Bcl-2 Messenger RNA Research Articles

Steamed daylily flower (Hemerocallis fulva L.) protected cardiac and hepatic cells against ethanol induced apoptotic and oxidative damage.

Our previous study examined the phytochemical composition and bio-activities of raw daylily flower (Hemerocallis fulva L.). However, this plant food is usually served via heat process such as cooking in a soup. This study aimed to investigate the phytochemical profile and biofunctions of steamed daylily flower (SDF). The content of total phenolic acids, total flavonoids, total carotenoids, total anthocyanins and total triterpenoids in SDF aqueous extract was assessed. Normal cardiac and hepatic cells, H9c2 and L-02 cells, were used to evaluate the protective effects of SDF against ethanol. SDF concentrations of 0.25%, 0.5%, and 1% were applied to treat H9c2 or L-02 cells for 48 h at 37 °C initially, followed by exposure to ethanol at 150 mM for 24 h at 37 °C. Results showed that the content of assessed phytochemicals was in the range of 1019-2045 mg/100 g dry weight. Flavonoids and triterpenoids were two major detected phytochemicals in SDF. SDF treatments at 0.5% and 1% increased the viability of H9c2 cells, but at three concentrations enhanced the survival of L-02 cells. SDF at 0.5% and 1% up-regulated Bcl-2 messenger RNA (mRNA) expression and down-regulated Bax mRNA expression. Ethanol increased reactive oxygen species production, decreased glutathione content, as well as lowered glutathione peroxidase and catalase activities. SDF treatments reversed these changes. SDF at 0.5% and 1% reduced the activity of cytochrome P450 2E1 and nicotinamide adenine dinucleotide phosphate oxidase, limited p47phox mRNA expression, as well as enhanced factor E2-related factor 2 and heme oxygenase-1 mRNA expression. SDF at three concentrations decreased gp91phox mRNA expression. In conclusion, these novel findings indicated that SDF aqueous extract was rich in phytochemicals and provided anti-apoptotic and anti-oxidative actions to protect cardiac and hepatic cells against ethanol. Thus, SDF might be considered as a functional food with multiple bio-activities.

Urolithin B inhibits proliferation and migration and promotes apoptosis and necrosis by inducing G2/M arrest and targeting MMP-2/-9 expression in osteosarcoma cells.

Osteosarcoma (OS) is the most prevalent primary bone cancer, with a high morbidity and mortality rate. Over the past decades, therapeutic approaches have not considerably improved patients' survival rates, and further research is required to find efficient treatments for OS. Data from several studies have shown that urolithin B (UB), the intestinal metabolite of polyphenolic ellagitannins, is emerging as a new class of anticancer compounds, yet its effect on OS cancer cells remains elusive. Herein, we investigated UB's antimetastatic, antiproliferative, and apoptotic effects on the MG-63 OS cell line. Cell viability assay, annexin V/propidium iodide staining, cell cycle arrest analysis, determination of the gene expression of MMP-2, MMP-9, Bax, Bcl-2, and p53 messenger RNA (mRNA), evaluation of reactive oxygen species (ROS) generation and migration, and MMP-2 and MMP-9 protein expression assessments were performed. UB caused late apoptosis, necrosis, G2/M arrest, and ROS generation in MG-63 cells. It increased the mRNA expression of the p53 tumor suppressor and Bax proapoptotic genes. UB also inhibited the migration and metastatic behavior of MG-63 OS cells by downregulating mRNA and MMP-2 and MMP-9 protein expression. In general, although further in vivo investigations are warranted, the current results showed that UB might be utilized as a potential novel natural compound for OS therapy due to its nontoxic, antiproliferative, and antimetastatic nature.

METTL3 Affects Spinal Cord Neuronal Apoptosis by Regulating Bcl-2 m6A Modifications After Spinal Cord Injury.

Spinal cord injury (SCI) is a severe type of neurological trauma. N6-methyladenosine (m6A) modification is one of the most common internal modifications of RNA. The role of METTL3, the predominant methylation enzyme of m6A modification, in SCI remains unclear. This study aimed to investigate the role of methyltransferase METTL3 in SCI. After establishing the oxygen-glucose deprivation (OGD) model of PC12 cells and rat spinal cord hemisection model, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. The m6A modification was identified on B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) by bioinformatics analysis, and m6A-RNA immunoprecipitation and RNA immunoprecipitation. In addition, METTL3 was blocked by the specific inhibitor STM2457 and gene knockdown, and then apoptosis levels were measured. In different models, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. After inducing OGD, inhibition of METTL3 activity or expression increased the mRNA and protein levels of Bcl-2, inhibited neuronal apoptosis, and improved neuronal viability in the spinal cord. Inhibition of METTL3 activity or expression can inhibit the apoptosis of spinal cord neurons after SCI through the m6A/Bcl-2 signaling pathway.

