TdT-mediated dUTP Nick-end Labeling Staining Research Articles

Circulating Exosomal miR-1-3p from Rats with Myocardial Infarction Plays a Protective Effect on Contrast-Induced Nephropathy via Targeting ATG13 and activating the AKT Signaling Pathway.

Rationale: With the widespread development of the interventional technique for cardiovascular diseases and the widespread use of contrast medium (CM), the incidence of contrast-induced nephropathy (CIN) has been increasing, which is associated with poor prognosis for cardiovascular diseases. This study aims to explore the effect of circulating exosomal microRNA from patients with myocardial infarction (MI) on CIN and related molecular mechanism. Methods: A rat MI model was established by ligating the left anterior descending coronary artery. Circulating exosomes were isolated from control (Exo-NC) and MI rats (Exo-MI) using a commercial kit. The in vivo and in vitro models of CIN were created using iodixanol. Reverse transcription quantitative PCR (RT-qPCR) was utilized to detect the expression of miR-1-3p. Western blot (WB) was used to detect the expression of exosomal surface markers, and apoptosis-related and autophagy-related proteins. The apoptosis rate was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry (FC). Transmission electron microscopy (TEM) was utilized to observe the exosomes and autophagosomes. Rat kidney injury was assessed by hematoxylin and eosin (H&E) staining and kidney injury molecule-1 (KIM-1) immunohistochemical staining. Renal function of rats was assessed by detecting the levels of blood urea nitrogen (BUN) and serum creatinine (Cr). The dual luciferase reporter assay was performed to identify the target gene of miR-1-3p. Results: The treatment of CM induced NRK-52E cell damage, which manifested as enhanced cell autophagy and enhanced apoptosis. The Exo-MI treatment significantly inhibited the CM-induced autophagy and apoptosis of NRK-52E cells. Furthermore, the Exo-MI treatment increased the Bcl-2 expression, but decreased the Bax expression and the ratio of LC3II/LC3I. Furthermore, the results of the TUNEL staining and FC showed that Exo-MI can reduce apoptotic rate. Through TEM, it was found that Exo-MI reduced the number of autophagosomes in NRK-52E cells. The rescue experiments revealed that the function of Exo-MI is to inhibit the CM-induced autophagy and apoptosis of NRK-52E cells, which can be inhibited by the miR-1-3p inhibitor. Furthermore, it was found that the overexpression of miR-1-3p can also inhibit the CM-induced autophagy and apoptosis of NRK-52E cells. Through dual luciferase reporter assay, ATG13 was found to be the target of miR-1-3p. In addition, the overexpression of miR-1-3p significantly reversed the CM-induced decrease in phosphorylation level of AKT. Furthermore, ATG13 silencing can also inhibit the CM-induced autophagy and apoptosis of NRK-52E cells. In vivo, Exo-MI significantly alleviated the renal injury, reduced the renal fibrosis, and improved the renal function of CIN rats. Conclusion: The circulating exosomal miR-1-3p after MI inhibited the CM-induced apoptosis and autophagy of renal tubular epithelial cells, and improved the renal function of rats by targeting ATG13 and activating the AKT signaling pathway.

Heat-killed Salmonella Typhimurium protects mice against carbon ion radiation.

ObjectivePatients receiving carbon-ion radiation therapy and astronauts exploring outer space are inevitably exposed to heavy ion radiation. The aim of this study was to develop radioprotectors to minimize the injuries induced by carbon ion radiation.MethodsHeat-killed Salmonella Typhimurium (HKST) was administered to mice by gavage prior to irradiation with a 12C6+ heavy ion accelerator. Hematoxylin and eosin staining and immunofluorescence TdT-mediated dUTP Nick-End Labeling staining were used to assess the radioprotective effect of HKST on organ damage and levels of apoptosis, respectively, in mice. To investigate the mechanism underlying the radioprotective effect of HKST, levels of the pro-apoptotic proteins BAX and caspase 3 as well as interferon-regulatory factor (IRF) 3/7 in the femur, testis and intestine were assessed using immunofluorescence.ResultsInjuries induced by carbon ion radiation were significantly eased by pretreatment with HKST. Both apoptosis and high expression levels of pro-apoptotic proteins induced by heavy ion radiation were inhibited by HKST pretreatment. The radioprotective effect of HKST was associated with stimulation of Toll-like receptor signaling mediated by enhanced IRF3 and IRF7 signaling.ConclusionHKST was an effective radioprotector alleviating damage to multiple organs caused by heavy ion radiation.

