Effects of intracellular expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein on proliferation and apoptosis of mouse macrophages

AIM：To establish Mycobacterium tuberculosis culture filtrate protein 10 (CFP10)-early secretory antigenic target 6 (ESAT6) eukaryotic expression vector and investigate the effect of intracellular expression of CFP10-ESAT6 fusion protein on the proliferation and apoptosis of mouse macrophages (RAW264.7 cells). METHODS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was constructed by inserting CFP10-ESAT6 fusion gene into eukaryotic expression vector pEGFP-N1, and then transfected into RAW264.7 cells to express CFP10-ESAT6 fusion protein. The viability of RAW264.7 cells was measured by MTT assay. Flow cytometry was applied to measure the apoptotic rate and Toll-like receptor 2 (TLR2) expression in RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein or staurosporine. RESULTS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was successfully constructed and transfected into RAW264.7 cells. Compared with the control cells, intracellular expression of CFP10-ESAT6 fusion protein did not affect the viability of RAW264.7 cells, but could inhibit the apoptosis of RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein. Moreover, CFP10-ESAT6-expressing macrophages had markedly lower expression of TLR2 on the surface. CONCLUSION：Intracellular expression of CFP10-ESAT6 fusion protein has no cytotoxicity on mouse macrophages, but can inhibit the apoptosis of the macrophages treated with Mycobacterium tuberculosis 19 kD lipoprotein through down-regulating the expression of TLR2.     

Adipophilin induces expression of inflammatory factors through ERK1/2-AP-1 signaling pathway

AIM: To observe the effects of adipose differentiation-related protein (adipophilin) on the expression of inflammatory factors in RAW264.7 macrophage and to clarify the related mechanism. METHODS: The cell models with high expression and low expression of adipophilin were constructed by transfecting PA317 packaging cells with stable high or low expression adipophilin retroviral vectors into the RAW264.7 cells. The concentrations of IL-6, MCP-1 and TNF-α in the cell culture medium were detected by ELISA. The protein levels of AP-1, p-AP-1, ERK1/2 and p-ERK1/2 were measured by Western blot. The protein levels of adipophilin, p-ERK1/2 and p-AP-1 and the releases of the inflammatory factors in the RAW264.7 cells treated with or without ERK1/2 inhibitor PD98059 or AP-1 inhibitor curcumin were determined. RESULTS: The RAW264.7 cells with high expression of adipophilin had higher levels of IL-6, MCP-1 and TNF-α, and higher protein levels of p-AP-1 and p-ERK1/2 than those in the cells with low expression of adipophilin. ERK1/2 inhibitor had no significant effect on the expression of adipophilin, but the protein expression of ERK1/2 and AP-1 was significantly inhibited (P<0.05). The administration of AP-1 inhibitor curcumin had no significant effect on the protein expression of adipophilin and ERK1/2, but the protein expression of AP-1 was significantly inhibited (P<0.05). At the same time, the releases of inflammatory factors IL-6, MCP-1 and TNF-α were significantly decreased. CONCLUSION: Adipophilin may regulate the expression of inflammatory factors through ERK1/2-AP-1 pathway in RAW264.7 macrophages.     

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.     

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.     

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.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

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.     

Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7

AIM: To establish a cell line of stable silencing of P2X₇ receptor (P2X₇R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X₇R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X₇R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X₇R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X₇R at mRNA and protein levels was down-regulated by 80% in sh P2X₇R group compared with negative control (NC) plasmid transfection. In addition, P2X₇R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X₇R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X₇R expression was successfully established. P2X₇R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.     

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。     

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.     

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.     

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.     

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 autophagy inhibitor bafilomycin A1 on macrophages polarization

AIM:To investigate the effect of bafilomycin A1 (Baf A1) on polarization in mouse macrophages RAW264.7. METHODS:The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS:The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α (TNF-α) was increased significantly after Baf A1 intervention (P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR (M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group (P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group (P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated (P<0.05). For autophagy activator rapamycin (Rapa) treated group, autophagosome formation was also significant increased (P<0.05), but the expression of P62 was notable down-regulated (P<0.05). CONCLUSION:Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.     

Dengue virus type 2 induces apoptosis of RAW264.7 cells and effect of apoptosis on virus replication

AIM:To explore the apoptotic pathway in dengue virus type 2 (DENV2)-infected RAW264.7 cells and to analyze the effect of apoptosis on virus replication. METHODS:RAW264.7 cells were infected with DENV2. MTT assay was used to detect the cell viability. Apoptosis was assessed by Hoechst 33342 staining and Annexin V-FITC/PI staining. The expression levels of caspase-3 and caspase-8 were determined by Western blotting. The activity of caspase-9 was measured with a colorimetric kit. Mitochondrial membrane potential was evaluated using JC-1 fluorescent staining. TCID50 was used to estimate the infectious virion concentration after using Z-VAD-FMK to inhibit apoptosis. RESULTS:The viability of RAW264.7 cells decreased after DENV2 infection at 24 h, 36 h and 48 h. Karyopyknosis in dengue virus-infected cells was observed. The protein levels of caspase-3 and caspase-8 and the activity of caspase-9 increased in the apoptotic cells after dengue virus infection. Mitochondrial membrane potential was reduced after dengue virus infection. There was a higher virion concentration in the cell culture medium after inhibition of apoptosis. CONCLUSION: Dengue virus induces apoptosis of RAW264.7 cells. Apoptotic inhibition of RAW264.7 cells facilitates the production of dengue virus.     

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.     

LRRK2/NF-κB signaling modulates inflammatory responses in BCG-infected RAW264.7 macrophages

AIM: To investigate the biological function and potential mechanism of leucine-rich repeat kinase 2 (LRRK2) in RAW264.7 macrophages during Mycobacterium tuberculosis infection. METHODS: The bacillus Calmette-Guerin (BCG)-infected RAW264.7 cell model was established. Colony-forming unit (CFU) analysis was used to determine the mycobacterial viability. The releases of interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) in the RAW264.7 cells were detected by ELISA. qPCR and Western blot were used to measure the mRNA and protein expression levels, respectively. RESULTS: LRRK2 was robustly enhanced in the RAW264.7 cells in response to BCG infection. Additionally, silencing of LRRK2 suppressed intracellular growth of mycobacteria during BCG challenge. Moreover, silencing of LRRK2 dramatically attenuated the accumulation of inflammatory cytokines IL-1β, IL-6 and IFN-γ induced by BCG infection. More importantly, LRRK2 modulated BCG-induced inflammatory responses by positively regulating the nuclear factor-κB (NF-κB) signaling pathway. CONCLUSION: LRRK2/NF-κB signaling pathway positively modulates inflammatory responses during BCG infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.     

Hydrogen inhibits C/EBP homologous protein-mediated macrophage apoptosis by activating autophagy

AIM: To investigate the protective effect of hydrogen (H₂) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H₂-saturated medium was added to murine RAW264.7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine (3-MA) and 3 μmol/L rapamycin (Rap) for 1 h, and then treated with ox-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining, respectively. The activity of lactate dehydrogenase (LDH) in medium was detected. The protein levels of beclin-1 (a molecular marker of autophagy) and C/EBP homologous protein (CHOP, a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability, LDH leakage, apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation, and LC3 granulation, and this promotion effect of hydrogen was inhibited by 3-MA (an autophagy inhibitor) and further enhanced by Rap (an autophagy inducer). Moreover, the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis, reduction of cell viability and CHOP upregulation were also blocked by 3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages, as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation, and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression, and the upstream mechanism may partially involved in the activation of autophagy.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.