Bax mRNA Expression as A Potential Biomarker of Placental Apoptosis in Early-onset Preeclampsia

BACKGROUND: Early-onset preeclampsia is characterized by higher oxidative stress and apoptosis level than late-onset one. Studies comparing the expression of the Bcl-2 family protein in early and late-onset preeclampsia are still lacking and show inconclusive evidence. This study aimed to compare the expression of Bax and Bcl-2 messenger RNA (mRNA) as a biomarker of placental apoptosis between early-onset and late-onset preeclampsia.METHODS: A cross-sectional study was conducted using formalin-fixed, paraffin-embedded preeclamptic placental samples and dividing them into early-onset and late-onset preeclampsia groups. Bax and Bcl-2 mRNA expressions were assessed using the quantitative real-time polymerase chain reaction method. Apoptosis was assessed through DNA fragmentation examination by the ligation-mediated real-time polymerase chain reaction method.RESULTS: Thirty early-onset and 30 late-onset preeclamptic placental samples were included. The mean fold change Bax mRNA in early-onset was higher than in late-onset preeclampsia (6.02±3.59 vs. 2.82±1.97; p=0.00). The mean fold change Bcl-2 mRNA early-onset was not different from late-onset preeclampsia (31.20±17.94 vs. 31.01±27.60; p=0.98). The mean DNA fragmentation cycle threshold in early-onset preeclampsia was lower than in late-onset preeclampsia (28.07±0.64 vs. 30.63±0.96; p=0.00). A weak negative correlation exists between fold change Bax mRNA and DNA fragmentation cycle threshold (r=-0.30; p=0.02).CONCLUSION: Bax mRNA showed significant correlation in DNA fragmentation compared to Bcl-2 mRNA; hence, might show more role in apoptotic pathway. Early-onset preeclampsia has higher Bax mRNA relative expression and apoptosis than late-onset preeclampsia. Therefore, Bax mRNA can be potential biomarker in early-onset preeclampsia.KEYWORDS: mRNA, Bax, Bcl-2, apoptosis, DNA fragmentation, early-onset, preeclampsia

Distinct microRNA profiles for complete hydatidiform moles at risk of malignant progression

MicroRNAs are small noncoding RNAs with important regulatory functions. Although well-studied in cancer, little is known about the role of microRNAs in premalignant disease. Complete hydatidiform moles are benign forms of gestational trophoblastic disease that progress to gestational trophoblastic neoplasia in up to 20% of cases; however, there is no well-established biomarker that can predict the development of gestational trophoblastic neoplasia. This study aimed to investigate possible differences in microRNA expression between complete moles progressing to gestational trophoblastic neoplasia and those regressing after surgical evacuation. Total RNA was extracted from fresh frozen tissues from 39 complete moles collected at the time of uterine evacuation in Brazil. In the study, 39 cases achieved human chorionic gonadotropin normalization without further therapy, and 9 cases developed gestational trophoblastic neoplasia requiring chemotherapy. Total RNA was also extracted from 2 choriocarcinoma cell lines, JEG-3 and JAR, and an immortalized normal placenta cell line, 3A-subE. MicroRNA expression in all samples was quantified using microRNA sequencing. Hits from the sequencing data were validated using a quantitative probe-based assay. Significantly altered microRNAs were then subjected to target prediction and gene ontology analyses to search for alterations in key signaling pathways. Expression of potential microRNA targets was assessed by quantitative real-time polymerase chain reaction and western blot. Finally, potential prognostic protein biomarkers were validated in an independent set of formalin-fixed paraffin-embedded patient samples from the United States (15 complete moles progressing to gestational trophoblastic neoplasia and 12 that spontaneously regressed) using quantitative immunohistochemistry. In total, 462 microRNAs were identified in all samples at a threshold of <1 tag per million. MicroRNA sequencing revealed a distinct set of microRNAs associated with gestational trophoblastic neoplasia. Gene ontology analysis of the most altered transcripts showed that the leading pathway was related to response to ischemia (P<.001). Here, 2 of the top 3 most significantly altered microRNAs were mir-181b-5p (1.65-fold; adjusted P=.014) and mir-181d-5p (1.85-fold; adjusted P=.014), both of which have been shown to regulate expression of BCL2. By quantitative real-time polymerase chain reaction, BCL2 messenger RNA expression was significantly lower in the complete moles progressing to gestational trophoblastic neoplasia than the regressing complete moles (-4.69-fold; P=.018). Reduced expression of BCL2 was confirmed in tissue samples by western blot. Immunohistochemistry in the independent patient samples revealed significantly lower cytoplasmic expression of BCL2 in the villous trophoblasts from cases destined for progression to gestational trophoblastic neoplasia compared with those that regressed, both with respect to staining intensity (optic density 0.110±0.102 vs 0.212±0.036; P<.001) and to the percentage of positive cells (16%±28% vs 49.4%±28.05%; P=.003). Complete moles progressing to gestational trophoblastic neoplasia are associated with a distinct microRNA profile. miR-181 family members and BCL2 may be prognostic biomarkers for predicting gestational trophoblastic neoplasia risk.

