-LDL uptake, were examined by real-time PCR and Western blotting, respectively. RESULTS：Pretreatment with QUE (20, 40 and 80 mmol/L) significantly attenuated ox-LDL-induced lipid accumulation in RAW264.7 cells and foam cell formation in a dose-dependent manner. Ox-LDL induced LDH release in RAW264.7 cells. This cytotoxic effect was significantly inhibited by QUE pretreatment. Compared with ox-LDL group, the intracellular ROS content and MDA level in culture medium decreased markedly in QUE group. In addition, pretreatment with QUE attenuated ox-LDL-induced up-regulation of CD36 at mRNA and protein levels. CONCLUSION: QUE inhibits ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages and the mechanism may partially involve its ability to down-regulate CD36 expression.     

ox-LDL increases endothelial lipase in RAW264.7 macrophages

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the expression of endothelial lipase (EL) in murine RAW264.7 macrophages. METHODS: RAW264.7 cells were incubated with ox-LDL at concentrations of 0~100 mg/L for 24 h.On the other hand, the cells were incubated with or without PDTC (NF-κB inhibitor) for 30 min and then with ox-LDL (50 mg/L) for 24 h. The expression of EL and p65 was detected by Western blotting. RESULTS: The level of EL was significantly increased after ox-LDL incubation in RAW264.7 cells (P<0.05). NF-κB was activated by ox-LDL at concentration of 50 mg/L for 15~30 min in RAW264.7 cells. The increase in EL induced by ox-LDL was markedly inhibited by a NF-κB inhibitor PDTC (P<0.05). CONCLUSION: ox-LDL significantly increases the expression of EL in RAW264.7 macrophages, which is possibly related to NF-κB activation.     

Effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia

AIM:To investigate the effects of sodium ferulate on cholesterol and triglyceride metabolism in atherosclerosis with hyperlipidemia. METHODS:The rabbit model of atherosclerosis was produced by feeding high lipid forages. RAW264.7 foam cell and HepG2 injured cell models were established by incubation with oxidized low density lipoprotein (ox-LDL). The atherosclerotic plaque area was measured, and serum lipids were detected. The cellular lipid accumulation was examined by oil red O staining. The cellular contents of total cholesterol and cholesterol ester were quantified by high performance liquid chromatography. The hepatic lipase (HL) mRNA expression was determined by RT-PCR. RESULTS:(1) Compared with hyperlipid group, the aorta atherosclerosis plaque area and the serum triglyceride level were significantly decreased in sodium ferulate-treated rabbits, but the serum cholesterol level showed little change. (2) Compared with ox-LDL group, the HL mRNA expression in HepG2 cells was enhanced significantly in sodium ferulate-treated group, but the cellular contents of total cholesterol and cholesterol ester in RAW264.7 foam cells showed little change. CONCLUSION:Sodium ferulate inhibits the formation of atherosclerotic plaque in high-cholesterol-fed rabbits aorta. This antiatherosclerotic function may reduce serum triglyceride level through enhancing the expression of HL mRNA without influencing serum cholesterol level and foam cell formation.     

Ethanol extract of propolis protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting caspase-12

AIM: To investigate the inhibitory effect of ethanol extract of propolis (EEP) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with EEP (7.5, 15 and 30 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium (DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The activity of superoxide dismutase (SOD), and the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the cells were measured. The protein levels of caspase-12, a proapoptotic molecule under endoplasmic reticulum stress (ERS), were examined by Western blot analysis. RESULTS: Like PBA (an ERS inhibitor), EEP protected RAW264.7 macrophages from ox-LDL-induced injury in a dose-dependent manner, as assessed by the increased cell viability and the decreased apoptotic rate. The decrease in cell viability and increase in apoptotic rate induced by TM, an ERS inducer, were also attenuated by EEP. Moreover, EEP suppressed ox-LDL-induced oxidative stress as revealed by the decreased generation of ROS and MDA as well as elevated SOD activity, which were similar to DPI, an oxidative stress inhibitor. Furthermore, EEP significantly suppressed ox-LDL- or TM-induced activation of caspase-12. Similar results were observed in the cells pretreated with PBA or DPI and then treated with ox-LDL. CONCLUSION: EEP may protect RAW264.7 macrophages from ox-LDL-induced apoptosis and the mechanism is at least partially involved in the ability of EEP to suppress oxidative stress and subsequent activation of caspase-12.     

