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.     

Dithiothreitol induces apoptosis of BRL-3A cells by activation of calpain-2/caspase-12 signaling pathway

AIM: To investigate the expression changes of glucose-regulated protein 78 (GRP78), calpain-2, caspase-12 and caspase-3 in dithiothreitol (DTT)-induced endoplasmic reticulum stress in rat normal liver cell line BRL-3A, and to explore their effects on apoptosis of BRL-3A cells. METHODS: BRL-3A cells were treated with 2.5 mmol/L DTT to induce endoplasmic reticulum stress. The mRNA expression of GRP78, calpain-2, caspase-12 and caspase-3 was detected by real-time PCR. The protein expression of GRP78, calpain-2, caspase-12 and caspase-3 was detected by cell immunofluorescence technique. The protein levels of cleaved caspase-12 and cleaved caspase-3 were determined by Western blot. The apoptosis of BRL-3A cells was analyzed by flow cytometry. RESULTS: After treatment with DTT for 12 h and 24 h, the mRNA expression of GRP78, calpain-2 and caspase-12 in the BRL-3A cells was obviously increased compared with normal control group (P<0.01), and no significant change of caspase-3 mRNA after treatment with DTT for 12 h and 24 h was observed. The results of immunofluorescence technique and Western blot showed that the protein levels of GRP78, calpain-2, caspase-12, cleaved caspase-12, caspase-3 and cleaved caspase-3 were obviously elevated after treatment with DTT for 12 h and 24 h(P<0.05). In addition, the increased apoptotic rate was also found in the BRL-3A cells treated with DTT for 12 h and 24 h (P<0.05). CONCLUSION: The activation of calpain-2/caspase-12 signaling pathway may be involved in apoptosis of BRL-3A cells induced by DTT.     

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.     

Effects of fructose sodium diphosphate on expression of CHOP and JNK in endoplasmic reticulum stress and islet apoptosis in rats with type 2 diabetes

AIM: To study the effects of fructose sodium diphosphate (FDP) on the expression of CHOP and c-Jun N-terminal binase(JNK) in endoplasmic reticulum stress and islet apoptosis in the rats with type 2 diabetes mellitus (T2DM). METHODS: T2DM model was established in male Wistar rats by feeding of high lipid diet and injection of streptozotocin. The rats were divided into 4 groups (n=8):normal control group, T2DM model group, T2DM+low-dose FDP (2 mL穔g^-1穌^-1, ip) group and T2DM+high-dose FDP (5 mL穔g^-1穌^-1, ip) group. The rats in the treatment groups received FDP for 8 weeks. The levels of fasting blood glucose (FBG), fasting serum insulin (FINS) and insulin sensitivity index (ISI) were measured. TUNEL was used to detect the islet apoptosis. The protein levels of CHOP and JNK were determined by the method of immunohistochemistry. RESULTS: (1) Compared with normal control group, FBG, FINS, the expression of CHOP and JNK, and apoptosis in T2DM model group were significantly increased (P<0.01). The level of ISI was significantly decreased. (2) Compared with T2DM model group, the levels of FBG and FINS, the expression of CHOP and JNK, and apoptosis in high-dose FDP group were significantly decreased. The level of ISI was significantly increased (P<0.01). However, the level of FBG, the expression of CHOP and JNK, and apoptosis in low-dose FDP group were significantly decreased. Compared with low-dose FDP group, the levels of FBG and FINS, the expression of CHOP and JNK, and apoptosis in high-dose FDP group were significantly decreased. The level of ISI was significantly increased (P<0.01 or P<0.05). CONCLUSION: FDP may prevent islet cells from apoptosis in T2DM rats by decreasing the expression of CHOP and JNK.     

Suberoylanilide hydroxamic acid induces apoptosis of HepG2 cells by endoplasmic reticulum stress apoptotic pathway

AIM: To investigate the effect of suberoylanilide hydroxamic acid (SAHA) on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells and to explore its possible mechanism. METHODS: HepG2 cells were treated with SAHA at different concentrations for 48 h. The proliferation of HepG2 cells was detected by real-time cellular analysis. The protein levels of acetylated histones H3K9 and H3K27, glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and p-PERK were determined by Western blot. The cell apoptosis was analyzed by flow cytometry. RESULTS: Compared with control group, treatment with SAHA at 0.1 μmol/L and 1 μmol/L for 48 h showed no significant inhibitory effect on the proliferation of HepG2 cells, while SAHA at 6 μmol/L and 12 μmol/L significantly inhibited the proliferation of HepG2 cells (P<0.05). The results of Western blot showed that the protein levels of acH3K9, acH3K27, GRP78 and p-PERK increased significantly after treated with SAHA at diffe-rent concentrations for 48 h, while the protein level of PERK was decreased significantly (P<0.05). The results of flow cytometry analysis showed that the apoptotic rates of the HepG2 cells increased with the increase in SAHA concentration. CONCLUSION: SAHA up-regulates the acetylation of H3K9 and H3K27 in the HepG2 cells and induces apoptosis of HepG2 cells by activating the endoplasmic reticulum stress-related apoptosis pathway.     

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.     

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.     

