The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3β and ERK1/2 signaling pathway

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.     

Interleukin-6 and Lipopolysaccharide Modulate Hepcidin mRNA Expression by Hepg2 Cells

Iron homeostasis is controlled by hepcidin (Hpc) as well as other ways. Hpc expression is regulated by iron (Fe) storage and by inflammation, but the joint effect of both stimuli remains unclear. We studied the modulatory role of inflammatory agents (IL6 and LPS) over Hpc and DMT1 mRNA expression in HepG2 cells preloaded with Fe. HepG2 cells were preloaded with different Fe concentrations (holo-Tf or Fe-NTA) and then incubated with IL6 or LPS. We measured intracellular Fe levels by AAS with graphite furnace, transferrin receptor (TfR) by ELISA and mRNA relative abundance of Hpc and DMT1 by qRT-PCR. The maximum effect on Fe uptake was observed in cells incubated with 30?ng/ml IL6 (p?<?0.01) and 500?ng/ml LPS (p?<?0.05). In HepG2 cells preloaded with holo-Tf or Fe-NTA and challenged with IL6 and LPS, we observed a decreased: (a) Hpc mRNA relative abundance (two-way ANOVA: p?<?0.05 and p?<?0.001, respectively), (b) DMT1 mRNA relative abundance and TfR1 protein levels (two-way ANOVA: p?<?0.001), and (c) intracellular Fe concentration (two-way ANOVA: p?<?0.001 and p?<?0.01, respectively) compared to control cells incubated only with Fe (holo-Tf or Fe-NTA). Our results support the idea that Fe storage and inflammation act together to regulate Fe homeostasis and suggest a negative regulation in this hepatic cellular model to prevent excessive increases in Hpc.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Involvement of hydrogen peroxide in mistletoe lectin-II-induced apoptosis of myeloleukemic U937 cells

Mistletoe lectin-II, a major component of Korean mistletoe (Viscum album var. coloratum) induces apoptotic death in cancer cells. In this study, we demonstrated that lectin-II induced the generation of pro-oxidants and thus resulted in the apoptotic death of human myeloleukemic U937 cells. We observed that lectin-II-induced apoptotic death was inhibited by antioxidants including reduced glutathione (GSH), N-acetylcysteine (NAC), ebselen, mnTBP, catalase and pyrrolidine dithiocarbamate (PDTC). GSH and NAC also abolished the apoptotic DNA ladder pattern fragmentation of U937 cells after lectin-II stimulation. Obviously, lectin-II treatment of cells resulted in a remarkable generation of intracellular hydrogen peroxide (H2O2) as an early event, which was monitored fluorimetrically using scopoletin-horse radish peroxidase (HRP) assay and peroxide-sensitive fluorescent probe, DCF-DA. In addition, antioxidants inhibited the activation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) as well as cytosolic release of cytochrome c by mistletoe lectin-II. Moreover, lectin-II-induced activation of caspase-9 and 3-like protease and cleavage of poly(ADP-ribose) polymerase (PARP) were inhibited by pretreatment of cells with thiol antioxidants, GSH and NAC. Taken together, these results suggest that Korean mistletoe lectin-II is a strong inducer of pro-oxidant generation such as H2O2, which mediates the JNK/SAPK activation, cytochrome c release, activation of caspase-9 and caspase 3-like protease, and PARP cleavage in human myeloleukemic U937 cells.     