The Effect of Indocyanine Green Antimicrobial Photothermal/Photodynamic Therapy on the Expression of BCL-2 and BAX Messenger RNA Levels in Human Gingival Fibroblast Cells.

Antimicrobial photothermal/photodynamic therapy (PTT/PDT) with indocyanine green (ICG) is an adjuvant therapeutic approach in the treatment of periodontitis. To explore whether PTT/PDT with ICG causes cell death by apoptosis in human gingival fibroblast (HGF) cells, BAX and BCL-2 genes expression as key events for apoptosis were evaluated in this study. HGF cells were treated with: 1) different concentrations (500-2000 µg/mL) of ICG alone, 2) Diode laser irradiation alone with a fluency of 39.06 J/cm2; 3) PTT/PDT combined different concentrations (500-2000 µg/mL) of ICG with an 808 nm diode laser with a fluency of 39.06 J/cm2, and 4) controls (untreated cells). After that, BAX and BCL-2 messenger RNA levels were evaluated by real-time quantitative reverse transcription PCR. PTT/PDT with 500 µg/mL of ICG caused significant increases in the expression of the BAX gene, with an 8.5-fold increase, which was approximately 7- and 8.5-fold higher than PTT/PDT with ICG for 1500 and 2000 µg/mL of ICG, respectively, indicating induction of apoptosis in HGF cells. ICG (in different test concentrations), diode laser, and PTT/PDT with ICG (1500 and 2000 µg/mL of ICG) treatment displayed insignificant increases in expression levels of BAX (all p>0.05). Our experiments showed an insignificant increase (1.1-1.6-fold) in the expression of BCL-2 after ICG, diode laser, and PTT/PDT with ICG treatment (all p>0.05). This study suggests that various concentration of ICG can be the diverse expression of BAX responses to PTT/PDT on HGF cells.

Protective effect of epigallocatechin-3-gallate against neuroinflammation and anxiety-like behavior in a rat model of myocardial infarction.

ObjectiveIndividuals who experience myocardial infarction (MI) often experience anxiety. Green tea has potent antioxidative properties and, epigallocatechin‐3‐gallate (EGCG), which is a primary component of tea polyphenols, has advantageous effects on anxiety and depression. However, its mechanism of action regarding the inhibition of anxiety‐like symptoms after MI remains unclear. This study examined whether EGCG alleviated anxiety‐like behavior in MI rats and its possible mechanism.Material and MethodsRats were administered a daily gavage of EGCG (50 mg/kg) 7 days before and 14 consecutive days after the MI procedure. The open‐field test and light/dark shuttle box were performed to evaluate anxiety‐like behavior. Serum and hippocampus interleukin (IL)‐6 levels were tested using ELISA. Caspase 3, caspase 8, caspase 9 and bcl‐2 messenger RNA levels in the hippocampus were determined using quantitative polymerase chain reaction, and STAT3 protein was detected by Western blot.ResultsResults of the open field test and light/dark shuttle box task demonstrated that the MI procedure induced anxiety‐like behavior in the animals, and this impairment was improved by EGCG. Daily EGCG administration significantly decreased the level of IL‐6 both in serum and hippocampus after MI. The administration of EGCG also significantly moderated the expression of caspases 3, 8, and 9 mRNA, which was related to apoptosis in the hippocampus. Furthermore, EGCG also downregulated the expression of STAT3, which was related to the activity of IL‐6. These results suggest that EGCG alleviated anxiety‐like behavior by inhibiting increases in neuroinflammation and apoptosis in the rat hippocampus. In addition, EGCG reversed alterations of IL‐6 and STAT3 in the brain to alleviate apoptosis in the hippocampus.ConclusionsThus, EGCG reversed anxiety‐like behavior through an anti‐inflammation effect to alleviate apoptosis in neurons and may be a useful therapeutic material for anxiety‐like behavior after MI.

