TdT-mediated dUTP-biotin Nick End Labeling Research Articles

C-MYC-induced long noncoding RNA MEG3 aggravates kidney ischemia\u2013reperfusion injury through activating mitophagy by upregulation of RTKN to trigger the Wnt/\u03b2-catenin pathway

Ischemia–reperfusion injury (IRI)-induced acute kidney injury (AKI) is a life-threatening disease. The activation of mitophagy was previously identified to play an important role in IRI. Maternally expressed 3 (MEG3) can promote cerebral IRI and hepatic IRI. The present study was designed to study the role of MEG3 in renal IRI. Renal IRI mice models were established, and HK-2 cells were used to construct the in vitro models of IRI. Hematoxylin–eosin staining assay was applied to reveal IRI-triggered tubular injury. MitoTracker Green FM staining and an ALP kit were employed for detection of mitophagy. TdT-mediated dUTP-biotin nick-end labeling assay was used to reveal cell apoptosis. The results showed that renal cortex of IRI mice contained higher expression of MEG3 than that of sham mice. MEG3 expression was also elevated in HK-2 cells following IRI, suggesting that MEG3 might participate in the development of IRI. Moreover, downregulation of MEG3 inhibited the apoptosis of HK-2 cells after IRI. Mitophagy was activated by IRI, and the inhibition of MEG3 can restore mitophagy activity in IRI-treated HK-2 cells. Mechanistically, we found that MEG3 can bind with miR-145-5p in IRI-treated cells. In addition, rhotekin (RTKN) was verified to serve as a target of miR-145-5p. MEG3 upregulated RTKN expression by binding with miR-145-5p. Further, MEG3 activated the Wnt/β-catenin pathway by upregulation of RTKN. The downstream effector of Wnt/β-catenin pathway, c-MYC, served as the transcription factor to activate MEG3. In conclusion, the positive feedback loop of MEG3/miR-145-5p/RTKN/Wnt/β-catenin/c-MYC promotes renal IRI by activating mitophagy and inducing apoptosis, which might offer a new insight into the therapeutic methods for renal IRI in the future.

LncRNA XIST sponges miR-199a-3p to modulate the Sp1/LRRK2 signal pathway to accelerate Parkinson's disease progression.

In vitro and in vivo models of Parkinson’s disease were established to investigate the effects of the lncRNA XIST/miR-199a-3p/Sp1/LRRK2 axis. The binding between XIST and miR-199a-3p as well as miR-199a-3p and Sp1 were examined by luciferase reporter assay and confirmed by RNA immunoprecipitation analysis. Following the Parkinson’s disease animal behavioural assessment by suspension and swim tests, the brain tissue injuries were evaluated by hematoxylin and eosin, TdT-mediated dUTP-biotin nick end labelling, and tyrosine hydroxylase stainings. The results indicated that miR-199a-3p expression was downregulated, whereas that of XIST, Sp1 and LRRK2 were upregulated in Parkinson’s disease. Moreover, miR-199a-3p overexpression or XIST knockdown inhibited the cell apoptosis induced by MPP+ treatment and promoted cell proliferation. The neurodegenerative defects were significantly recovered by treating the cells with shXIST or shSp1, whereas miR-199a-3p inhibition or Sp1 and LRRK2 overexpression abrogated these beneficial effects. Furthermore, the results of our in vivo experiments confirmed the neuroprotective effects of shXIST and miR-199a-3p against MPTP-induced brain injuries, and the Parkinson’s disease behavioural symptoms were effectively alleviated upon shXIST or miR-199a-3p treatment. In summary, the results of the present study showed that lncRNA XIST sponges miR-199a-3p to modulate Sp1 expression and further accelerates Parkinson’s disease progression by targeting LRRK2.

Effect of lactoferrin on murine sperm apoptosis induced by intraperitoneal injection of lipopolysaccharide.