CREB1 regulates glucose transport of glioma cell line U87 by targeting GLUT1

Chemical modification of chitosan is a promising method for the improvement of biological activity. In this study, chitosan-caffeic acid (CCA) was prepared and its in vitro hepatoprotective ability against hydrogen peroxide-induced hepatic damage in liver cells was evaluated. Treatment with CCA (50–400 µg/mL) did not show cytotoxicity and also significantly (p < 0.05) recovered cell viability against 650 µM hydrogen peroxide-induced hepatotoxicity. CCA treatment attenuated reactive oxygen species generation and lipid peroxidation in addition to increasing cellular glutathione level in cultured hepatocytes. To validate the underlying mechanism, antioxidant and phase II detoxifying enzyme expressions, which are mediated by NF-E2-related factor 2 (Nrf2) activation, were analyzed and CCA treatment was found to increase the expression of superoxide dismutase-1 (SOD-1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and NAD(P)H:quinine oxidoreductase 1 (NQO1). CCA treatment resulted in increased Nrf2 nuclear translocation. The phosphorylation of extracellular regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) by CCA treatment contributed to Nrf2 activation. Pharmacological blockade of ERK, JNK, and p38 MAPK revealed that SP600125 (JNK inhibitor) and PD98059 (ERK inhibitor) treatment reduced Nrf2 translocation into the nucleus while SB203580 (p38 inhibitor) exhibited weak inhibition. Collectively, CCA protects liver cells against hydrogen peroxide-induced injury and this ability is attributed to the induction of antioxidants and phase II detoxifying enzymes that are mediated by Nrf2 translocation via JNK/ERK signaling.     

Effects of Selenium and L-Carnitine on Oxidative Stress in Blood of Rat Induced by 2.45-GHz Radiation from Wireless Devices

The levels of blood lipid peroxidation, glutathione peroxidase, reduced glutathione, and vitamin C were used to follow the level of oxidative damage caused by 2.45 GHz electromagnetic radiation in rats. The possible protective effects of selenium and L-carnitine were also tested and compared to untreated controls. Thirty male Wistar Albino rats were equally divided into five groups, namely Groups A₁ and A₂: controls and sham controls, respectively; Group B: EMR; Group C: EMR + selenium, Group D: EMR + L-carnitine. Groups B–D were exposed to 2.45 GHz electromagnetic radiation during 60 min/day for 28 days. The lipid peroxidation levels in plasma and erythrocytes were significantly higher in group B than in groups A₁ and A₂ (p?<?0.05), although the reduced glutathione and glutathione peroxidase values were slightly lower in erythrocytes of group B compared to groups A₁ and A₂. The plasma lipid peroxidation level in group A₂ was significantly lower than in group B (p?<?0.05). Erythrocyte reduced glutathione levels (p?<?0.01) in group B; erythrocyte glutathione peroxidase activity in group A₂ (p?<?0.05), group B (p?<?0.001), and group C (p?<?0.05) were found to be lower than in group D. In conclusion, 2.45 GHz electromagnetic radiation caused oxidative stress in blood of rat. L-carnitine seems to have protective effects on the 2.45-GHz-induced blood toxicity by inhibiting free radical supporting antioxidant redox system although selenium has no effect on the investigated values.     

Manganese Alters Rat Brain Amino Acids Levels

Manganese (Mn) is an essential element and it acts as a cofactor for a number of enzymatic reactions, including those involved in amino acid, lipid, protein, and carbohydrate metabolism. Excessive exposure to Mn can lead to poisoning, characterized by psychiatric disturbances and an extrapyramidal disorder. Mn-induced neuronal degeneration is associated with alterations in amino acids metabolism. In the present study, we analyzed whole rat brain amino acid content subsequent to four or eight intraperitoneal injections, with 25?mg MnCl₂/kg/day, at 48-h intervals. We noted a significant increase in glycine brain levels after four or eight Mn injections (p?<?0.05 and p?<?0.01, respectively) and arginine also after four or eight injections (p?<?0.001). Significant increases were also noted in brain proline (p?<?0.01), cysteine (p?<?0.05), phenylalanine (p?<?0.01), and tyrosine (p?<?0.01) levels after eight Mn injections vs. the control group. These findings suggest that Mn-induced alterations in amino acid levels secondary to Mn affect the neurochemical milieu.     