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.     

Foam cells can be induced by oxidized low density lipoprotein in human monocyte-derived dendritic cells

AIM: The purpose of this study is to investigate the mechanisms related to oxidized low-density lipoprotein (ox-LDL) and dendritic cells (DCs) in the process of atherosclerosis.METHODS: Human DCs were prepared from human CD14+ peripheral blood monocytes using rhGM-CSF (100 μg/L) and rhIL-4 (40 μg/L).Cells were incubated with 100 mg/L native or oxidized LDL for 72 h.The formation of foam cells was investigated by electron microscopy and oil red O staining.Phenotypic and immune functional assays were used with FACS, FITC-dextran phagocytosis, allogeneic mixed T lymphocytes reaction and secretion of Th1/Th2 (IL-12/IL-2) cytokines were also conduced.RESULTS: DCs treated with ox-LDL, but not native LDL were induced into foam cells after cultured for 72 h.Compared with native LDL, ox-LDL-treated DCs were less potent in FITC-dextran phagocytosis.ox-LDL promoted allogeneic T cells proliferation.Moreover, ox-LDL upregulated CD80 (72.4± 9.6 vs 89.5±10.1, P<0.01), CD86 (67.2±8.8 vs 80.2±11.6, P<0.01), HLA-DR (80.6±9.8 vs 86.6±10.8, P<0.01) and CD1a (40.2±10.3 vs 60.2±9.3, P<0.01) expressions, increased IL-12 secretion ［(44.3±8.9)ng/L vs (65.1±10.4)ng/L, P<0.05］ in DCs.However, the secretion of IL-2 was decreased ［(43.6±7.8)ng/L vs (10.0±4.5 )ng/L, P<0.01］ significently.CONCLUSION: DCs were induced into foam cells by ingesting ox-LDL with some functional characteristic of mature DC.DCs seem to be a new source of foam cells and play a key role in immunopathogenesis of atherosclerosis.     

Inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on scavenger receptor A1 in macrophage-derived foam cells

AIM:To investigate the inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on the scavenger receptor A1 (SR-A1) in macrophage-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL). METHODS:RAW264.7 cells were pretreated with different concentrations (12.5, 25 and 50 mg/L) of D-4F or 50 mg/L inactive control peptide scrambled D-4F (sD-4F) for 1 h or endoplasmic reticulum stress (ERS) inhibitor 4-phenylbutyric acid (5 mmol/L) for 30 min, followed by the treatment with 100 mg/L ox-LDL for 12 h. In addition, the cells were pretreated with 50 mg/L D-4F or sD-4F for 1 h, and then stimulated with 2 mg/L tunicamycin (TM; an ERS inducer), for 4 h. The viability of the cells was measured by MTT assay, and the content of intracellular total cholesterol (TC) was measured by a tissue/cell TC assay. The protein and mRNA levels of SR-A1 and glucose-regulated protein 78 (GRP78) were analyzed by Western blotting and quantitative real-time PCR, respectively. The fluorescence intensity of DiI-ox-LDL in the cells was detected by a multifunctional microplate reader. RESULTS:D-4F significantly reduced ox-LDL-induced macrophage injury and intracellular cholesterol accumulation, and attenuated the ox-LDL-induced expression of SRA1 and GRP78 in a dose-dependent manner. Additionally, D-4F significantly inhibited the TM-induced protein expression of SR-A1 and GRP78, and attenuated the uptake of ox-LDL by macrophages. CONCLUSION: D-4F reduces ox-LDL-induced macrophage cholesterol accumulation and injury by inhibiting SR-A1 expression. The mechanism may be related to the inhibition of ERS signaling pathway mediated by GRP78.     

