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.     

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.     

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.     

DENV-2 induces apoptosis of EA.hy926 cells through mitochondrial pathway

AIM：To explore the effect of dengue virus type 2 (DENV-2) infection on the change of mitochondrial membrane potential (Δψm) in EA.hy926 cells. METHODS：The inhibitory effect of DENV-2 infection on EA.hy926 cell growth was examined by MTT assay. The changes of Δψm were analyzed by flow cytometry or observed under fluorescence microscope with JC-1 staining. The activity of caspase-9 was measured by a colorimetric kit. RESULTS：Infection of DENV-2 for 24 h, 36 h and 48 h inhibited the viability of EA.hy926 cells. After DENV-2 infection, the changes of Δψm in EA.hy926 cells were observed. Compared with the normal control cells, Δψm in DENV-2-infected EA.hy926 cells was notably decreased. The activity of caspase-9 increased at early stage after infection of DENV-2 and maintained at a high level at least to 48 h. CONCLUSION：DENV-2 infection decreases the mitochondrial membrane potential and increases the activity of caspase-9 in EA.hy926 cells in the early stage of proliferation, thus promoting the process of apoptosis.     

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.     

Effect of AMD3100 on dengue virus type 2-induced apoptosis in Eahy926 cells

AIM: To investigate the role of AMD3100 (an inhibitor of CXCR4) in dengue virus type 2 (DV2)-induced apoptosis in human umbilical vein endothelial cell line Eahy926. METHODS: The expression of factor Ⅷ in Eahy926 cells was examined by immunohistochemistry staining. The cells were divided into untreated group, DV2 infection group and DV2+AMD3100 group. Flow cytometric analysis was used to detect the expression of CXCR4 in Eahy926 cells 24 h, 36 h, 48 h and 60 h after DV2 infection. In addition, the percentage of apoptotic cells was also analyzed by flow cytometry. Immunofluorescence was performed to detect the phosphatidylserine (PS) on the surface of Eahy926 cells.RESULTS: Eahy926 cells were factor Ⅷ-positive. Compared with untreated group, the expression of CXCR4 increased in DV2 infection group, most markedly 48 h after infection (66.13%±10.30%, P<0.05). The percentage of apoptotic Eahy926 cells after DV2 infection was the highest at 36 h (29.85%±15.78%, P<0.05). The percentage of DV2-induced apoptotic cells in DV2+AMD3100 group was higher than that in DV2 infection group. The green fluorescence-labeled cells in DV2 infection group and DV2+AMD3100 group were more than those in untreated group. CONCLUSION: DV2 infection induces apoptosis and increases the expression of CXCR4 in Eahy926 cells. AMD3100, the inhibitor of CXCR4, may be a promoter of apoptosis in Eahy926 cells after DV2 infection.     

Dengue virus type 2 induces apoptosis and expression of death receptor in hepatic veno-endotheliocyte ED25

AIM: To investigate apoptosis and the expression of death receptors of TRAIL,TNF and Fas on hepatic veno-endotheliocyte ED25 cell strain induced by dengue virus type 2(DV2).METHODS: Flow cytometric analysis was used to detect the number of apoptotic cells and the expression levels of TRAILR1-4 ，TNFR1-2，Fas on ED25 cells before/after DV2 infection.RESULTS: The numbers of apoptotic cells of ED25 increased after DV2 infection,there were only about 5.7%±1.2% of apoptotic cells before virus infection while there were approximately 27.3%±1.6% of apoptotic cells after virus infection.At the same time the expression level of Fas also increased,before virus infection about 44.3%±2.2% of ED25 cells expressed Fas while 63.0%±2.3% of ED25 cells expressed Fas after virus infection.CONCLUSION: DV2 infection can induce apoptosis of ED25 cells,and it suggests strongly that Fas/FasL may be involved in the apoptotic signal transduction.     

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.     

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.     

Zoledronic acid inhibits proliferation and induces apoptosis in U937 cells

AIM: To study the antiproliferation and proapoptotic effects of zoledronic acid(ZOL) on human acute myeloid leukemia cell line U937. METHODS: The viability of U937 cells was detected by CCK-8 assay. The cell cycle of the U937 cells was analyzed by flow cytometry with PI staining. Apoptotic rate was assessed by flow cytometry with Annexin V-PI and Hoechst 33342 staining. Mitochondrial membrane potential was detected by JC-1 assay. Methylcellulose was used to assess colony formation. The protein levels of p21, Bcl-2 and Bax were determined by Western blot. RESULTS: ZOL inhibited the viability of U937 cells. ZOL induced S-phase cell cycle arrest in the U937 cells. The results of flow cytometry analysis with Annexin V-PI and Hoechst 33342 staining showed that ZOL also induced apoptosis in a dose- and time-dependent manner. Mitochondrial membrane potential assay was also used to verify the apoptosis. The apoptotic rate was consistent with the reduction of mitochondrial membrane potential. Colony formation assay showed that ZOL significantly inhibited the colony formation capacity of the U937 cells. This was achieved by the induction of S-phase cell cycle arrest, and up-regulation of Bax and p21, and down-regulation of Bcl-2. CONCLUSION: ZOL inhibits cell proliferation by regulating the expression of cell cycle-related protein, and induces apoptosis via the mitochondrial apoptotic pathway.     

