Effect of progesterone on SH-SY5Y cells injured by ATP

AIM: To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS: The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca²⁺ concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X₇ receptor was assessed by Western blot.RESULTS: The viability of the SH-SY5Y cells was significantly decreased (P<0.05) and YO-PRO-1 uptake was obviously increased (P<0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1, 3, 5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3, 10 and 30 nmol/L for 30 min (P<0.05) as compared with ATP group. YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P<0.05) by progesterone (30 nmol/L) or P2X₇ receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min, and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca²⁺ fluorescence intensity in normal group was a little, but that in ATP group was increased (P<0.05). Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca²⁺ induced by ATP (P<0.05). However, no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X₇ receptor in ATP group was significantly higher than that in control group (P<0.05), and progesterone inhibited ATP-induced P2X₇ receptor expression (P<0.05).CONCLUSION: Progesterone inhibits P2X₇ receptor expression, membrane pore formation, intracellular Ca²⁺ increase and cell death induced by ATP, so progesterone may protect SH-SY5Y cells against ATP-induced injuries.     

Effects of P2X4 receptor in spinal microglia on rrTNF-induced pathological pain

AIM: To investigate the effects of P2X₄ receptor on peri-sciatic administration of recombinant rat TNF-α (rrTNF)-induced mechanical allodynia. METHODS: Male Sprague-Dawley rats (180~200 g) were used in the experiments. The levels of P2X₄ receptor on day 3, day 7 and day 14 after peri-sciatic administration of rrTNF were examined by Western blot, and the location of P2X₄ receptor in the spinal dorsal horn was observed by double immunofluorescence staining. The changes of 50% paw-withdrawal thresholds of the rat were detected by behavioral test, and the level of TNF-α in the spinal dorsal horn was also examined by Western blot when TNP-ATP was intrathecally injected before the administration of rrTNF. RESULTS: Compared with control group, the expression of P2X₄ receptor in the spinal dorsal horn on the ipsilateral side significantly increased on day 3, day 7 and day 14 (P<0.01) after rrTNF (100 ng/L) administration. P2X₄ receptor was co-localized only with microglia, but not with neurons or astrocytes. Intrathecal injection of TNP-ATP before rrTNF administration prevented mechanical allodynia induced by rrTNF and inhibited the upregulation of TNF-α in the spinal dorsal horn. CONCLUSION: P2X₄ receptors in microglia may be involved in rrTNF-induced mechanical allodynia by the upregulation of TNF-α in the spinal dorsal horn.     

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.     

Protective effect of hydrogen sulfide on PC12 cells injured by ATP

AIM: To prove the purinergic signaling mechanism of the neuroprotective action of hydrogen sulfide by observing the effects of sodium hydrosulfide (NaHS), a donor of hydrogen sulfide, on the cell viability, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the change of membrane permeability in the PC12 cells injured by adenosine triphosphate (ATP). METHODS: PC12 cells in logarithmic growth phase were randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treated with ATP after cultured for 24 h. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before treated with ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, and the other treatments were as the same as those in NaHS+ATP group. The cell viability was assessed by MTT assay. The [Ca²⁺]_i was detected by Fura-2/AM staining. The membrane permeability was observed by staining with fluorescent dye YO-PRO-1.RESULTS: ATP at concentration of 0.3 mmol/L showed no injury effect on the cells. However, the cell viability was dropped gradually in a dose-dependent manner as the ATP at doses of 1, 3, 5 and 10 mmol/L. The decline of cell viability by ATP was obviously reversed by 200 μmol/L of NaHS in the PC12 cells (P<0.05), but exasperated by 800 μmol/L of NaHS (P<0.05). At the same time, ATP evoked the increase in [Ca²⁺]_i in a dose-dependent manner, which was inhibited by NaHS (P<0.05). Furthermore, the YO-PRO-1 uptake induced by ATP in a dose-dependent and time-dependent manner was also reduced by NaHS (P<0.05). CONCLUSION: Hydrogen sulfide has protective effect on the PC12 cells injured by ATP. The mechanism may be related to the reverse of the increased [Ca²⁺]_i and YO-PRO-1 uptake.     