Low density lipoprotein from patients of diabetes mellitus induces apoptosis of murine macrophages via activating caspase-12 pathway

AIM:To explore the effect of low density lipoprotein from the patients of diabetes mellitus (DM-LDL) on the activation of caspase-12 an important molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, in the murine macrophages, and to clarify the underlying molecular mechanisms of apoptosis. METHODS:Murine macrophage RAW264.7 was exposed to DM-LDL (25, 50 and 100 mg/L), normal low density lipoprotein (n-LDL, 50 mg/L), or tunicamycin (TM, 4 mg/L) for 24 h. Additionally, RAW264.7 macrophages were precultured with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then exposed to DM-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were detected by MTT assay and flow cytometry with Annexin V-FITC/propidium iodide staining, respectively. Lactate dehydrogenase (LDH) activity in the media was measured by assay kit. The protein level of caspase-12 was determined by Western blot. RESULTS:Similar to TM (an ERS inducer), treatment with DM-LDL caused significantly decrease in the viability and increase in LDH activity in the media and apoptotic rate of the RAW264.7 macrophages (P<0.05). Additionally, DM-LDL induced activation of caspase-12 especially at the dose of 50 and 100 mg/L (P<0.01). However, the ERS inhibitor PBA protected RAW264.7 macrophages from DM-LDL-induced decrease in viability and increase in LDH activity and apoptosis (P<0.05). Furthermore, PBA attenuated DM-LDL-induced activation of caspase-12 (P<0.05). CONCLUSION:DM-LDL may induce apoptosis in RAW264.7 macrophages, and the mechanism may be related to the activation of caspase-12.     

Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting caspase-12

AIM: To investigate the effect of D4F, an apolipoprotein A-I mimetic peptide, on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium (DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respectively. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in the cells and the activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined. The protein level of caspase-12 was examined by Western blot analysis. RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM (an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner. Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced oxidative stress, as expressed by the decreased generation of ROS and MDA (P <0.01), the increased activity of SOD and the decreased activity of NADPH oxidase (P <0.05). Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner (P <0.05). Furthermore, D4F also inhibited the caspase-12 activation induced by TM (P <0.05). CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.     

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid (PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of lactate dehydrogenase (LDH) in the medium and caspase-3 in the cells were determined by detection kits. The protein levels of beclin-1 (a molecular marker of autophagy), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein (CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis) were examined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope.RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability, and dramatic elevation in LDH leakage, cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autophagy inducer). ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap. Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL. Moreover, PBA (endoplasmic reticulum stress inhibitor) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3. CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages, and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment, the formation of foam cells was examined by oil red O staining. The cholesterol efflux, cholesterol level, free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay, total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins, including CD36, scavenger receptor class A （SR-A）, ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）, were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5, 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）, and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level, cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）, promoted efflux of intracellular cholesterol （P<0.01）, and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L, Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux, and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.     

Oxidized low-density lipoprotein induces autophagy in macrophages via CD36-mediated oxidative stress

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on autophagy in macrophages and the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with 2 mg/L anti-CD36 monoclonal antibody (anti-CD36 mAb), 5 μmol/L diphenyleneiodonium (DPI), 3 mmol/L 3-methyladenine (3-MA) or 1 μmol/L rapamycin for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The viability of the cells was measured by MTT assay. The activities of lactic dehydrogenase (LDH) in the medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD) in the cells as well as the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were determined to characterize the membrane integrity and the oxidative stress, respectively. The protein levels of beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), 2 important molecular markers of autophagy, were examined by Western blotting. RESULTS: ox-LDL induced autophagy in RAW264.7 macrophages as assessed by upregulation of beclin-1 and LC3-II. Similar to 3-MA, an autophagy inhibitor, anti-CD36 mAb significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Anti-CD36 mAb suppressed the ox-LDL-induced oxidative stress as revealed by decreased NADPH oxidase activation, ROS and MDA generation as well as increased SOD activity. Similar results were observed in the cells pretreated with DPI, a NADPH oxidase inhibitor. Moreover, DPI significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Inaddition, the decrease in the cell viability and increase in LDH release induced by ox-LDL were promoted by 3-MA and blocked by rapamycin (an autophagy inducer). CONCLUSION: ox-LDL induces autophagy in RAW264.7 macrophages, which may be involved in CD36-mediated ox-LDL uptake and subsequent activation of oxidative stress, and moderate activation of autophagy may protect macrophages from ox-LDL-induced injury.     