Genital bacterial infection is one of the most important causes of infertility, however, bacteria frequently exist in seminal fluid. Sperm express Toll-like receptors (TLRs) on their cell surfaces and bacterial recognition by TLRs induces sperm apoptosis. In this study, we examined the lactoferrin (LF) potentiality on sperm apoptosis induced by bacterial lipopolysaccharide (LPS). The TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay indicated that TUNEL-positive sperm cells were scarce in the group treated with LF and LPS (LF/LPS group) compared to the group treated with LPS only (LPS group). In addition, real-time RT-PCR detected lower mRNA expression levels of apoptosis-associated genes in the LF/LPS group compared to the LPS group. These results indicate that LF treatment of semen might decrease LPS-induced apoptosis of sperm.

Single high-dose peroral caffeine intake inhibits ultraviolet radiation-induced apoptosis in human lens epithelial cells in vitro.

The aim of the present study was to determine whether caffeine concentrations in human lens epithelial cells (LECs) achieved from acute peroral caffeine intake inhibit ultraviolet radiation-induced apoptosis in vitro. Patients were planned for cataract surgery of both eyes with a caffeine abstinence of 2weeks in total, starting 1week before surgery of the first eye. The second eye was scheduled 1week after the first eye. At the day of the second eye surgery, patients were given coffee containing 180mg caffeine shortly before surgery. Lens capsules including LEC, harvested after capsulorhexis, were transferred to a cell culture dish and immediately exposed to close to threshold ultraviolet radiation (UVR). At 24hr after UVR exposure, apoptotic LECs were analysed by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were detected in UVR-exposed lens capsules both after caffeine intake and in controls. The mean difference in TUNEL-positive cells between caffeine intake and contralateral controls (no caffeine) resulted in a 95% CI 15.3±10.4% (degrees of freedom: 16). Peroral caffeine consumption significantly decreased UVR-induced apoptosis in LEC supporting epidemiological findings that caffeine delays the onset of cataract.

Long Non-coding RNA TUG1 Modulates Expression of Elastin to Relieve Bronchopulmonary Dysplasia via Sponging miR-29a-3p.

Objective: Multiple studies have highlighted that long non-coding RNAs (lncRNAs) may exert paramount roles in relieving bronchopulmonary dysplasia (BPD). The aim of our investigation is to probe the role and mechanism of lncRNA taurine upregulated gene 1 (TUG1) in BPD.Methods: The current mouse model of BPD was simulated by induction of hyperoxia, and hyperoxia-induced mouse type II alveolar epithelial (MLE-12) (MLE-12) cells were established as a cellular model. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine relative expressions of TUG1, miR-29a-3p, and elastin (ELN). We assessed cell apoptosis by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining. Western blot was used for detection of apoptosis-related proteins. Moreover, cell viability was tested by cell counting kit-8 (CCK-8) assay. Inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter (DLR) assay was employed to confirm relationship between genes.Results: Upregulation of miR-29a-3p was found in lung tissues of BPD mice compared with lung tissues without BPD, while downregulations of TUG1 and ELN were discovered in BPD tissues in comparison with tissues without BPD. Increasing TUG1 was shown to alleviate lung injury of BPD mice and promote proliferation of hyperoxia-induced MLE-12 cells. Meanwhile, TUG1 inhibited inflammatory response and cell apoptosis in lung tissues of BPD mice and hyperoxia-induced MLE-12 cells. miR-29a-3p was targeted by TUG1 and negatively modulated by TUG1. ELN was inversely regulated by miR-29a-3p. Meantime, suppressive effects of TUG1 on apoptosis and inflammation were reversed by decreasing ELN or increasing miR-29a-3p in hyperoxia-induced MLE-12 cells.Conclusion: lncRNA TUG1 relieved BPD through regulating the miR-29a-3p/ELN axis, which provided a therapeutic option to prevent or ameliorate BPD.

Induction of apoptosis in indole-3-carbinol-treated lung cancer H1299 cells via ROS level elevation.