Exosomes derived from regulatory T cells ameliorate acute myocardial infarction by promoting macrophage M2 polarization.

Acute myocardial infarction (AMI) is a serious ischemic heart disease. Regulatory T cells (Tregs) participate in AMI. This article aims to investigate the mechanism of action of Tregs in AMI. We constructed AMI mouse model. Then, AMI mouse and mouse macrophages (RAW264.7) were treated with Tregs or Treg-derived exosomes. The cardiac function of mice was detected. Triphenyl-tetrazolium chloride and TdT-mediated dUTP nick-end labeling staining were performed to detect the myocardial infarct size or apoptosis. The proportions of macrophages were analyzed by flow cytometry. Enzyme linked immunosorbent assay and quantitative real-time PCR was performed to estimate the levels of cytokines and genes. We found that Tregs ameliorated cardiac function, reduced myocardial infarct size and inhibited apoptosis of myocardial cells in AMI mice. Moreover, Treg-derived exosomes reduced myocardial infarct size and repressed apoptosis of myocardial cells in AMI mice. Furthermore, Treg-derived exosomes suppressed the expression of M1 macrophage markers, and promoted the expression of M2 macrophage markers in myocardial tissues of AMI mice and RAW264.7 cells. In conclusion, our work demonstrates that exosomes derived from Tregs ameliorate AMI by promoting macrophage M2 polarization. Thus, Tregs may be an essential cell for AMI treatment.

MiR-28-5p mediates the anti-proliferative and pro-apoptotic effects of curcumin on human diffuse large B-cell lymphoma cells.

ObjectiveTo investigate the anti-proliferative and pro-apoptotic effects of curcumin on diffuse large B-cell lymphoma (DLBCL) cells and explore the mechanism.MethodsOCI-LY7 cells were treated with curcumin (2.5, 5, 10, 20, and 40 μM) for 24, 48, or 72 hours. Cell viability and apoptosis were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide assay and TdT-mediated dUTP nick-end labeling staining, respectively. MiR-28-5p expression was detected via qRT-PCR. The binding site of miR-28-5p was predicted using online databases and verified using the dual-luciferase reporter assay. MiR-28-5p overexpression and inhibition were achieved via transfection with an miR-28-5p mimic and inhibitor, respectively.ResultsCurcumin decreased the viability of OCI-LY7 cells in a concentration- and time-dependent manner, and these effects were attenuated by miR-28-5p inhibition. MiR-28-5p expression was upregulated by curcumin. Curcumin increased the numbers of apoptotic cells and upregulated cleaved caspase-3 expression, and these effects were attenuated by miR-28-5p inhibition. The dual-luciferase reporter assay confirmed that miR-28-5p directly targets the 3′-untranslated region of BECN1. Curcumin downregulated BECN1 and microtubule-associated protein 1 light chain 3 beta-II/I expression and upregulated p62 expression.ConclusionsOur results described the curcumin exerted anti-proliferative and pro-apoptotic effects on OCI-LY7 cells through a mechanism potentially involving miR-28-5p.

Therapeutic potential of adenovirus-encoding brain-derived neurotrophic factor for spina bifida aperta by intra-amniotic delivery in a rat model.

Spina bifida aperta is a type of neural tube defect (NTD). Although prenatal fetal surgery has been an available and effective treatment for it, the neurological functional recovery is still need to be enhanced. Our previous results revealed that deficiencies of sensory, motor, and parasympathetic neurons were primary anomalies that occurred with the spinal malformation. Therefore, we emphasized that nerve regeneration is critical for NTD therapy. We delivered an adenoviral construct containing genes inserted for green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) into the amniotic fluid to investigate its prenatal therapeutic potential for rat fetuses with spina bifida aperta. Using immunofluorescence, TdT-mediated dUTP nick-end labeling staining, and real-time polymerase chain reaction analysis, we assessed cell apoptosis in the defective spinal cord and Brn3a positive neuron survival in the dorsal root ganglion (DRG); a protein array was used to investigate the microenvironmental changes of the amniotic fluid. We found that most of the overexpressed BDNF was present on the lesions of the spina bifida fetuses, the number of apoptosis cells in Ad-GFP-BDNF-transfected spinal cords were reduced, mRNA levels of Bcl2/Bax were upregulated and Casp3 were downregulated compared with the controls, the proportion of Brn3a positive neurons in DRG were increased by activating the BDNF/TrkB/Akt signaling pathway, and most of the significant changes in cytokines in the amniotic fluid were related to the biological processes of regulation of apoptotic process and generation of neurons. These results suggest that intra-amniotic Ad-GFP-BDNF gene delivery might have potential as a supplementary approach to treat congenital malformations of neural tubes.

