Protective effect of paeoniflorin on nerve cells in APP/PS1 mice and its mechanism

AIM: To investigate the therapeutic and preventive effects of paeoniflorin (PF) on APP/PS1 mice, and to explore the possible mechanism. METHODS: Fifteen male 5-month-old APP/PS1 non-dominant mice were chosen as normal control group, 15 male 5-month-old APP/PS1 double transgenic mice were used as model group, and 15 male 5-month-old APP/PS1 double transgenic mice treated with 5 mg/kg PF by intraperitoneal injection were allocated in administation group. The learning and memory ability of the mice in each group was detected by Morris water maze. The apoptosis was assessed by TUNEL fluorescence staining. The protein expression of PI3K, Akt, p-PI3K, p-Akt, caspase-3, caspase-9, Bcl-2 and Bax in cerebral cortex and hippocampus was detected by Western Blot. The protein expression levels and distribution of caspase-3 and caspase-9 were detected by immunohistochemistry. RESULTS: (1) Compared with normal control group, the learning and memory ability declined in APP/PS1 model group. Compared with APP/PS1 model group, PF obviously improve the ability of learning and memory in mice. (2) Compared with normal control group, the apoptosis of nerve cells in APP/PS1 model group significantly increased and distributed in wider areas, while that in PF group was reduced (P<0.05). (3) Compared with APP/PS1 model group, PF could significantly lower pro-apoptotic factors, caspase-3, caspase-9 and Bax (P<0.05), and increase the expression of anti-apoptotic factors, p-PI3K, p-Akt and Bcl-2 (P<0.05). CONCLUSION: PF can up-regulate the expression of Bcl-2 and down-regulate the expression levels of caspase-9, caspase-3 and Bax via the activation of PI3K/Akt pathway, thereby inhibiting the nerve cell apoptosis and protecting the nerve cells, so as to treat neurodegenerative diseases.     

Effects of tangeretin on growth and invasion of human non-small-cell lung cancer cells

AIM: To study the influences of tangeretin (TGN) on the growth and invasion of non-small-cell lung cancer (NSCLC) cells, and to explore the molecular mechanisms. METHODS: The A549 cells were treated with different concentrations of TGN in vitro. The relative cell activity was determined by MTT assay. The apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI staining. The number of the invasive cells was measured by Transwell assay. The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR, and the protein levels of Ki67, Cyt C, caspase-3, cleaved caspase-3, MMP-2, MMP-9, Akt, p-Akt and p-PI3K were determined by Western blotting analysis. RESULTS: TGN inhibited the proliferation of A549 cells in a dose-dependent manner (P<0.05) along with the low expression level of proliferation biomarker Ki67. TGN up-regulated the protein levels of Cyt C, caspase-3 and cleaved caspase-3 (P<0.01) and promoted the apoptosis of A549 cells in a dose-dependent manner. Moreover, TGN down-regulated the invasion-related molecules MMP-2 and MMP-9 at the mRNA and protein levels, and the number of invasive cells reduced with the increase in the concentration of TGN. The protein levels of p-Akt and p-PI3K in the A549 cells was reduced (P<0.05), and no difference of the cell viability in the cells treated with different concentrations of TGN was observed after blocking PI3K/Akt signaling pathway using LY294002. CONCLUSION: TGN inhibits the growth and invasion of A549 cells and promotes the cell apoptosis by potentially inhibiting PI3K/Akt signaling pathway activation. Therefore, this study will provide a new target for the prevention and control of NSCLC.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Effects of CARHSP1 gene expression on viability,apoptosis and immune factors of vascular endothe-lial cells induced by hypoxia in atherosclerosis

AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.     