Tet3 is required for normal in vitro fertilization preimplantation embryos development of bovine.

DNA methylation is a central epigenetic event that regulates cellular differentiation, reprogramming, and pathogenesis. DNA demethylation occurs in preimplantation embryos and primordial germ cells. Recent studies suggest that TET3-mediated oxidation of 5-methylcytosine (5-mC) contributes to genome-wide loss of DNA methylation, yet the mechanism of this process in bovine preimplanted embryos has remained unknown. In this study, we analyzed the expression of Tet gene family at different stages of embryo development. The results revealed that Tet3 was highly expressed in bovine oocytes and in vitro fertilization preimplantation embryos. Knockdown of Tet3 by injection of siRNA in germinal vesicle oocytes was used to assess its role in epigenetic remodeling and embryo development. The results showed that knockdown of Tet3 significantly inhibited oocyte development, maturation, fertilization, and decreased subsequently cleavage and blastocyst rates. Tet3 knockdown significantly increased 5-mC levels, whereas the 5-hmC levels slightly declined. The quantitative polymerase chain reaction data showed that expression levels of the pluripotency genes (POU5F1 and NANOG) were significantly decreased, but the imprinted gene H19 did not change in the Tet3 knockdown group. In addition, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) promoter regions showed hypermethylation in the Tet3 knockdown group, except the imprinted gene H19. Furthermore, the percentage of apoptotic cells and the expression levels of the proapoptotic gene BAX were significantly increased, whereas the antiapoptotic gene BCL-2 messenger RNA levels were decreased in the Tet3 knockdown group. Our results indicated that Tet3 could influence the expression level of the pluripotency genes through regulating the methylation status of the promoter region, thus affect embryonic development.

Evaluation of the renoprotective effect of nano turmeric against toxic dose of copper sulfate: Role of vascular cell adhesion molecule-1, kidney injury molecule-1, and signal transducer and activator of transcription 3 protein expressions.

The aim of this study was to compare the potential renoprotective effects of turmeric (TM) and nano turmeric (NTM) with those of desferrioxamine (DSM) against copper sulfate (CS)-induced toxicity. Rats were administered a toxic dose of CS with TM, NTM, and DSM for 1 week. Next, serum-urea creatinine, uric acid, interleukin (IL)-10, c-reactive protein (CRP), and caspase-3 levels; renal nitric oxide (NO), glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), vascular cell adhesion molecule-1 (VCAM-1), kidney injury molecule (KIM)-1, signal transducer and activator of transcription 3 (STAT-3) protein expression; and nuclear factor (NF)-κB and B-cell lymphoma -2 (Bcl-2) messenger RNA expression levels were estimated. Administration of the investigated antioxidants downregulated the marked increase in urea, creatinine, uric acid, CRP, caspase-3, NO, MDA, VCAM-1, kidney injury molecule (KIM-1), STAT-3, NF-κB, and DNA fragmentation, and increased Bcl-2, IL-10, GSH, and SOD levels induced by CS. The histopathological examination confirmed the effects of the antioxidants on the investigated biochemical parameters. Interestingly, NTM exhibited a superior renoprotective effect, which was comparable with that of DSM. In conclusion, NTM was shown to be a promising candidate against CS-induced toxicity, and several molecular mechanisms were implicated in the CS-induced renotoxicity as well as the treatment effects of NTM.

Expression of miR-204 in pediatric retinoblastoma and its effects on proliferation and apoptosis of cancer cells.