Effect of Zhengtian pills on P2X3 receptor expression in trigeminal gan-glion of migraine rats

AIM: To explore the effect of Zhengtian pills on P2X₃ receptor expression in trigeminal ganglion (TG) of migraine rat. METHODS: Sprague-Dawley (SD) rats were randomly divided into control group, migraine group, Zhengtian pills (ZTP) group and A-317491 group. After given corresponding drugs for 7 d, migraine rat model was established by subcutaneously injection of nitroglycerin (10 mg/kg), while the control rats were injected with saline. The beha-vioral manifestations of the rats were observed. The expression of P2X₃ receptor in rat TG was detected by the methods of immunofluorescence, Western blot and rea-time PCR. RESULTS: About 5 min after subcutaneousl injection, the behavioral manifestations such as scratching head and climbing cage were observed. The behavioral manifestations were observed within the first 30 min in control group, but the erythroid ears did not appear. After 2 h of molding, the behavioral manifestations disappeared in ZTP group and A-317491 group, while those in migraine group lasted for 3 h. Immunofluorescence results showed that the expression of P2X₃ receptor in TG was positive in each group. The expression level in migraine group was significantly higher than that in other groups. The P2X₃ receptor protein and mRNA levels in the TG of migraine group were higher than those in control group (P<0.01), while those in ZTP group were lower than those in migraine group (P<0.01). No difference of the P2X₃ receptor expression between ZTP group and A-317491 group was observed. CONCLUSION: Zhengtian pills may effectively alleviate migraine by inhibiting the expression of P2X₃ receptor in TG.     

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.     

Brilliant blue G attenuates hippocampal injury of rats with acute carbon monoxide poisoning

AIM:To explore the role of ligand-gated ion channel purinergic P2X₇ receptor (P2X₇R) in acute carbon monoxide poisoning (ACMP)-induced brain injury, and to observe the effects of brilliant blue G (BBG) on ACMP-induced hippocampal injury of rats. METHODS:Male Sprague-Dawley rats (n=80) were divided into 4 groups including control group, ACMP group, ACMP +BBG group and BBG group. ACMP model was established by intraperitoneal injection of CO at 100 mL/kg. The degree of CO poisoning was determined by measuring the concentration of carboxyhemoglobin (HbCO) in venous blood. RT-qPCR was utilized to determine the mRNA level of P2X₇R. Wet-dry weight ratio was calculated to evaluate the edema in the hippocampus. ELISA was used to examine the concentrations of pro-inflammatory factors including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6. Morris water maze test was used to evaluate the learning and memory abilities. The morphological changes of the neurons located in hippocampal CA1 region were observed by HE staining. RESULTS:The rat survival rate in ACMP group (55%) was significantly reduced compared with control group (100%), while the mRNA expression of P2X₇R and the levels of pro-inflammatory factors including IL-1β, TNF-α and IL-6 in ACMP group were significantly increased (P<0.05). Moreover, ACMP administration increased the water content of hippocampal tissues and the escape latency of the rats, while decreased the exploration time in the target quadrant. All the effects induced by ACMP were reversed by BBG treatment. Additionally, ACMP treatment for 3 d resulted in cell disarrangement and morphologyical defect, and the nucleus was deeply dyed and condensed. However, BBG obviously alleviated the toxic effects of ACMP. CONCLUSION:P2X₇R is involved in ACMP-induced neuronal damage in hippocampus. BBG increases the survival rate, reduces the release of pro-inflammatory cytokines and the edema in the hippocampus, improves the learning and memory abilities, and attenuates neuronal damage in the CA1 region under ACMP.     

黄瓜种质资源苗期耐低温性评价

以43份不同类型的黄瓜品种(品系)为试验材料,在黄瓜苗期进行低温处理(昼/夜温度为12℃/9℃),以冷害指数作为鉴定指标对其耐低温性进行评价。最终鉴定出7份耐低温材料、10份低温敏感型材料。对黄瓜低温处理后的冷害指数(Y)与叶绿素降低率(X1)、可溶性蛋白增高率(X2)、MDA增高率(X3)、脯氨酸增高率(X4)、POD增高率(X5)进行相关性分析,并建立了鉴定黄瓜耐低温性的回归方程:Y=0.433+0.671X1+0.005X2+0.121X3-0.733X4+0.248X5。     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

Effects of Aβ1-42-induced microglial cells on survival of neural stem cells in vitro

AIM: To explore the effects of β-amyloid protein 1-42 (Aβ_1-42)-induced microglia on the survival of cultured neural stem cells (NSCs) in vitro . METHODS: Using the Transwell chambers to build a coculture system of NSCs and microglia, we detected the proliferation, differentiation and apoptosis of the NSCs with the microglia before and after induction by Aβ_1-42. RESULTS: Compared with non-intervention group, the proliferation rate of NSCs in Aβ_1-42 intervention coculture group decreased, as well as the positive expression rates of microtubule-associated protein 2 (MAP-2) and choline acetyltransferase. CONCLUSION: The inflammation mediated by Aβ_1-42 inhib their the proliferation of NSCs and induces their apoptosis. Inflammation also significantly reduces the ratio of NSCs differentiating to neurons, especially to cholinergic neurons.     