Effect of miR-25 on apoptosis of H9c2 cells induced by hypoxia/reoxygenation

AIM:To investigate the role and mechanism of microRNA-25 (miR-25) in apoptosis of H9c2 cells induced by hypoxia/reoxygenation. METHODS:The H9c2 cells with over-expression of miR-25 were treated with hypo-xia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1 (HMGB1) mRNA. Western blot was performed to examine the protein expression levels of HMGB1, Bcl-2 and cleaved caspase-3. Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9c2 cells. Moreover, the H9c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9c2 cells. RESULTS:Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3, and increased the expression of Bcl-2 and the entrance into S phase in H9c2 cells induced by hypoxia/reoxygenation (P<0.01). The result of dual-luciferase reporter assay showed that compared with the control group, transfection with HMGB1-3' UTR-psi-CHECK2+miR-25 mimic strongly inhibited the luciferase activity (P<0.05). After the H9c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation, the expression of Bcl-2 was up-regulated, the expression of cleaved caspase-3 was down-regulated, and the cells in S phase were increased (P<0.05). CONCLUSION:miR-25 reduces apoptosis of H9c2 cells induced by hypoxia/reoxygenation, and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3'-UTR.     

Reversing effect of naringin on cisplatin resistance in human lung cancer A549/DDP cells

AIM: To investigate the effect of naringin (NRG) on cisplatin (DDP) resistance in human lung cancer A549/DDP cells and its possible mechanism. METHODS: A549/DDP cells were cultured in vitro and treated with NRG and/or DDP at different concentrations for 24 h, and then the cell viability were measured by CCK-8 assay. The combination index (CI) of NRG and DDP were analyzed by Chou-Talalay method. The apoptosis rate was analyzed by flow cytometry. Western blot was performed to detect the protein levels of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), p-Akt, CXC chemokine receptor 4 (CXCR4), cleaved caspase-3, Bcl-2 and Bax.RESULTS: The protein levels of P-gp, MRP1, p-Akt and CXCR4 in the A549/DDP cells were higher than those in the A549 cells (P<0.05). The cell viability was remarkably reduced in a dose-dependent manner when A549/DDP cells were exposed to NRG and/or DDP (P<0.05), and the IC₅₀ values of NRG and DDP were 36.92 μmol/L and 129.77 μmol/L, respectively. When the inhibition rate exceeded 15%, NRG in combination with DDP produced a synergistic effect (CI<1). Combination treatment with NRG and DDP significantly induced apoptosis (P<0.05), up-regulated the protein levels of cleaved caspase-3 and Bax, and down-regulated the protein level of Bcl-2 (P<0.05). Meanwhile, NRG remarkably down-regulated the protein levels of P-gp, MRP1, p-Akt and CXCR4 in a dose-dependent manner (P<0.05). CONCLUSION: NRG may enhance the sensibility of A549/DDP cells to DDP most likely via up-regulating the protein level of Bax and down-regulating the protein levels of Bcl-2, P-gp, MRP1, p-Akt and CXCR4.     

Effects of aspirin on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To investigate the effects of aspirin on the apoptosis of nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance and its potential mechanism. METHODS: The effects of aspirin on the cell viability, apoptosis, and protein levels of procaspase-3, cleaved caspase-3, procaspase-9, procaspase-12, PARP and cleaved PARP, PI3K p110α, Akt, Bcl-2, Bax and p27 in the CNE2R cells and CNE2 cells were detected by the methods of MTT assay, flow cytometry and Western blot. RESULTS: Aspirin inhibited the viability of homologous nasopharyngeal carcinoma cell lines CNE2 and CNE2R (with the IC₅₀ to CNE2 cells of 6.18, 3.92 and 3.06 mmol/L for 24 h, 48 h and 72 h, respectively; and with the IC₅₀ to CNE2R cells of 7.05, 3.90 and 2.20 mmol/L for 24 h, 48 h and 72 h, respectively). After treated with aspirin for 48 h, the apoptotic rate of CNE2R cells was higher than that of CNE2 cells (P<0.05). After treated with aspirin for 48 h, the protein levels of procaspase-3, procaspase-9, procaspase-12 and PARP were decreased, the protein levels of cleaved caspase-3, cleaved PARP and p27 were increased, and the protein levels of PI3K p110α, Akt and Bcl-2/Bax were decreased. CONCLUSION: Aspirin inhibits the viability of homologous nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance. Aspirin also induces the apoptosis of CNE2 and CNE2R cells, which is more effective in CNE2R cells. The underlying mechanisms may be involved in affecting PI3K/Akt signaling pathway, Bcl-2/Bax and p27 expression.     