苹果MLP家族基因鉴定及非生物胁迫响应分析

在苹果中鉴定了13个Major Latex Protein(MLP)家族基因Md MLP。序列比对及构建蛋白同源模型发现,Md MLP蛋白含有Bet_v1典型的Gly-rich loop区域结构,且为MLP家族特有的Gxxxxx G结构。经多物种MLP系统发育及共线性比较分析,Md MLP与其他蔷薇科物种MLP具有相似基因结构和蛋白质保守结构域。q RT-PCR分析表明,Md MLP在‘新疆1号’苹果14个器官组织中均有不同程度的表达,对ABA、Na Cl、PEG、低温(4℃)和高温(40℃)有一定响应,且同一亚族基因表达情况呈现相似趋势。String构建蛋白互作网络发现,Md MLP可能通过与PRSP、SNRK1/2、b HLH等应激、ABA相关转录蛋白互作,参与苹果对非生物胁迫的防御。     

Cordyceps militaris polysaccharide promotes reverse cholesterol transport in CETP-tg mice

AIM To verify whether Cordyceps militaris polysaccharide （CMPS） has the effect of promoting reverse cholesterol transport （RCT） in vitro and in vivo, and to explore the underlying mechanism. METHODS For in vivo experiments, RCT efficiency was detected in cholesterol ester transporter transgene （CETP-tg） mice by isotope tracer technique, and the plasma lipid levels were measured by enzyme method. For in vitro experiments, the residual lipid content after cholesterol efflux in RAW264.7 macrophage-derived foam cells was tested by oil red O staining and total cholesterol （TC） kit. Western blot was used to analyze the protein expression of the molecules involved in cholesterol transport, uptake and transformation in the foam cells and mice liver. RESULTS After 4 weeks of intragastric administration of CMPS, the concentrations of TC, low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C） in the plasma of CETP-tg mice were reduced by 24%, 23% and 22%, respectively. RCT efficiency of CETP-tg mice was accelerated and the appearance of ³H-cholesterol tracer in plasma, bile, intestine and feces was significantly increased in CMPS group. Meanwhile, the expression levels of cholesterol receptors scavenger receptor B1 （SR-B1） and LDL receptor （LDLR）, and cholesterol converting rate-limiting enzyme cholesterol 7α-hydroxylase A1 （CYP7A1） were upregulated by 105%, 71% and 58% in the liver of CMPS group, respectively. The results of in vitro experiments showed that CMPS preincubation promoted cholesterol efflux, decreased intracellular lipid and TC levels, and up-regulated the expression of peroxisome proliferator-activated receptor γ （PPARγ）-liver X receptor α （LXRα）-ATP-binding cassette transporter A1 （ABCA1）/ABCG1 signaling pathway related proteins in macrophage-derived foam cells. CONCLUSION CMPS promotes excess cholesterol efflux from peripheral macrophage-derived foam cells and accelerates its discharge through liver pathway. PPARγ-LXRα-ABCA1/ABCG1 pathway may be involved in the mechanism.     