Cross talk of CCK8 and EGF in mouse neurons

AIM: To explore the mechanism of signaling transduction and cross talk between cholecystokinin octapeptide (CCK₈) and epidermal growth factor (EGF) in mouse neurons and to observe the effect of CCK₈ in coordination with EGF on neuron growth and cell viability. METHODS: For determining which kind of CCK receptor mediated the phosphorylation of EGF receptor, the cultured neurons were randomly divided into control group, CCK₈ stimulation group, CCK_A receptor antagonist group, CCK_B receptor antagonist group, and CCK_A+CCK_B receptor antagonist group. Control and stimulation groups were stimulated with DMEM and CCK₈ (10^-7 mol/L) for 5 min, respectively, while antagonist groups were pre-incubated with different types of receptor antagonists (10^-8 mol/L) for 10 min and followed by stimulating the neurons with CCK₈. For observing the effect of CCK₈ and EGF on the phosphorylation of EGFR in neurons and on neuron growth and cell viability, the cultured neurons were randomly divided into control group, CCK₈ stimulation group, EGF stimulation group and CCK₈+EGF stimulation group, which were stimulated with DMEM, CCK₈ (10^-7 mol/L), EGF (40 μg/L) and CCK₈+EGF for 5 min, respectively. Reactions were terminated by freezing the neurons in liquid nitrogen and the phosphorylated EGFR was detected by Western blotting. Meanwhile, the viability of the neurons was observed by MTT method after stimulated for 24 h, 48 h, 72 h and 96 h. RESULTS: The phosphorylation levels of EGFR were decreased in the neurons treated with either of the two CCK receptor antagonists, and more obvious decrease was observed when the two CCK receptor antagonists were used in combination. Compared with control group, the phosphorylation levels of EGFR in the neurons were significantly increased(P<0.05) after stimulated with CCK₈ or EGF, and the increase was more remarkable in CCK₈+EGF stimulation group. CCK₈ or EGF improved the viability and prolonged the life span of the neuron, and synergism of these two reagents was observed. CONCLUSION: Both CCK_A and CCK_B receptors are involved in the phosphorylation of EGFR in the neurons stimulated by CCK₈, and the type A receptor may play a more important role. There is cross-talk between CCK₈ and EGF signaling pathways in neurons. The signaling cross-talk between CCK₈ and EGF may be the underlying molecular mechanism responsible for the synergistic effect on the neuron growth and viability in vitro.     

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.     

Activation of TLR9 affected chemotherapeutic sensitivity of doctaxel in human non-small cell lung cancer in vitro

AIM: To probe whether CpG oligodeoxyribonucleotides 7909 (CpG ODN7909) combined with Toll like receptor (TLR)9 affected the chemotherapeutic sensitivity of doctaxel (DOC) in human lung cancer A549 and H520 cell lines.METHODS: Sequences of TLR9 siRNAs were designed. A549 and H520 cells were transfected with TLR9 siRNA by lipofectamine. The expression of TLR9 was detected by Western blot. The cell activity was measured by CCK-8 assay. The experiments were divided into blank control group, control siRNA group and TLR9 siRNA interference group. The cell cycle distribution and cell apoptosis were analyzed by flow cytometry. The expression of P38 and Bax was determined by Western blot. The cells in each group were exposed to CpG ODN7909 and/or DOC.RESULTS: In A549 cells and H520 cells, CpG ODN7909 alone had no obvious effect on the cell activity, G₂/M phase arrest and apoptosis, but increased the protein expression of P38 and Bax (P<0.01). In addition, there was no significant changes of the above indexes in CpG ODN7909 treated-TLR9 siRNA group was observed. DOC alone significantly inhibited the cell activity, higher the G₂/M phase fractions, apoptotic rates and Bax expression (P<0.01), but didn't affect the expression of P38 in all 3 groups. Compared with the cells treated with DOC alone, the cells treated with CpG ODN7909 combined with DOC exhibited lower cell activity, higher G₂/M phase fractions, apoptosis rates and more Bax expression (P<0.01), showed no significant change of P38 expression. In addition, there was no significant change of the above indexes in CpG ODN7909 combined with DOC treated-TLR9 siRNA group was observed.CONCLUSION: CpG ODN7909 may enhance the chemotherapeutic sensitivity of DOC in human lung cancer cells by combining with TLR9. The mechanism might be related to enhancing the inhibitory effect and apoptosis of DOC on the cell activity in vitro, arresting the cells at G₂/M phase of the cells.     