Astragalosides inhibit autophagy and apoptosis of rat cardiomyocytes induced by H2O2 through mTOR signaling pathway

AIM: To investigate the effects of astragalosides on autophagy and apoptosis of rat cardiomyocytes induced by hydrogenperoxide (H₂O₂).METHODS: The injury model of H9c2 cells induced by H₂O₂ was established, and the cells in astragalosides group and rapamycin group were treated with 20 mg/L astragalosides and 0.1 mg/L rapamycin, respectively. The apoptotic rate was detected by flow cytometry. The autophagy was observed by acridine orange staining. Western blot was used to detect the protein levels of p-mTOR, P70S6K, LC3 and caspase-3. RESULTS: Compared with H₂O₂ group and rapamycin group, the viability of H9c2 cells in astragalosides group was significantly increased (P<0.05). The shape of the H9c2 cells in astragalosides group was complete, the nuclei were stained with yellow-green fluorescence, and the chromatin was distributed evenly. The protein levels of p-mTOR and P70S6K in the H9c2 cells of astragalosides group were significantly increased (P<0.05), whereas the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H9c2 cells of astragalosides group were decreased significantly (P<0.05). CONCLUSION: Astragalosides enhance the viability, inhibit the apoptosis, increase the protein levels of p-mTOR and P70S6K, and decrease the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H₂O₂-induced rat myocardial H9c2 cells. The mechanism is related to the mTOR signaling pathway.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM:To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer HeLa cells, and to explore the possible role of PI3K/Akt/mTOR signaling pathway in these processes. METHODS:The HeLa cells were treated with shikonin, and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the HeLa cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor Z-DEVD-FMK, the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3 (LC3) and cleaved caspase-3 in the HeLa cells were determined by Western blot. The protein levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were also determined by Western blot. RESULTS:Shikonin significantly inhibited the viability of HeLa cells (P<0.05). Compared with control group, shikonin significantly induced apoptosis of HeLa cells (P<0.05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the HeLa cells after shikonin treatment, while the autophagosomes were rarely observed in control group. Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin+3-MA group (P<0.05). Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin+Z-DEVD-FMK group (P<0.05). Compared with control group, shikonin significantly decreased the protein levels of p-PI3K, p-Akt and p-mTOR (P<0.05). CONCLUSION:The apoptosis and autophagy of the HeLa cells are induced by shikonin, these two processes are complementary. The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.     

Lycium barbarum polysaccharides attenuate oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide

AIM: To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide (H₂O₂). METHODS: The EA.hy926 cell model of oxidative stress injury was established by H₂O₂ treatment. The EA.hy926 cells were divided into 5 groups:control group, damage (H₂O₂ at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H₂O₂), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA.hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA.hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide (AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS: LBP at concentration of 100 mg/L significantly attenuated the injury of EA.hy926 cells induced by H₂O₂, as indicated by improved cell viability (P<0.05) and decreased apoptosis (P<0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA.hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA.hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION: LBP has protective effect on H₂O₂ -induced EA.hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.     

Resveratrol inhibits chondrosarcoma via mitochondrial and PI3K/Akt signaling pathways

AIM：To investigate the inhibitory effects of resveratrol on chondrosarcoma and the relation with mitochondrial and PI3K/Akt pathways. METHODS：Chondrosarcoma SW1353 cells were treated with resveratrol at concentrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h. The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK8 assay and Hoechst 33258 staining, respectively. The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting. The cell migration ability was determined by wound scratch assay. RESULTS：Exposure of the cells to resveratrol resulted in a decrease in the cell viability in a dose- and time-dependent manner (P<0.05). visible nuclei with apoptotic characteristics in resveratrol group were observed. The protein levels of activated caspase-3 and Bax were increased, and Bcl-2 and p-Akt were decreased compared with control group. The total Akt were not significantly changed. Resveratrol also significantly reduced the migration of tumor cells. CONCLUSION：Resveratrol induces apoptosis of chondrosarcoma, which plays a role of part through mitochondrial and PI3K/Akt signaling pathways.     

Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells.     

Advanced glycation end products induce rat chondrocyte injury by modulating oxidative stress

ATM: To explore the possibility that advanced glycation end products (AGEs) induces rat chondrocyte injury by modulating oxidative stress. METHODS: Primarily cultured rat chondrocytes were identified. The viability of the chondrocytes was measured by CCK-8 assay. The intracellular levels of reactive oxygen species (ROS) were detected by DCFH-DA staining. The number of apoptotic cells was determined by Hoechst 33342 nuclear staining and flow cytometry. RT-PCR was performed to measure the mRNA levels of Bax, Bcl-2, caspase-3, MMP3, MMP13 and COL2 in the chondrocytes. Western blotting was used to evaluate the protein levels of cleaved caspase-3, MMP3, MMP13 and COL2. RESULTS: Compared with control group, the intracellular levels of ROS in the chondrocytes treated with AGEs were significantly increased (P<0.05), and pretreatment with N-acetyl-L-cysteine (NAC) suppressed the formation of ROS (P<0.05). Besides, NAC inhibited AGEs-induced apoptosis of the chondrocytes, as indicated by reduceing the levels of Bax/Bcl-2 and caspase-3, decreased the expression of MMP3 and MMP13, and reduced the loss of COL2.CONCLUSION: AGEs induce chondrocyte injury by activating oxidative stress.     

Role of aloperine on ischemia-reperfusion-induced injury and inflammatory response in H9c2 cardiomyocytes

AIM: To explore the role of aloperine in ischemia-reperfusion (I/R)-induced H9c2 cardiomyocyte injury and inflammation.METHODS: The H9c2 cardiomyocytes were cultured under hypoxia and re-oxygenation conditions to simulate ischemia-reperfusion (SI/R) injury. After treatment with aloperine at various doses, the cell viability was measured by MTT assay. The cell apoptosis was analyzed by flow cytometry. Simultaneously, the levels of lactate dehydrogenase (LDH), malonaldehyde (MDA) and caspase-3 activity were detected by the commercial kits. The levels of inflammatory cytokines were also detected by ELISA. Moreover, the effects of aloperine on the activation of PI3K/AKT signaling pathway were determined by Western blot.RESULTS: Pre-treatment with aloperine remarkably abated the inhibitory effect of SI/R on H9c2 cell viability, and decreased the elevations of LDH and MDA triggered by SI/R (P<0.05). Pre-treatment with aloperine dramatically suppressed the cell apoptosis induced by SI/R treatment (P<0.05), concomitant with the decrease in caspase-3 activity and increase in Bcl-2/Bax ratio (P<0.05). In contrast to SI/R group, aloperine treatment notably restrained the concentrations of pro-inflammatory cytokines, including interleukin-6, tumor necrosis factor-α and interleukin-1β (P<0.05). Furthermore, aloperine remarkably increased the protein levels of p-PI3K and p-AKT. While blocking the PI3K/AKT pathway with its specific inhibitor LY294002, the viability-promoting, anti-apoptotic and anti-inflammatory effects of aloperine on the H9c2 cells were obviously attenuated (P<0.05).CONCLUSION: Alope-rine protects against cardiomyocytes from I/R injury and inhibits inflammatory responses by activating the PI3K/AKT signaling pathway, implying a potential benefic role of aloperine against myocardial I/R injury.     

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.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Berberine promotes anti-proliferative effects of doxorubicin in T24 bladder cancer cells by enhancing apoptosis

AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.