Expression, clinical significance and molecular mechanism of miR-204 in human retinoblastoma (RB) and para-carcinoma tissues were investigated. A total of 110 cases of RB tissues preserved after ophthalmectomy in the First Affiliated Hospital of Hunan Normal University (People's Hospital of Hunan Province) from April 2013 to June 2017 were collected along with 100 cases of para-carcinoma normal tissues. The expression of miR-204 in RB tissues was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and its associations with clinicopathological features were analyzed. Y79 cells were transfected with miR-204 mimics. A total of 80 pmol/l miR-204 mimics and 10 µl Lipofectamine 2000 were added into the experimental group. Cell proliferation was detected via methyl thiazolyl tetrazolium (MTT) assay at 24, 48, 72 and 96 h, apoptosis was detected via flow cytometry at 48 h after transfection, and the relative expression levels of B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) and Sirt1 mRNA were detected via RT-qPCR. The results of MTT assay revealed that the measured value of the optical density (OD) in the experimental group at 48 h was obviously lower than that in the negative control group (p<0.001). The proportion of apoptotic cells in the experimental group was remarkably higher than that in the negative control group (p<0.001). Compared with those in the negative control group, the relative expression levels of Bcl-2 and Sirt1 mRNAs in the experimental group were significantly decreased (p<0.001). miR-204 may be involved in the occurrence and development of RB, which is significantly associated with clinical tissue differentiation, neural infiltration and lymph node metastasis in patients. miR-204 may inhibit proliferation and promote apoptosis of RB cells through downregulating the expression of Bcl-2 and Sirt1 in RB. Therefore, miR-204 may become a new biological index for early diagnosis, prognosis evaluation and biotherapy of RB.

Implantation of Endothelial Cells with Mesenchymal Stem Cells Accelerates Dental Pulp Tissue Regeneration/Healing in Pulpotomized Rat Molars

IntroductionThis study aimed to examine whether the implantation of mesenchymal stem cells (MSCs) with endothelial cells (ECs) accelerates pulp tissue regeneration/healing and induces dentin bridge formation in a rat model of molar coronal pulp regeneration. MethodsThe maxillary first molars of Wistar rats were subjected to pulpotomy. Then, pulp chambers were implanted with biodegradable hydrogel-made scaffolds carrying MSCs together or without dermal microvascular ECs, and the cavities were sealed with mineral trioxide aggregate. After 14 days, pulp samples were analyzed by immunohistochemistry; messenger RNA expression of B-cell lymphoma 2 (Bcl-2), chemokine (C-X-C motif) ligand 1 (Cxcl1), CXC receptor 2 (Cxcr2), and dentin sialophosphoprotein (Dspp) by quantitative polymerase chain reaction, and protein expression of nestin and vascular endothelial growth factor by Western blotting. ResultsTeeth coimplanted with MSCs and ECs showed pulp healing with complete dentin bridge formation, whereas those implanted with MSCs alone had incomplete dentin bridges. Bcl-2, Cxcl1, Cxcr2, and Dspp messenger RNA levels were significantly up-regulated in the pulp of MSC/EC-implanted teeth compared with those in MSC-implanted teeth. Immunohistochemical analysis revealed the expression of nestin in odontoblastlike cells under dentin bridges in the MSC/EC coimplanted group. The density of CD31-expressing ECs and the expression of nestin and vascular endothelial growth factor proteins were significantly up-regulated in the MSC/EC-implanted pulp compared with the MSC-implanted pulp. ConclusionsThe implantation of ECs with MSCs accelerated pulp tissue regeneration/healing and dentin bridge formation, up-regulated the expression of proangiogenic factors, and increased the density of ECs in pulpotomized rat molars.

Effects of Different Levels of Calcium Intake on Brain Cell Apoptosis in Fluorosis Rat Offspring and Its Molecular Mechanism.

The purpose of the investigation is to reveal the influence of dietary calcium on fluorosis-induced brain cell apoptosis in rat offspring, as well as the underlying molecular mechanism. Sprague-Dawley (SD) female rats were randomly divided into five groups: control group, fluoride group, low calcium, low calcium fluoride group, and high calcium fluoride group. SD male rats were used for breeding only. After 3months, male and female rats were mated in a 1:1 ratio. Subsequently, 18-day-old gestation rats and 14- and 28-day-old rats were used as experimental subjects. We determined the blood/urine fluoride, the blood/urine calcium, the apoptosis in the hippocampus, and the expression levels of apoptosis-related genes, namely Bcl-2, caspase 12, and JNK. Blood or blood/urine fluoride levels and apoptotic cells were found significantly increased in fluorosis rat offspring as compared to controls. Furthermore, the Bcl-2 messenger RNA (mRNA) expression levels significantly decreased, and caspase 12 mRNA levels significantly increased in each age group as compared to controls. Compared with the fluoride group, the blood/urine fluoride content and apoptotic cells evidently decreased in the high calcium fluoride group, Bcl-2 mRNA expression significantly increased and caspase 12 mRNA expression significantly decreased in each age group. All results showed no gender difference. Based on these results, the molecular mechanisms of fluorosis-induced brain cell apoptosis in rat offspring may include the decrease in Bcl-2 mRNA expression level and increase in caspase 12 mRNA expression signaling pathways. High calcium intake could reverse these gene expression trends. By contrast, low calcium intake intensified the toxic effects of fluoride on brain cells.