This study was focused on investigating the anticancer potential of indole-3-carbinol (I3C) against lung cancer H1299 cells via an increase in ROS levels. To investigate the induction of growth arrest and/or cell death in H1299 cells, a cell cycle arrest assay, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay, and reactive oxygen species (ROS) detection assay were performed. Through the TUNEL assay, we detected I3C-induced DNA fragmentation. Fluorescence-activated cell sorting (FACS) analysis showed that I3C induced an increase in ROS levels and apoptotic rate in a dose- and time-dependent manner in H1299 cells. Western blotting demonstrated that activated forms of caspase-3, caspase-7, caspase-9, and poly (ADP-ribose) polymerase (PARP) were increased in I3C-treated H1299 cells following treatment with I3C. Furthermore, protein expression levels of FOXO3, bim, bax, and phosphorylated ERK and JNK were increased, while those of pAkt, Bcl-xL, and Bcl-2 were decreased by I3C treatment of H1299 cells. To confirm the relationship between cell apoptosis and ROS generation, H1299 cells were treated with I3C simultaneously with N-acetylcysteine (NAC), and it was shown that ROS levels decreased and viability increased. Moreover, in western blot analysis, expression of anti-apoptotic proteins (thioredoxin1, peroxiredoxin-1, Bcl-2, and Bcl-xL) in I3C-treated cells was evidently downregulated and pro-apoptotic proteins (active ASK1 and cleaved PARP) were upregulated compared to cells co-treated with NAC. The study showed that I3C induced downregulation of ROS regulator proteins and elevation of ROS, thus activating apoptotic signaling cascades in human lung cancer H1299 cells.

MicroRNA‐203–mediated inhibition of doublecortin underpins cardioprotection conferred by sevoflurane in rats after myocardial ischaemia‐reperfusion injury

Myocardial ischaemia‐reperfusion (I/R) injury is a serious illness with high morbidity and mortality. Mounting evidence indicates the utility of sevoflurane (SEV) in the treatment of myocardial I/R injury. This study aimed to explore the molecular mechanisms underlying the protective action of SEV against myocardial I/R injury. A rat model of myocardial I/R injury was established, and I/R rats were treated with different concentrations of SEV. MicroRNA‐203 (miR‐203) and doublecortin (DCX) expression levels were determined using reverse transcription‐quantitative polymerase chain reaction. Putative target relationship between miR‐203 and DCX was explored using dual‐luciferase reporter gene assay and RNA‐binding protein immunoprecipitation assay. Ischaemia‐reperfusion rats were treated with SEV, miR‐203 antagomir or sh‐DCX, followed by determination of oxidative stress‐ and inflammation‐related factor levels using nitrite and enzyme‐linked immunosorbent assays, and that of apoptosis‐related factors using Western blot analysis. The apoptotic rate of myocardial tissues was determined using TdT‐mediated dUTP‐biotin nick end labeling (TUNEL) staining, and the infract area was evaluated using triphenyltetrazolium chloride staining. The results showed miR‐203 was poorly expressed and DCX was highly expressed in myocardial tissues of I/R rats. Sevoflurane was found to elevate miR‐203, and miR‐203, in turn, could target and reduce DCX expression. Sevoflurane, miR‐203 overexpression or DCX silencing resulted in declined oxidative stress, inflammation, apoptosis and infarct area, ultimately alleviating myocardial I/R injury. Collectively, these findings showed that SEV‐activated miR‐203 exhibited suppressive effects on myocardial I/R injury in rats and highlighted the SEV/miR‐203/DCX axis as a promising therapeutic target for myocardial I/R injury management.