Group IV cytosolic phospholipase A2 mediates arachidonic acid release in H9c2 rat cardiomyocyte cells in response to hydrogen peroxide

Damaging reactive oxygen species are released during episodes of ischemia and reperfusion. Some cellular adaptive responses are triggered to protect the injured organ, while other cascades are triggered which potentiate the damage. In these studies, we demonstrate that rat cardiomyocte H9c2 cells release arachidonic acid in response to hydrogen peroxide. In H9c2 cells, arachidonic acid release is attenuated by methyl arachidonyl fluorophosphonate (MAFP) and pyrrophenone, indicating that a phospholipase A2 Group IV enzyme mediates arachidonic acid mobilization. Moreover, hydrogen peroxide alters the cellular morphology of the H9c2 cells, causing drastic cell shrinkage. Because MAFP and pyrrophenone prevent the morphological alterations caused by hydrogen peroxide, these studies show that phospholipase A2 Group IV activity is likely integrally involved in the damage initiated by hydrogen peroxide.     

Selenium Reduces Mobile Phone (900 MHz)-Induced Oxidative Stress,Mitochondrial Function,and Apoptosis in Breast Cancer Cells

Exposure to mobile phone-induced electromagnetic radiation (EMR) may affect biological systems by increasing free oxygen radicals, apoptosis, and mitochondrial depolarization levels although selenium may modulate the values in cancer. The present study was designed to investigate the effects of 900 MHz radiation on the antioxidant redox system, apoptosis, and mitochondrial depolarization levels in MDA-MB-231 breast cancer cell line. Cultures of the cancer cells were divided into four main groups as controls, selenium, EMR, and EMR?+?selenium. In EMR groups, the cells were exposed to 900 MHz EMR for 1 h (SAR value of the EMR was 0.36?±?0.02 W/kg). In selenium groups, the cells were also incubated with sodium selenite for 1 h before EMR exposure. Then, the following values were analyzed: (a) cell viability, (b) intracellular ROS production, (c) mitochondrial membrane depolarization, (d) cell apoptosis, and (e) caspase-3 and caspase-9 values. Selenium suppressed EMR-induced oxidative cell damage and cell viability (MTT) through a reduction of oxidative stress and restoring mitochondrial membrane potential. Additionally, selenium indicated anti-apoptotic effects, as demonstrated by plate reader analyses of apoptosis levels and caspase-3 and caspase-9 values. In conclusion, 900 MHz EMR appears to induce apoptosis effects through oxidative stress and mitochondrial depolarization although incubation of selenium seems to counteract the effects on apoptosis and oxidative stress.     

Human OXR1 maintains mitochondrial DNA integrity and counteracts hydrogen peroxide-induced oxidative stress by regulating antioxidant pathways involving p21

The oxidation resistance gene 1 (OXR1) prevents oxidative stress-induced cell death by an unknown pathway. Here, depletion of human OXR1 (hOXR1) sensitized several human cell lines to hydrogen peroxide-induced oxidative stress, reduced mtDNA integrity, and increased apoptosis. In contrast, depletion of hOXR1 in cells lacking mtDNA showed no significant change in ROS or viability, suggesting that OXR1 prevents intracellular hydrogen peroxide-induced increase in oxidative stress levels to avoid a vicious cycle of increased oxidative mtDNA damage and ROS formation. Furthermore, expression of p21 and the antioxidant genes GPX2 and HO-1 was reduced in hOXR1-depleted cells. In sum, these data reveal that human OXR1 upregulates the expression of antioxidant genes via the p21 signaling pathway to suppress hydrogen peroxide-induced oxidative stress and maintain mtDNA integrity.     