Tumor suppressor miRNA-204-5p promotes apoptosis by targeting BCL2 in prostate cancer cells

Prostate cancer (PCa) is a leading cause of cancer-related death in men, which emphasizes the need for novel therapeutic approaches. Targeting microRNA (miRNA) has been considered as a therapeutic strategy against cancers. Human miR-204-5p potentially targeting BCL2 has been reported to be downregulated in various cancers. We hypothesized that miR-204-5p overexpression induces cancer cell apoptosis by repressing BCL2 expression. A vector harboring mature miR-204-5p was constructed and delivered into human PCa cells. The expression level of miR-204-5p was determined by miRNA quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the function of mature miR-204-5p and its direct binding to BCL2 transcripts. The expression levels of BCL-2 messenger RNA (mRNA) and protein samples were measured by QPCR and Western blot, respectively. Cell viability was detected by WST-1 assays. Induction of apoptosis was determined by increased levels of cleavage caspase 3 and caspase 3/7 activity. The expression levels of miR-204-5p were downregulated in PCa cells compared with normal prostate epithelial cells. Transfection of pSM-204 resulted in up to 6.2-fold higher expression of miR-204-5p when compared with pSM control. The mRNA levels of several potential target genes of miR-204-5p were decreased in pSM-204-transfected PC3 and Rv1 cells. BCL2 mRNA and protein expression decreased in miR-204-5p-transfected cells, which led to cytochrome C release from mitochondria. It subsequently increased cleaved caspase 3 and caspase 3/7 activities and reduced cell viability. Cotransfection of a reporter vector harboring the BCL2 3'-untranslated region to compete with endogenous transcripts partially rescued miR-204-5p-induced apoptosis. Human miR-204-5p targets BCL2 in PCa cells. Restoration of miR-204-5p in PCa could therefore be considered as a novel strategy by targeting antiapoptotic BCL2.

Valproic acid-mediated myocardial protection of acute hemorrhagic rat via the BCL-2 pathway.

Hemorrhage is a major cause of morbidity and mortality among trauma patients. The pathophysiologic changes following acute severe hemorrhage and tissue hypoxia lead to an imbalance of protein acetylation. Histone deacetylase inhibitors (HDACIs) were reported to restore the acetylation imbalance and serve as potential drugs for treating severe hemorrhage. However, the molecular mechanism of HDACI-mediated cytoprotection remains unclear. In this study, we examined the myocardial protective effects and respective mechanism of the HDACI valproic acid (VPA) administered during hemorrhagic and hypoxic stress in vivo and in vitro. In vivo, the therapeutic effect of VPA was evaluated in acute severe hemorrhagic rats, and the expressions of BCL-2 signal pathway molecules were observed in rat heart tissues. To explore the molecular mechanism of VPA-mediated myocardial protection, a cobalt chloride (CoCl2)-induced hypoxia model of rat H9c2 cardiomyoblasts was applied to mimic hypoxic injury raised by acute hemorrhage. VPA administration significantly improved the 4-hour survival rate of hemorrhagic animals from 55% to 100% and protected H9c2 cells against CoCl2-induced hypoxic injury at a dose of between 12.5 μM and 100 μM. Increased expression of BCL-2 messenger RNA was observed following VPA treatment in the heart tissues of hemorrhagic rats (approximately 4.9-fold) and in H9c2 cells that survived CoCl2-induced hypoxia (approximately 4.9-fold). Western blot analysis showed a concomitant increase in BCL-2 protein expression and Akt phosphorylation following VPA treatment. The cytoprotective activity of VPA was diminished by triciribine-mediated inhibition of Akt activation and by silencing of BCL-2 gene expression. These findings suggest that VPA protects myocardial cells from hemorrhagic and hypoxic stress through the Akt/BCL-2 survival pathway, indicating a potential use of HDACIs for acute severe hemorrhage treatment.