Prion peptide-mediated calcium level alteration governs neuronal cell damage through AMPK-autophagy flux

BackgroundThe distinctive molecular structure of the prion protein, PrPsc, is established only in mammals with infectious prion diseases. Prion protein characterizes either the transmissible pathogen itself or a primary constituent of the disease. Our report suggested that prion protein-mediated neuronal cell death is triggered by the autophagy flux. However, the alteration of intracellular calcium levels, AMPK activity in prion models has not been described. This study is focused on the effect of the changes in intracellular calcium levels on AMPK/autophagy flux pathway and PrP (106–126)-induced neurotoxicity.MethodsWestern blot and Immunocytochemistry was used to detect AMPK and autophagy-related protein expression. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells. Calcium measurement was employed using fluo-4 by confocal microscope.ResultsWe examined the effect of calcium homeostasis alterations induced by human prion peptide on the autophagy flux in neuronal cells. Treatment with human prion peptide increased the intracellular calcium concentration and induced cell death in primary neurons as well as in a neuronal cell line. Using pharmacological inhibitors, we showed that the L-type calcium channel is involved in the cellular entry of calcium ions. Inhibition of calcium uptake prevented autophagic cell death and reduction in AMP-activated protein kinase (AMPK) activity induced by human prion peptide.ConclusionOur data demonstrated that prion peptide-mediated calcium inflow plays a pivotal role in prion peptide-induced autophagic cell death, and reduction in AMPK activity in neurons. Altogether, our results suggest that calcium influx might play a critical role in neurodegenerative diseases, including prion diseases.3p5PCBHnn1RWkUoaQCoeATVideo

MiR-808 inhibits cardiomyocyte apoptosis and expressions of caspase-3 and caspase-9 in rats with myocardial infarction by regulating TGF-β1 signaling pathway.

To investigate the effects of micro ribonucleic acid (miR)-808 on cardiomyocyte apoptosis and expressions of caspase-3 and caspase-9 in rats with myocardial infarction (MI) by regulating the transforming growth factor-β1 (TGF-β1) signaling pathway. A total of 24 specific pathogen-free female Sprague-Dawley rats were enrolled and randomly divided into normal group, model group, and miR-808 group, 8 rats in each group. In the model group and miR-808 group, MI model was prepared by ligation of the left anterior descending coronary artery in the rats. The miR-808 group was transfected with miR-808 lentivirus after the model was established. After one week of intervention, the expression of TGF-β1 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The cardiac function of rats was determined by echocardiography. The myocardium of rats was observed by Masson staining. The cardiomyocyte apoptosis of rats was examined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. The expression levels of caspase-3 and caspase-9 were detected by Western blotting. The expression of TGF-β1 mRNA was higher in the model group than that in the normal group (p<0.05), but compared with that in the model group, it was lower in the miR-808 group. The myocardial function and cardiomyocyte survival rate in the miR-808 group was better and higher than those in the model group (p<0.05). The expression levels of caspase-3 and caspase-9 in the miR-808 group were lower than those in the model group (p<0.05). MiR-808 can inhibit cardiomyocyte apoptosis in rats with MI by down-regulating TGF-β1 expression and inhibiting the expressions of caspase-3 and caspase-9.

Radiotheranostic Targeting Cancer Stem Cells in Human Colorectal Cancer Xenografts.