Effects of rosiglitazone on expression of SOCS1/SOCS3 and inflammatory reaction in RAW 264.7 cell-derived foam cells

AIM: To investigate whether perioxisome proliferator-activated receptor γ (PPARγ) ligand rosiglitazone regulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3 expression as well as pro-inflammatory/anti-inflammatory responses in RAW 264.7 cell-derived foam cells. METHODS: The concentrations of TNF-α, IL-6 and IL-10 in the cultured supernatant of RAW 264.7 cell-derived foam cells were detected by ELISA, and the ratios of TNF-α/IL-10 and IL-6/IL-10 were calculated. RT-PCR and Western blotting were used to analyze the effects of rosiglitazone on the expression of SOCS1 and SOCS3 at mRNA and protein levels. RESULTS: The concentrations of TNF-α, IL-6 and IL-10, and ratios of TNF-α/IL-10 and IL-6/IL-10 in foam cell group were obviously higher than those in control group, but the concentrations of the above factors in oxidized low-density lipoprotein (ox-LDL) +rosiglitazone group were apparently lower than those in foam cell group. The expression of SOCS1 and SOCS3 at mRNA and protein levels in oxLDL+rosiglitazone group was apparently higher than that in control and foam cell group. CONCLUSION: PPARγ ligand rosiglitazone up-regulates the expression of SOCS1 and SOCS3 at mRNA and protein levels and regulates the balance of pro-inflammatory/anti-inflammatory responses in RAW 264.7 cell-derived foam cells.     

Preparation of histones and their effects on macrophages

AIM: To prepare histones with different methods and to observe their effects on cell viability and cytokine release by macrophages in vitro.
METHODS: Prokaryotic expression plasmids of histone H3 and histone H4 were constructed by cloning methods. The histones containing GST-tag or His-tag (GST-H3, GST-H3, His-H3, His-H4) were expressed and purified. The histones from eukaryotic cells (RAW264.7 and 293F cells) were extracted with the high salt method. RAW264.7 cells were treated with histones (7.5~50 mg/L) for 4 h and the cell vitality was examined by MTT assay and flow cytometry. The concentrations of IL-6 and TNF-α in the culture supernatants of RAW264.7 cells treated with histones were also detected.
RESULTS: His-H3/H4 and the histones from eukaryotic cells significantly reduced the viability of RAW264.7 cells and induced apoptosis. The effects of histones from different sources on the releases of IL-6 and TNF-α were different.
CONCLUSION: His-H3/His-H4 expressed by prokaryotic plasmids and the histones extracted from eukaryotic cells affect the vitality of macrophages as well as induce late apoptosis and necrosis. Histone may involve in the inflammatory process by promoting the release of inflammatory cytokines.     

Effect of RIP3 expression on apoptosis of mouse macrophages infected with BCG

AIM:To investigate the function of receptor-interacting proteins 3 (RIP3) in regulating Bacillus Calmette-Guérin (BCG)-induced apoptosis of mouse macrophages (RAW264.7 cells). METHODS:The RIP3 adenovirus interference vector was constructed and used to infect the RAW264.7 cells, and then the RAW264.7 cells were infected with BCG. The cell viability was measured by MTT assay. The apoptotic rate, mitochondrial membrane potential and production of reactive oxygen species (ROS) were determined by flow cytometry analysis. The protein levels of RIP3 and apoptosis-associated proteins were examined by Western blot. RESULTS:The viability of RAW264.7 cells was decreased after BCG infection. In the meantime, the expression of RIP3 was up-regulated significantly (P<0.01). Compared with BCG infection group, the apoptotic rate and ROS level in BCG and RIP3 adenovirus interference vector co-infection group were significantly decreased (P<0.01). Importantly, RIP3 was able to further promote apoptosis in BCG-infected RAW264.7 cells in part by increasing mitochondrial membrane potential (P<0.01). In addition, Western blot analysis further demonstrated that RIP3 was involved in BCG-induced apoptosis partly through down-regulation of anti-apoptotic protein Bcl-2, and up-regulation of Bax and cleaved caspase-3 (P<0.01). CONCLUSION:RIP3 is involved in BCG-induced apoptosis of RAW264.7 cells, and this process may be achieved by the mitochondrial pathway.