Effect of component II of broccoli polypeptide on glioma cell apoptosis

AIM：To explore the effect of component II of broccoli polypeptide on the apoptosis in glioma cells. METHODS：Human glioma SHG-44 cells were cultured and divided into control group and 3, 10, 30 and 100 mg/L component II of broccoli polypeptide groups. Cell viability was detected by MTT assay. The apoptotic rates were examined by Annexin V/PI staining. The morphological changes of the cells were observed under inverted microscope. The protein expression of Bax and Bcl-2 was detected by immunocytochemistry and Western blotting. The protein level of caspase-3 was also examined by Western blotting. RESULTS：Treatment with component II of broccoli polypeptide for 24 h, 48 h or 72 h induced significant inhibition of viability of SHG-44 cells in a time- and dose-dependent manner. The results of Annexin V/PI staining showed that the apoptotic rates were increased in treatment groups in a dose-dependent manner. The density of glioma cells was decreased after treated with increasing concentrations of the drug, and the apoptotic bodies were observed under inverted microscope at 72 h. The results of immunocytochemistry and Western blotting showed that the expression of Bax protein was increased but Bcl-2 protein expression was decreased, and the ratio of Bax/Bcl-2 was increased significantly compared with control group (P<0.05 or P<0.01). The level of caspase-3 protein was increased in 30 and 100 mg/L component II of broccoli polypeptide groups compared with control group (P<0.01). CONCLUSION：The component II of broccoli polypeptide increases the ratio of Bax/Bcl-2 and activates caspase-3 protein, thus inducing the apoptosis of glioma cells.     

Mycoplasma pneumoniae induces IL-1β production through activating NLRP3 inflammasome by ROS in RAW264.7 cells

AIM: To investigate whether Mycoplasma pneumoniae (Mp)-induced interleukin-1β (IL-1β) production in RAW264.7 cells is through the activation of NLRP3 inflammasome via reactive oxygen species (ROS). ME-THODS: RAW264.7 cells were randomly divided into 3 groups. In normal group, RAW264.7 cells were treated without Mp. In model group, RAW264.7 cells were treated with 1∶ 10 multiplicity of infection (MOI) of Mp. In NAC group, RAW264.7 cells were pretreated with N- acetylcysteine (NAC) at a concentration of 5 mmol/L for 30 min before infection with Mp. The RAW264.7cells were infected with Mp (1∶ 10 MOI) for 4, 8, 16 and 24 h in model group and NAC group, respectively. The intracellular ROS level was analyzed by flow cytometry. The mRNA expressions of NLRP3, ASC and caspase-1 were detected by real-time PCR. The protein levels of NLRP3, ASC and caspase-1 p20 were determined by Western blot. The levels of pro-inflammatory cytokine IL-1β in the supernatant were measured by ELISA. RESULTS: Compared with normal group, the production of ROS were significantly increased at 4, 8, 16 and 24 h after infection, the mRNA expression of NLRP3, ASC and caspase-1 were increased at 8, 16 and 24 h after infection, the protein levels of NLRP3, ASC and caspase-1 p20 were increased at 16 and 24 h after infection, and the releases of IL-1β were increased at 24 h after infection in model group (P<0.01). Compared with the model group, the level of ROS in NAC group decreased, so as the expression of NLRP3, ASC and caspase-1 at mRNA and protein levels and the releases of IL-1β in the supernatant at the corresponding time points. CONCLUSION: Mp may stimulate the ROS production to activate NLRP3 inflammasome in RAW264.7 cells.     

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.     

Effect of arctigenin on apoptosis of human nasopharyngeal carcinoma cell line CNE-1

AIM: To investigate the effect of arctigenin on the apoptosis of human nasopharyngeal carcinoma cell line CNE-1 and its potential mechanism. METHODS: The inhibition of cell viability was analyzed by CCK-8 assay. The activity of caspase-3 and caspase-9 was analyzed by caspase-3 and caspase-9 activity kit. Apoptotic cell percentage was evaluated by Annexin V-PI staining. The expression of PI3K/AKT/XIAP signal pathway-related molecules at mRNA and protein levels was analyzed by real-time PCR and Western blot. RESULTS: Arctigenin inhibited the cell activity in a dose- and time-dependent manner after treatment with arctigenin at concentrations of 10, 20, 40 and 80 μmol/L for 24 h, 48 h and 72 h (P<0.01). Arctigenin also increased the activity of caspase-3 and caspase-9 and the apoptotic rate (P<0.05), and down-regulated the mRNA and protein expression of PI3K/AKT/XIAP signal pathway-related molecules (P<0.05).CONCLUSION: Arctigenin induces the apoptosis of CNE-1 cells through PI3K/AKT/XIAP signal pathway.     

Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay. The apoptotic rate of HepG2 cells was analyzed by flow cytometry. The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining. The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot. RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner. TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h. The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually. CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.     

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.     

Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial BEAS-2B cells

AIM: To explore the effect of hexokinase 2 (HK2) on lipopolysaccharide (LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms.METHODS: BEAS-2B cells were treated with LPS to induce cell injury, and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot. The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment.RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner (P<0.01). The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with z-LEHD-fmk, but not z-IETD-fmk, reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression, the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression.CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells.     

Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.