The aim of this study was to perform radiotheranostics with radioiodinated monoclonal antibodies (mAbs) for targeting cancer stem cells (CSCs) in human colorectal cancer xenografts and evaluate the relative advantage of a cocktail containing both [131I]CD133 mAb and [131I]CD44 mAb. Tumor-bearing mice were randomly divided into eight groups: [131I]CD133mAb, [131I]CD44 mAb, [131I]IgG isotype control, radioiodinated mAb cocktail, CD133 mAb, CD44 mAb, unradioiodinated mAb cocktail, and saline groups. In vivo single photon emission computed tomography (SPECT) imaging was used to monitor dynamically changes in the CSC population after treatment. The radioactivity uptake of tumors was quantified ex vivo. The expression of CD133 and CD44 after treatment was also assessed by immunohistochemistry and western blot. Tumor growth curves and survival curves were generated to assess treatment efficacy. Cell apoptosis and proliferation in xenografts 30days after treatment were measured by TdT-mediated dUTP-biotin nick end labeling (aka, TUNEL) and Ki67 staining. The expression levels of biomarkers in xenografts 30days after treatment were measured by flow cytometry. The radioimmunoimaging (RII) with in vivo SPECT showed that the CSC-targeting radioimmunotherapy (RIT) groups ([131I]CD133 mAb, [131I]CD44 mAb, and radioiodinated mAb cocktail groups) had intense accumulations of radiolabeled agents in the tumor areas. The ex vivo biodistribution confirmed these findings. In the CSC-targeting RIT groups, immunohistochemistry and western blot indicated significant reduction of specific target expression in the xenografts. The tumor growth curves and survival curves showed that the CSC-targeting RIT significantly inhibited tumor growth and prolonged mean survival, respectively. Significantly increased apoptosis and decreased proliferation in xenografts further confirmed the therapeutic efficacy of CSC-targeting RIT. Flow cytometry showed that the decreases in CSCs correlated with the presence of the corresponding antibodies. Our results suggest that the CSC-targeting RIT can effectively reduce CSCs which consequently inhibits tumor development. The radioiodinated mAb cocktail may generate enhanced CSC-targeting specificity.

MiR-22 protect PC12 from ischemia/reperfusion-induced injury by targeting p53 upregulated modulator of apoptosis (PUMA)

ABSTRACT MicroRNAs have been implicated as critical regulatory molecules in many cerebrovascular diseases. Recent studies demonstrated miR-22 might provide a potential neuroprotective effect. However, the neuroprotective effect of miR-22 in ischemia/reperfusion (I/R) injury has not been thoroughly elucidated. In this study, the PC12 cells were subjected to 4 h oxygen and glucose deprivation (I) and 24 h reoxygenation (R). The PC12 cells were pre-transfected with miR-22 or anti-miR-22 or siRNA-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA)(PUMA siRNA) or their controls at 24 h prior to exposure to I/R. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were employed to analyze mRNA and protein expression. PI and Annexin V assays and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to quantify the rate of apoptosis. We found that miR-22 expression was significantly downregulated in the PC12 cells subjected to I/R. Loss of function of miR-22 increased PC12 apoptosis after I/R, and overexpression of miR-22 decreases PC12 apoptosis after I/R. PUMA protein was upregulated in the I/R group as compared with the sham group. The increased PUMA protein expression and apoptosis induced by I/R was reversed by transfection with PUMA siRNA. We concluded that I/R enhanced apoptosis and PUMA expression in PC12 cells via downregulation of miR-22. Enhanced miR-22 expression reversed both PUMA expression and apoptosis induced by I/R in PC12 cells. miR-22/PUMA axis has important implications for their clinical applications.

Protective Effects of Ginsenoside Rg1 on Acute Myocardial Infarction

Context: Ginsenoside Rg1 (Rg1), as the active ingredient of Panax notoginseng, has protective effect on the cardiovascular system. While, the efficiency of Rg1 against acute myocardial infarction (MI) is not fully elucidated. The aim of this study was to investigate the cardio-protection and the underlying mechanism for Rg1. Objective: Myocardial infarction was induced by ligation of the left anterior descending coronary artery of wistar rats. 2,3,5-triphenyltetrazolium chloride (TTC) stain assay was used to detect infarct size; hematoxylin and eosin (HE) and phosphotungstic acid-hematoxilin (PTAH) stain assay were used to evaluate the cardiac structure; terminal deoxytransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to detect apoptosis; immunohistochemical stain was used to evaluate neutrophils and macrophages infiltration. Key Findings: Firstly, Extract Rg1 (P < 0.01) and Ferment Rg1 (P < 0.01) down-regulated myocardial infarct size and up-regulated superoxide dismutase (SOD) activity significantly. Then, HE and PTAH stain indicated the protection of Extract Rg1 and Ferment Rg1 on heart structure, especially on the integrity of sarcomere. Finally, both Extract Rg1 (P < 0.001) and Ferment Rg1 (P < 0.001) reduced the number of apoptosis cells, and further inhibited the infiltration of neutrophils into the infarct site of heart. Summary: For the first time, our present study verified the efficiency of Rg1 against acute MI, and provided experimental evidence that Ferment Rg1 holds similar cardio-protection with Extract Rg1. Rg1 holds potential therapeutic value for further drug development on heart disease.