Protective effect of quercetin on ER stress-related apoptosis induced by thapsigargin in macrophages

AIM: To investigate the effects and possible mechanisms of quercetin (Que) on endoplasmic reti-culum stress (ERS)-related apoptosis induced by thapsigargin (TG) in RAW264.7 cells. METHODS: ER stress of RAW264.7 cells were induced by TG at concentration of 1 μmol/L for 24 h. After treated with different concentrations of Que (80, 120 and 160 μmol/L), the cell viability was determined by MTT assay.The apoptotic rate and the changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) were determined by flow cytometry, and the cell apoptotic morphology was observed under laser scanning confocal microscope.The protein levels of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by Western blotting. The effect of Que on GRP78 and CHOP induced by TG with phosphatidylinositol 3-kinase (PI3K) inihibitor LY294002 at concentration of 15 nmol/L was measured by Western blotting. RESULTS: Que suppressed ER stress-related injury induced by TG in RAW264.7 cells. Compared with TG group, the cell viability increased (P<0.05), apoptotic rate and [Ca²⁺]_i decreased (P<0.05) and the changes of apoptotic morphology were alleviated. The increase in GRP78 and CHOP induced by TG as an ER stress marker was suppressed by Que (P<0.05). The suppressive effect of Que on GRP78 and CHOP was reproduced by LY294002 (P<0.05), but they failed to exhibit additive suppression. CONCLUSION: Que suppresses the ER stress induced by TG in RAW264.7 cells. The protective effect may be related to its suppression on PI3K signaling pathway.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Effects of diabetes on ischemic/reperfusion injury and cell apoptosis in rats myocardium via alterations in peroxynitrite

AIM: To determine the effects of different-term streptozotocin(STZ)-induced diabetes on ischemia/reperfusion (I/R) injury and cell apoptosis in rats myocardial via alterations in myocardial peroxynitrite.METHODS: The models of I/R injury were induced by occlusion and reperfusion of the left descending coronary artery (LDCA) in rats.I/R-induced infarct size was determined using triphenyltetrazolium chloride (TTC) staining.Quantified caspase-3 expression was used to represent apoptosis by Western blotting analysis.Peroxynitrite formation as indicated by nitrotyrosine level was measured by morphometric analysis.RESULTS: Two weeks after STZ treatment,infarct size (35.00%±3.00%) was smaller in 2 weeks diabetic hearts (2WKD) as compared with time-matched control group (2WKC) (51.00%±3.30%),whereas after 16 weeks of diabetes (16WKD),the infarct size (61.00%±3.00%) was bigger in the diabetic hearts as compared with the 16WKC group (50.00%±2.00%,P<0.05).After I/R,caspase-3 expression was lower in 2WKD+I/R group (A value：481±77) than that in 2WKC+I/R group (A value：1033±46),while caspase-3 was higher in the 16WKD+I/R group (A value：1206±78) than that in 16WKC+I/R group (A value：940±72,P<0.05).Nitrotyrosine was lower in 2WKD hearts (A value：211±13) than that in controls (A value：409±12),but was higher in 16WKD group (A value：506±37) compared with controls (A value：378±46,P<0.05).CONCLUSION: Short- and long-term STZ induced diabetes exerts opposite influences on myocardial I/R injury and cell apoptosis,and these contradictory influences may depend on different alterations in myocardial peroxynitrite.     

Effects of HDL from patients with coronary artery disease on lipid deposition and apoptosis in mouse peritoneal macrophages

AIM: To compare the effects of high-density lipoprotein (HDL) from healthy subjects (HDL_heathy) and HDL from the patients with coronary artery disease (HDL_CAD) on the lipid deposition and apoptosis in mouse peritoneal macrophages. METHODS: HDL was isolated from healthy subjects, stable CAD patients (HDL_SCAD) and acute myocar-dial infarction patients (HDL_AMI). The accumulation of intracellular lipids was determined by oil red O staining. The apoptosis of macrophages was measured by fluorescence microscopy with annexin-V/PI staining. DCHF-DA, a redox-sensitive dye, was used to detect intracellular reactive oxygen species (ROS) levels. The protein expression of ATP-binding cassette transporter (ABC) A1, ABCG1, Bcl-2 and Bax was determined by Western blot analysis. RESULTS: Lipid deposition in the macrophages was increased significantly after oxidized low-density lipoprotein (ox-LDL) treatment, and the expression of ABCA1 and ABCG1 was up-regulated (P<0.05). Compared with ox-LDL treatment alone, HDL_healthy decreased lipid deposition in the macrophages and up-regulated the expression of ABCA1 and ABCG1 (P<0.05), while treatment with HDL_SCAD or HDL_AMI further decreased lipid deposition in the macrophages and down-regulated the expression of ABCA1 and ABCG1 (P<0.05). Compared with HDL_SCAD treatment, lipid deposition in the macrophages was further increased after HDL_AMI treatment, and the expression of ABCA1 and ABCG1 was down-regulated (P<0.05). HDL_healthy decreased the levels of intracellular ROS and apoptosis by increasing the level of antiapoptotic protein Bcl-2 and reducing the expression of proapoptotic protein Bax. In contrast, HDL_SCAD and HDL_AMI had opposite effects on the intracellular ROS, the cell apoptosis and the expression of apoptosis-related proteins Bcl-2 and Bax. CONCLUSION: HDL_CAD promotes lipid accumulation in macrophages and induces macrophage apoptosis. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in CAD.