Ischemic postconditioning altered microRNAs in human valve replacement

BackgroundAlthough the involvement of microRNAs (miRNAs) has been intensively studied in myocardial infarction, there is no report on the regulation of miRNAs by ischemic postconditioning in patients undergoing cardiac surgery. We aim to explore the regulation of miRNAs by ischemic postconditioning in double valve replacement. Materials and methodsIn this prospective, controlled clinical study, consecutive 30 patients undergoing double valve replacement were enrolled. The patients were randomized into two groups, namely an ischemic postconditioning (IPO) group (n = 15) and a control (CON) group (n = 15). For ethical considerations, samples of right atrial muscle were harvested, respectively, 10 min before cardiopulmonary bypass (pre-CPB) and 5 min after aortic declamping (post-CPB) for analysis of miRNAs, genes and apoptosis. ResultsCompared with the CON group, miR-1 was downregulated, whereas miR-21 was upregulated, and BCL2 messenger RNA (mRNA) was upregulated, whereas BAX mRNA and programmed cell death 4 mRNA remained unchanged in the IPO group. Likewise, a significant increase in BCL2 protein and a striking decrease in BAX protein were observed in the IPO group when compared with those in the CON group. The IPO group showed a significantly smaller increase of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling-positive myocytes after CPB than CON group. ConclusionsIschemic postconditioning could regulate miR-1, miR-21, and downstream effectors and resulted in actual attenuation of apoptosis in patients undergoing valvular heart surgery.

Hyperbaric oxygen preconditioning inhibits skin flap apoptosis in a rat ischemia–reperfusion model

BackgroundHyperbaric oxygen (HBO) improves skin flap function and inhibits partial necrosis induced by ischemia–reperfusion (I/R) injury. Our study aimed to evaluate the mechanism underlying HBO regulation of the antiapoptosis factors associated with I/R injury of skin flaps. MethodsThe rats were divided into sham surgery, I/R, and HBO groups. Rats from the HBO group received HBO preconditioning followed by I/R surgery. Blood perfusion of the skin flaps was measured with laser Doppler flowmeters. Tissue morphology and apoptosis were subsequently assessed based on hematoxylin-eosinhe and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Protein expression of phosphorylated apoptosis signal-regulating kinase 1 (pASK-1), phosphorylated c-Jun N-terminal kinase (pJNK), B-cell lymphoma-2 (Bcl-2), and Bcl2-associated X protein (Bax) was examined by immunodetection, and Bcl-2 messenger RNA expression was detected by quantitative polymerase chain reaction. In addition, caspase-3 activity was also measured. ResultsThe result of microcirculation analysis showed that the survival and blood perfusion rates significantly increased in the skin flap after HBO exposure. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining revealed that cell apoptosis was significantly attenuated in the HBO group. Furthermore, HBO preconditioning increased the expression of Bcl-2 and inhibited pASK-1, pJNK, and Bax expression as determined by both immunohistochemistry and Western blot. Caspase-3 activity and the Bax/Bcl-2 ratio declined in the HBO group. ConclusionsHBO preconditioning effectively ameliorates I/R injury by regulating the apoptosis signal-regulating kinase 1 and/or c-Jun N-terminal kinase pathway and anti- and proapoptosis factors.