Interaction of PKCε with the CaR in hypoxic post-conditioning protects the cardiomyocytes in neonatal rat

AIM: To study the interaction of PKC_ε with the CaR in hypoxic post-conditioning for protecting the cardiomyocyte of neonatal rat. METHODS: The ventricular cardiomyocytes of Wistar neonatal rat (3-7 d after birth) were incubated for about 3-5 d, then randomly divided them into 7 groups: (1) Sham control group (N group); (2) Hypoxic/re-oxygenation group (H/Re group); (3) Hypoxic post-conditioning group (HPC group); (4) HPC+GdCl₃, NiCl₂, CdCl₂ group; (5) HPC+caffeine, GdCl₃, NiCl₂, CdCl₂ group; (6) HPC+PKC_ε inhibitor group (PKCI group); (7) HPC+PKCI+GdCl₃, NiCl₂, CdCl₂ group. The neonatal cells were incubated in the D-Hanks solution (pH=6.8) which was saturated with N₂ gas for 1 h at least and then re-incubated in the DMEM solution containing 20% new-born calf serum to establish a model of H/Re. The viability of cardiomyocytes was assayed by MTT, the activity of LDH and the content of MDA were determined, the expression of CaR and PKC_ε of the membrane in each group was analyzed by Western blotting, PKC_ε interaction with CaR in the membrane was detected by immunoprecipitation, and the concentration of intracellular calcium ([Ca²⁺]_i) was measured by laser confocal scanning microscope (LCSM), apoptotic cells were measured by TUNEL assay. RESULTS: The viability of cardiomyocytes in H/Re, GdCl₃ and PKCI groups was lower than that in N and HPC groups, while the activity of LDH and the content of MDA were significantly higher than those in N and HPC groups. Meanwhile, the quantitative expression of CaR in GdCl₃, caffeine and PKCI+GdCl₃ groups was higher than that in HPC and PKCI groups, and so were the [Ca²⁺]_i and the apoptosis index. The quantitative expression of PKCε in PKCI and PKCI+GdCl₃ groups was lower than that in H/Re, HPC, GdCl₃ and caffeine groups. Immunoprecipitation of cell membrane PKCε revealed the interaction of PKC_ε with CaR. CONCLUSION: In the cardiomyocytes of HPC, PKC_ε translocates to the membrane and interacts with CaR to reduce [Ca²⁺]_i, which protects the cardiomyocytes of neonatal rat during hypoxic/oxygenation.     

Effect of fucoxanthin on growth and apoptosis of HSC-T6 cells

AIM: To explore the effect of fucoxanthin (Fu) on the growth and apoptosis of HSC-T6 cells. METHODS: HSC-T6 cells were divided into blank control group, negative control group and drug groups (treated with different concentrations of Fu). The cell viability was detected by CCK-8 assay at 24 h, 48 h and 72 h after Fu treatment. The cell cycle distribution and apoptotic rate were analyzed by flow cytometry. The protein expression of Bcl-2 and Bax were detected by Western blot. RESULTS: Compared with blank control group, the viability of HSC-T6 cells was inhibited by Fu at concentrations of 15~75 μmol/L in a dose- and time-dependent manner (P < 0.01). The cell ratio of G₁ phase was significantly decreased (P < 0.01) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.01) in 60 μmol/L Fu group after 24 h. The cell ratio of G₁ phase was significantly decreased (P < 0.05) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.05) in 15 μmol/L and 30 μmol/L Fu groups in a dose-dependent manner after 48 h. The early cell apoptotic rates and total cell apoptotic rates were significantly increased in the Fu treatment groups in a dose-dependent manner (P < 0.05). The protein expression of Bax was significantly increased in the Fu treatment groups and the protein expression of Bcl-2 was significantly decreased in 30 μmol/L and 60 μmol/L Fu groups (P < 0.05).CONCLUSION: Fu inhibits the growth of HSC-T6 cells possiblely via arresting the cell cycle at S phase and G₂ phase. The apoptosis of HSC-T6 cells induced by Fu might be via down-regulating the protein expression of Bcl-2 and up-regulating the protein expression of Bax.     