Inhibition of T cell immunoglobulin and mucin-1 (TIM-1) protects against cerebral ischemia-reperfusion injury

BackgroundThe T cell Ig domain and mucin domain (TIM)-1 protein expressed on the surface of Th2 cells regulates the immune response by modulating cytokine production. The present study aimed to investigate the role and possible mechanism of TIM-1 in cerebral ischemia-reperfusion injury.MethodsWestern blot was used to detect TIM-1 and apoptosis-related protein expression, whereas TIM-1 mRNA was examined using quantitative real-time reverse transcription PCR. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells and a pathological examination was performed. The migration of neutrophils and macrophages was analyzed by immunohistochemistry.ResultsOur results suggest that TIM-1 expression was transiently increased 24 h or 48 h following middle cerebral artery occlusion (MCAO)/reperfusion. The infarct size was markedly increased in MCAO, whereas treatment with a TIM-1-blocking mAb could reduce the infarct size. TIM-1 blocking mAb effectively reduced the number of neutrophils, macrophage functionality, cytokine (i.e., IL-6, IL-1β, and TNF-α) and chemokine (i.e., CXCL-1 and CXCL-2) production in the brain tissue. The effect of in vitro T cell damage on neurons was significantly reduced following treatment with a TIM-1 blocking mAb or the knockdown of TIM-1 in co-cultured T cells and neurons.ConclusionTake together, these results indicated that TIM-1 blockade ameliorated cerebral ischemia-reperfusion injury. Thus, TIM-1 disruption may serve as a novel target for therapy following MCAO.

The Synthesis and Biological Function of a Novel Sandwich-Type Complex Based on {SbW9 } and Flexible bpp Ligand.

A novel sandwich-type complex [Na(H2 O)4 ][{Na3 (H2 O)5 }{Mn3 (bpp)3 } (SbW9 O33 )2 }]·8H3 O (MnSbW-bpp) (bpp = 1,3-bis(4-pyridyl) propane) is synthesized and characterized by elemental analysis, IR, thermogravimetric analysis, and single-crystal X-ray diffraction. The MnSbW-bpp compound is the first sandwich case bridged by a flexible ligand. Its biological function of MnSbW-bpp in antitumor activity is also determined in vitro and in vivo. The inhibitory proliferation and induction of apoptosis are performed by flow cytometry assay, S180 (sarcoma) tumor xenograft in ICR mice, the color Doppler ultrasound monitor, and TdT-mediated dUTP-biotin nick end labeling assay. The results show that the novel compound-MnSbW-bpp-is synthesized and identified by its physical and chemical characteristics, such as the fluorescent and paramagnetic activities. MnSbW-bpp indicates a potency inhibition of human cancer lines, such as SGC-7901, HT-29, HepG2, Hela, U2OS, SaoS2, and HMC cells. MnSbW-bpp also inhibits the growth of tumor xenograft in mice, induced cell apoptosis, and released cytochrome c in vivo and in vitro. Thus, MnSbW-bpp, as a new compound, possesses the potent inhibition of cancer cells, which indicates that the MnSbW-bpp has potential merit for the further evaluation of a novel antitumor agent.

Radix Kansui Stir-Fried with Vinegar Reduces Radix Kansui-Related Hepatotoxicity in Mice via Mitochondrial Pathway.