Effect of zinc on in vitro development of porcine embryos

This study aimed to investigate the effect of zinc on in vitro development of porcine embryos. We evaluated the effects of zinc on blastocysts formation and investigated gene expression at zinc-deficient and supplemented conditions. Zinc-deficient in vitro condition was induced by 10-μM N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN) (zinc chelator) treatment during IVC. On parthenogenetic activated embryos, this treatment significantly decreased cleavage rate and blastocyst formation compared with the control (0.0% and 0.0% vs. 69.0% and 36.0%, respectively). And time effect of the zinc deficiency exposure is observed. Blastocyst formation rate was significantly decreased as zinc-deficient time increases (54.1%, 31.0%, 9.0%, and 1.2% for zinc deficiency during 0, 3, 5, and 7 hours). However, zinc supplementation during IVC supported in vitro embryonic development. On parthenogenetic activated embryos, supplementation of 0.8 μg/mL of zinc during IVC significantly increased blastocyst formation compared with other groups (43.9%, 57.8%, 67.1%, 51.4%, and 52.6% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 μg/mL). In vitro–fertilized (IVF) embryos showed similar results. The blastocyst formation rate was significantly higher in the 0.8 μg/mL of zinc-supplemented group than in the other groups (21.3%, 24.1%, 36.1%, 25.9%, and 25.2% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 μg/mL). PCNA, POU5F1, and Bcl2 messenger RNA expressions were unregulated in IVF-derived blastocysts in the 0.8 μg/mL of zinc-supplemented group compared with the control. These results suggest that zinc is required for embryonic development, and supplementation with adequate zinc concentrations during IVC improved the viability of porcine embryos, possibly by increasing PCNA, POU5F1, and Bcl2 gene expression of embryos.

Synergistic effect of hyperbaric oxygen preconditioning and hydrogen-rich saline in ameliorating rat flap ischemia/reperfusion injury

Aim: This study was conducted to evaluate the synergistic effects of hyperbaric oxygen (HBO) preconditioning and hydrogen-rich saline (HRS) treatment on skin flap survival and apoptosis in a rat ischemia/reperfusion (IR) skin flap model. Methods: Male Sprague-Dawley rats were randomly divided into five groups: one sham surgery group (sham group) and four surgery groups (IR group, HBO group, HRS group, and HBO + HRS group). An extended epigastric adipocutaneous flap (6 cm × 9 cm) was raised over the abdomen in each animal of all five groups. The last four groups underwent 6 h of IR management and were treated, respectively, with normal saline, HBO, HRS (HRS, 0.8 mmol/L), or a combined approach (HBO and HRS). On the 3 rd postoperative day, flap survival rate and perfusion condition, apoptotic index, caspase-3 activity, protein expression of pASK1 and Bcl-2/Bax ratio, and Bcl-2 messenger RNA (mRNA) expression were assessed. Results: Prior studies have shown the protective effects of HBO and HRS, both of which have been associated with an increase in flap survival. Compared to the IR group, the flaps in the HBO, HRS, and HBO + HRS groups showed better perfusion and a larger survival area with a low number of apoptotic cells, low caspase-3 activity and pASK1 expression, and a high Bcl-2/Bax ratio and Bcl-2 mRNA expression. Of these groups, the HBO + HRS group showed the best flap survival. Conclusion: Both HBO and HRS treatments increase the rate of flap survival, while the synergistic application of HBO and HRS showed a higher survival rate as compared to individual treatments of each. The potential regulation of apoptosis with the use of these two modalities may improve skin flap survival.

MiR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer

Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC.

Estrogenic effects of ginsenoside Rg1 in endometrial cells in vitro were not observed in immature CD-1 mice or ovariectomized mice model

The present study was designed to determine whether ginsenoside Rg1 could exert selective estrogenic effects by using both cell lines and an animal model. The endometrial Ishikawa cells and preosteoblastic MC3T3-E1 cells were treated with a different dose of Rg1. Immature CD-1 mice and ovariectomized (OVX) C57BL/6J mice were used to study the short-term and long-term estrogenic effects of Rg1, respectively. Rg1 significantly increased estrogen receptor-dependent alkaline phosphatase activity, activated estrogen response element-luciferase activity, and induced the phosphorylation of mitogen-activated protein kinase kinase, extracellular-regulated kinase, and estrogen receptor-α in Ishikawa cells. In contrast, Rg1 did not induce any estrogenic responses in MC3T3-E1 cells. Administration of Rg1 to immature CD-1 mice did not alter their uterine weight or the estrogen-regulated gene expressions in the uterus. Treatment of OVX C57BL/6J mice with Rg1 via mini-osmotic pumps for 3 months did not alter the uterine weight or induce any transcriptional activation of estrogen receptor in the uterus. Rg1 induced Bcl-2 messenger RNA expression in the left ventricular tissue and striatum but failed to alter the bone mineral density in the femur and tibia of the OVX mice. Rg1 exerted potent estrogenic effects in endometrial cells in vitro as well as in heart and brain tissues in vivo. However, it did not exert any estrogenic effects on reproductive tissues in vivo, nor did it stimulate bone tissues in vitro or in vivo. Our results suggest that the estrogenic effects of Rg1 are distinct from those of estradiol and are cell type and tissue selective.