Non-steroidal anti-inflammatory drugs induce apoptosis in implanted hepatoma of mice

AIM: To study the mechanism of the effect of NSAIDs on apoptosis in mice hepatoma at anti-inflammatory doses. METHODS: Kunming breed mice were inoculated subcutaneously in the flank with mice hepatoma H22 cell line. The effects of ibuprofen, indomethacin, and nimesulide on apoptosis were determined by using electron microscopy, agarose gel electrophoresis, flow cytometry, and Western blot analysis of the expression of c-myc, bcl-x_L and bcl-2 proteins. RESULTS: NSAIDs induced apoptosis of mice implanted hepatoma, which includes the morphological changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the "ladder pattern" revealed by agarose gel electrophoresis of DNA. The apoptotic index was increased to 15%±1.0%, 29.7%±1.5%, 46.3%±3.5% from 3.3%±0.6% by detecting Sub-G₁ peaks on flow cytometry. Western blot analysis showed that levels of bcl-2 and bcl-x_L were significantly reduced by treatment with nimesulide. Ibuprofen and indomethacin decreased bcl-2 expression but increased bcl-x_L expression. C-myc wasn't changed in these groups. CONCLUSION: These results suggest that NSAIDs induces apoptosis of mice hepatoma, which may be due to their regulation on the expressions of bcl-2 family genes.     

Potentiation of the action of phorbol ester by heparin in human CNE2 cells: Association with enhanced expression of c-jun,p53 and p21

AIM: To measure the effect of addition of heparin to TPA on cell proliferation and apoptosis in CNE2 cells and investigate the possible molecular mechanisms underlying heparin and TPA interaction on cell proliferation and apoptosis. METHODS: Cell viability and cell cycle were determined by cell counting and flow cytometry.Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL)and agarose gel electrophoresis. The expression of c-jun, c-fos, p21 and p53 was examined by Western blot. RESULTS: TPA alone inhibited CNE2 cell proliferation and evoked apoptosis associated with typical morphological changes and DNA fragmentation,which was augmented when heparin was added. Compared with TPA or heparin alone, TPA plus heparin obviously enhanced the number of TUNEL-positive cells from 23%±1.2% to 51%±0.9%. After exposure to different concentrations of heparin (with or without TPA) for 24 h, CNE2 cells were accumulated G₀/G₁ phase. There was a decrease in the number of cells in S phase by the combined heparin and TPA treatment compared to heparin or TPA alone. Western blot analysis revealed that TPA induced the increases in c-jun and p53, p21 protein expression and the levels were remarkably increased following heparin in combination with TPA treatment,whereas no significant change in c-fos was detected. CONCLUSION: These results suggest that heparin synergistically potentiates the action of TPA in CNE2 cells, which may be associated with the increase in c-jun protein level and the upregulation of p21, p53 protein expression.     

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy. METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3 groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group. The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 mL/kg, suspended in corn oil, ig). After 24 h of operation, the rats from each group were sacrificed and their brains were collected. TTC staining and HE staining were used to define the infarct areas and brain structure. The autophagy-related proteins beclin-1, P62, LC3-Ⅱ/-I and Atg5 in the cortex in each group were compared by Western blot. In vitro, PC12 cells were divided into 3 groups:control group, CoCl₂ group and CoCl₂+EA pretreatment group. CoCl₂ at 800 μmol/L was added to the PC12 cells to induce an anoxic environment. The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay. The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining. MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively. The autophagy-related proteins in PC12 cells were also investigated. RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining. HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage. EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group. Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-Ⅱ/-I in the cortex were increased (P<0.01), and P62 protein expression was decreased (P<0.01) in HIE group. Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-Ⅱ/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01). In vitro, compared with CoCl₂ group, the PC12 cells in CoCl₂+EA pretreatment group showed a lower ROS level. Moreover, the cells in CoCl₂+EA pretreatment group exhibited higher mitochondrial membrane potential than that in CoCl₂ group. MDC staining in CoCl₂ group showed high value of fluorescence and increased number of autophagosomes. EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12 cells. Furthermore, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I in CoCl₂ group were higher (P<0.01), and the protein expression of P62 was lower (P<0.01) than those in control group. In CoCl₂+EA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with CoCl₂ group (P<0.01). CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.