To investigate the mechanism of Radix Kansui (RK) stir-fried with vinegar (VRK) decreased hepatotoxicity in mice. According to a random number table, 40 mice were randomly divided into negative control group (0.5% carboxymethylcellulose sodium, 20 mL/kg), positive control group (0.1% mixture of carbon tetrachloride in soybean oil, 20 mL/kg), RK group (the ethyl acetate extracts of RK, 250 g crude drug/kg) and VRK group (the ethyl acetate extracts of VRK, 250 g crude drug/kg) with 10 mice per group. All mice were administered orally by gavage daily for 7 continuous days. The morphology of liver tissues was examined to assess the liver injury by a transmission electron microscope. Hepatocyte apoptosis in vivo was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling (TUNEL) assay. Immunohistochemical technique was adopted to detect the expression of particular antiapoptotic and proapoptotic proteins in the mitochondrial pathways, including B-cell lymphoma (Bcl-2) and caspase-3, as well as the expression of inflammatory mediators, including nuclear factor kappa B (NF- κ B) and intercellular adhesion molecule-1 (ICAM-1). Liver injury and hepatocyte apoptosis were observed in RK mice, and the liver injury were significantly reduced in VRK-treated mice. In immunohistochemistry study, compared with the negative control group, RK inhibited dramatically the Bcl-2 protein expression and significantly increased the expression of caspase-3, NF- κ B and ICAM-1 (all P&lt;0.01). Compared with the RK group, VRK group induced significant increase on Bcl-2 protein expression, and decreased the caspase-3, NF- κ B and ICAM-1 protein expression (P&lt;0.05 or P&lt;0.01). The mechanism of reduced hepatotoxicity of VRK may be associated with the reduced inflammation, regulation of antiapoptotic and proapoptotic mediators in the mitochondrial pathway.

The apoptotic effects of bisphenol A exposure on the rat ovary: an experimental study.

Bisphenol A (BPA) is a key endocrine-disrupting chemical (EDC) in the manufacturing industry. It is found in the structure of compounds such as polycarbonate and epoxy in combination with other chemicals. Our objective was to investigate the effect of BPA on rat ovaries. A total of 32 female rats were divided into four equal groups: In group 1 (control), vehicle was administered; in group 2, BPA 50μg/day was administered intraperitoneally; in group 3, BPA 100mg/kg/day was administered intraperitoneally; and in group 4, BPA 100mg/kg/day and vitamin C (50mg/kg) were administered intraperitoneally, while vitamin E (50mg/kg) was administered intramuscularly. Thirty days after the treatment, the effects of BPA on the ovaries were evaluated by terminal deoxynucleotidyltransferase [TdT]-mediated dUTP-biotin nick end labeling (TUNEL) assay. There was no difference in the number of apoptotic cells between group 2 and group 4. In addition, there was no significant difference between control group and group 2, 4. However, the number of apoptotic cells per unit area was significantly increased in group 3 compared with all groups (p < 0.01, p < 0.05). In conclusion, this study showed that high doses of BPA (100mg/kg/day) have a toxic effect on the ovaries. The fact that the number of apoptotic cells in the group administered with high dose of BPA + 50mg/kg/day vitamin C + 50mg/kg/day vitamin E was lower than that of the high-dose BPA-administered group shows that these vitamins may have a protective effect.

LHX1-a multifunctional regulator in preoptic area-derived interneuron development.

LHX1-a multifunctional regulator in preoptic area-derived interneuron development.

Neuroprotective effect of Notch pathway inhibitor DAPT against focal cerebral ischemia/reperfusion 3 hours before model establishment.