Insulin ameliorates miR-1-induced injury in H9c2 cells under oxidative stress via Akt activation

Growing evidence indicates that aberrant upregulation of microRNA-1 (miR-1) occurs in ischemic myocardium. In addition, insulin elicits metabolism-independent cardioprotection against cardiovascular diseases. The aim of this study is to determine whether insulin ameliorates miR-1-induced injury in H9c2 cells under oxidative stress and to investigate the underlying mechanisms. By quantitative real-time RT-PCR (qRT-PCR), we show that miR-1 is upregulated in H9c2 cells after treatment with hydrogen peroxide (H(2)O(2)), and this effect is both dose- and time dependent. Furthermore, expression of miR-1 decreased significantly after insulin treatment (4.5?±?0.1 vs. 3.0?±?0.2, p?相似文献   

Pyrroloquinoline quinone inhibits oxygen/glucose deprivation-induced apoptosis by activating the PI3K/AKT pathway in cardiomyocytes

The purposes of this study were to examine the protective effect of pyrroloquinoline quinone (PQQ) on oxygen/glucose deprivation (OGD)-induced injury to H9C2 rat cardiomyocytes and to investigate the mechanism. Using H9C2 cells cultured in vitro, we examined changes in cell viability with an MTT assay at 12, 24, and 48 h after injury induced by OGD. Various concentrations of PQQ (1, 10, and 100 μM) were added, and the effect of PQQ on cell viability after OGD was assessed using the MTT assay. Thus, the optimal concentration of PQQ for the protection of cardiomyocytes against oxygen and glucose deprivation injury was determined. We also used flow cytometry analysis to examine the effect of PQQ on H9C2 cells with OGD-induced injury. The molecular probe 2′,7′-dichlorofluorescin diacetate was used to label the H9C2 cells, and flow cytometry was used to detect the effect of PQQ on reactive oxygen species (ROS) content. After labeling the H9C2 cells using a mitochondrial green fluorescent probe (Mito-Tracker Green), we measured the change in the mitochondrial content of PQQ-treated H9C2 cells. Western blotting was used to examine the effect of PQQ on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the H9C2 cells. The results of the MTT assay showed that 48 h of OGD significantly injured the H9C2 cells (p < 0.01) and that treatment with 100 μM PQQ effectively decreased the level of OGD-induced injury (p < 0.01). The results of the flow cytometry analysis showed that PQQ significantly reduced apoptosis in H9C2 cells subjected to OGD (p < 0.05). In addition, OGD significantly increased the ROS level in H9C2 cells (p < 0.01), and PQQ significantly inhibited this increase (p < 0.05). The results of the Mito-Tracker Green staining suggested that PQQ effectively inhibited the decrease in mitochondrial content caused by OGD (p < 0.05). Western blot analysis showed that PQQ partially reversed the decrease in Akt phosphorylation that was caused by OGD (p < 0.05). PQQ treatment dose-dependently protects H9C2 cells from OGD-induced injury by reducing apoptosis, decreasing intracellular ROS levels, and rescuing the OGD-induced decrease in mitochondrial content. The protective effect of PQQ may be related to its effects on the PI3K/Akt pathway.     

Involvement of group VIA calcium-independent phospholipase A2 in macrophage engulfment of hydrogen peroxide-treated U937 cells

Hydrogen peroxide-induced apoptosis of U937 cells results in substantial hydrolysis of membrane phospholipids by calcium-independent group VIA phospholipase A(2) (iPLA(2)-VIA). However, abrogation of cellular iPLA(2)-VIA neither delays nor decreases apoptosis, suggesting that, beyond a mere destructive role, iPLA(2)-VIA may serve other specific roles. In this study, we report that phagocytosis of apoptosing U937 cells by macrophages is blunted if the cells are depleted of iPLA(2)-VIA by treatment with an inhibitor or an antisense oligonucleotide, and it is augmented by overexpression of iPLA(2)-VIA in the dying cells. Thus, the magnitude of macrophage phagocytosis correlates with the level of iPLA(2)-VIA activity of the dying cells. Eliminating by antisense oligonucleotide technology of cytosolic group IVA phospholipase A(2) does not attenuate phagocytosis of U937 dying cells by macrophages. Incubation of U937 cells with different fatty acids has no effect on either the extent of hydrogen peroxide-induced apoptosis or the degree of phagocytosis of the dying cells by macrophages. However, preincubation of the macrophages with lysophosphatidylcholine before exposing them to the dying cells blocks phagocytosis of the latter. These results indicate that formation of lysophosphatidylcholine by iPLA(2)-VIA in hydrogen peroxide-treated U937 cells to induce apoptosis directly contributes to their efficient clearance by macrophages.     

Serum Trace Element Levels in Febrile Convulsion

Febrile convulsion is the most common disorder in childhood with good prognosis. There are different hypotheses about neurotransmitters and trace element changes in biological fluids which can have a role in pathogenesis of febrile convulsion. In this study, serum selenium, zinc, and copper were measured by atomic absorption spectrometry in the children with febrile convulsion (n?=?30) and in the control group (n?=?30). The age and sex of the subjects were registered. Selenium and zinc were found to be significantly lower in febrile convulsion cases than in the control group (p?<?0.0001 and p?<?0.0001, respectively). There was no significant difference in the value of copper between the two groups (p?=?0.16). While selenium and zinc levels were 44.92?±?10.93 μg/l and 66.13?±?18.97 μg/dl in febrile convulsion, they were found to be 62.98?±?9.80 μg/l and 107.87?±?28.79 μg/dl in healthy children. Meanwhile, copper levels were 146.40?±?23.51 μg/dl in the patients and 137.63?±?24.19 μg/dl in the control group, respectively. This study shows that selenium and zinc play an important role in the pathogenesis of febrile convulsion.     

Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O₂ ^?) and hydrogen peroxide (H₂O₂), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H₂O₂), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O₂ ^? formation.     

Generation of reactive oxygen species during cryopreservation may improve Lilium × siberia pollen viability

During biotic and abiotic stress in plants, reactive oxygen species (ROS) may play two very different roles: high ROS concentrations can exacerbate damage, whereas low concentrations can activate defense responses. The aim of this study was to investigate the relationship between ROS generation and pollen viability after cryopreservation. ROS generation was detected from ‘Siberia’ (Lilium?×?siberia) pollen using flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. Pollen viability was determined by 2,3,5-triphenyltetrazolium chloride staining. ROS generation was slightly increased by rapid cooling (26.13?±?4.74 vs. 15.80?±?2.30 for fresh pollen) and significantly increased by vitrification (49.74?±?1.43; P?<?0.01). Pollen viabilities after rapid cooling and vitrification were significantly increased (58.88?±?3.76% and 70.35?±?2.90%, respectively) over that of fresh pollen (46.65?±?1.61%; P?<?0.01). No significant differences in ROS generation were associated with cold acclimation at different temperatures before rapid cooling. However, sharp decreases in viability were observed with cold acclimation at 4°C and ?20°C relative to rapid cooling without acclimation (P?<?0.01). We observed nonsignificant decreases in ROS generation among vitrification treatments that omitted different steps and a significant decrease when the unloading step was omitted (P?<?0.05). Pollen viabilities were significantly reduced when the loading or dehydration steps were omitted (P?<?0.01). No significant differences were observed in ROS generation or pollen viability among the treatments when 200 U/ml catalase was added to different solutions used in the vitrification process. Comprehensive analysis of all data indicated a positive correlation between ROS generation and pollen viability (r?=?0.651, P?<?0.001). Therefore, increasing ROS generation during cryopreservation may improve the viability of ‘Siberia’ pollen.     

Expression, Purification, and Characterization of a Dentin Phosphoprotein Produced by Escherichia coli, and Its Odontoblastic Differentiation Effects on Human Dental Pulp Cells

To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p?<?0.05). Cell viability was also significantly increased by rhDPP at 5?days (p?<?0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.     

αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H₂O₂)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H₂O₂ to establish a model of H₂O₂ injury to cells. H₂O₂ was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H₂O₂-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H₂O₂-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins’ expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H₂O₂ treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H₂O₂-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H₂O₂-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H₂O₂-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H₂O₂-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H₂O₂ injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H₂O₂-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.