As an inhibitor of the Notch signaling pathway, N-[N-(3,5-difluorohenacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester (DAPT) may protect brain tissue from serious ischemic injury. This study aimed to explore neuroprotection by DAPT after cerebral ischemia/reperfusion (I/R) injury. DAPT was intraperitoneally injected 3 hours before the establishment of a focal cerebral I/R model in the right middle cerebral artery of obstructed mice. Longa scores were used to assess neurological changes of mice. Nissl staining and TdT-mediated dUTP-biotin nick-end labeling staining were used to examine neuronal damage and cell apoptosis in the right prefrontal cortex, while immunofluorescence staining was used to detect glial fibrillary acidic protein- and Notch1-positive cells. Protein expression levels of Hes1 and Hes5 were detected by western blot assay in the right prefrontal cortex. Our results demonstrated that DAPT significantly improved neurobehavioral scores and relieved neuronal morphological damage. DAPT decreased the number of glial fibrillary acidic protein- and Notch1-positive cells in the right prefrontal cortex, while also reducing the number of apoptotic cells and decreasing interleukin-6 and tumor necrosis factor-α contents, and simultaneously downregulating Hes1 and Hes5 protein expression. These findings verify that DAPT alleviates pathological lesions and strengthens the anti-inflammatory response after cerebral I/R injury. Thus, DAPT might be developed as an effective drug for the prevention of cerebral I/R injury.

Indole-3-Carbinol Induces Apoptosis in Human Osteosarcoma MG-63 and U2OS Cells.

This study was focused on investigating the anticancer potential of indole-3-carbinol (I3C) against osteosarcoma MG-63 and U2OS cells. A wound healing assay indicated that IC3 inhibited migration of MG-63 and U2OS cells. MTT, WST-1, and colony formation assays revealed that treatment of MG-63 and U2OS cells with I3C decreased cell viability. Fluorescence-activated cell sorting (FACS) analysis showed that I3C induced apoptosis in a dose- and time-dependent manner in MG-63 and U2OS cells. Moreover, via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP-biotin nick-end labeling (TUNEL) assay, we detected that I3C induced DNA fragmentation. Western blotting demonstrated that activated forms of caspase-3, caspase-7, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP) were increased in MG-63 and U2OS cells, following treatment with I3C. Furthermore, protein expression levels of FOXO3, Bax, and Bim extra-large form were increased while those of Akt, JNK, p38, phosphorylated ERK, and Bcl-xL were decreased by I3C treatment in MG-63 and U2OS cells. Thus, the study indicates that I3C may induce apoptosis in human osteosarcoma MG-63 and U2OS cells via the activation of apoptotic signaling pathways by FOXO3.

MicroRNA-874 inhibition targeting STAT3 protects the heart from ischemia-reperfusion injury by attenuating cardiomyocyte apoptosis in a mouse model.

MicroRNAs (miRs) were involved in numerous cardiovascular diseases, especially ischemic heart diseases, but the miR changes during cardiac ischemia-reperfusion (I/R) injury following sevoflurane (SEV) preconditioning are still unknown. This study aims to investigate the effect of miR-874 on cardiac I/R injury in mouse models pretreated with SEV. Following establishment of mouse models with myocardial I/R injury, mice were pretreated with SEV. The functional mechanism of miR-874 in I/R injury was explored when miR-874 and the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway were inhibited. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP biotin nick-end labeling (TUNEL) staining was used to detect cardiomyocyte apoptosis and dual luciferase reporter gene assay to identify the targeting relationship between miR-874 and STAT3. Expression of the JAK2/STAT3 signaling pathway and apoptosis-related genes was determined. Initially, upregulated miR-874 was observed in I/R mice. Then, miR-874 inhibition improved cardiac function of I/R mice, inhibited cardiomyocyte apoptosis (also shown as decreased Bcl-2 associated X protein B [Bax] and increased B-cell lymphoma-2 [Bcl-2]), and activated the JAK2/STAT3 signaling pathway. STAT3, a target gene of miR-874, was upregulated following miR-874 inhibition. Finally, we also observed that the effect of miR-874 was lost when the JAK2/STAT3 signaling pathway was blocked. The findings indicate miR-874 as a contributory role in cardiac I/R injury, with miR-874 inhibition alleviating cardiac I/R injury in mice following SEV pretreatment by targeting STAT3 through the JAK2/STAT3 signaling pathway.