Neuroprotective effect of Longshengzhi capsule following permanent middle cerebral artery occlusion in rats

Longshengzhi capsule (LSZC) is an optimized preparation based on the traditional Chinese Medicine formula Buyanghuanwu Decoction (BYHWD), and is approved by the China Food and Drug Administration for treating stroke-induced disability and vascular diseases. Herein, we examined the pharmacodynamics, anti-apoptotic and anti-oxidant actions, and potential mechanisms of action of LSZC following stroke in rats. Permanent middle cerebral artery occlusion (MCAO) was used as an ischemic stroke model. LSZC was administered intragastrically. We examined the survival rate, bodyweight, and neurological deficits in stroke rats. Brain infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. Brain pathology was examined using hematoxylin and eosin staining, Nissl staining, and TdT-mediated dUTP Nick-End Labeling staining. Malondialdehyde, catalase, superoxide dismutase, and glutathione levels were examined by commercial kits. Expression of Nrf2, heme oxygenase-1, Bax, Bcl-2, cleaved caspase-3, and caspase-3 proteins in brain tissue was measured by Western blot. LSZC markedly improved the survival rate and bodyweight, and reduced infarct volume and neurological deficit scores, in MCAO stroke rats. LSZC also significantly attenuated oxidative stress, as indicated by decreased expression of malondialdehyde, and upregulation of Nrf2, heme oxygenase-1, catalase, superoxide dismutase, and glutathione. Moreover, LSZC significantly decreased apoptosis, including a decrease in Bax and cleaved caspase-3 expression, and an increase in Bcl-2, as well as a reduction in numbers of apoptotic neurons. LSZC treatment is neuroprotective against ischemic stroke, potentially via reducing oxidative stress and apoptosis. The Nrf2 and apoptotic signaling pathways may play important roles in the antioxidant and anti-apoptotic actions of LSZC.

The role of sperm associated antigen 6 gene in morphological changes of inner ear development and signal regulation of auditory organs in mice

AimIn order to analyze the effects of the deletion of the Sperm Associated Antigen 6 (Spag6) gene on the inner ear and the auditory system of mice. MethodClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology was utilized, and the Spag6 gene knockout mouse model was constructed. Self-breeding was carried out to obtain the F1 generation Spag6 homozygous mouse (Spag−/−), the Spag6 heterozygous mouse (Spag+/−) and the Spag6 wild mouse (Spag+/+) were obtained. Polymerase Chain Reaction (PCR) technology was used to verify the three genotypes of mice. The mean hearing threshold of Spag−/− mice, Spag+/− mice and Spag+/+ mice after different intensity of sound stimulation was detected. The inner ear cochlea tissues of Spag−/− mice and Spag+/+ mice were collected and paraffin sections were made. Morphological differences of inner ear tissues in mice were analyzed by hematoxylin – eosin (HE) staining. Immunofluorescence staining was used to analyze the number of hair cells in the inner ear of mice. Apoptosis of mouse inner ear hair cells was analyzed by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining ResultIt was found that the hearing of Spag−/− mice decreased significantly compared with that of Spag+/+ mice (P < 0.01), and the hearing of Spag+/− mice decreased significantly compared with that of Spag+/− mice (P < 0.05). After HE staining and immunofluorescence staining, hair cells in the inner ear cochlea of Spag−/− mice were found to be defective. The apoptosis detection results of TUNEL staining indicated that the number of apoptosis of hair cells in inner ear cochlea of Spag−/− mice was significantly higher than that of Spag+/+ mice, and the average optical density of Corti apparatus of Spag−/− mice was significantly higher than that of Spag+/+ mice (P < 0.01). ConclusionThe results showed that the loss of the Spag6 gene accelerated the apoptosis of hair cells in the cochlear tissue of the inner ear of mice, thereby affecting the auditory system of mice. The significance of this study is to lay the foundation for future studies on the effects of Spag6 on deafness.

18F-ML-10 PET/CT imaging in early evaluation of doxorubicin-induced cardiotoxicity

Objective To investigate the feasibility of early monitoring doxorubicin (DOX)-induced cardiotoxicity by apoptosis molecular imaging of 2-(5-[18F]fluoro-pentyl)-2-methyl-malonic acid (18F-ML-10) PET/CT. Methods Forty-seven BALB/c mice were randomly divided into the chemotherapy group (n=30) and the control group (n=17) according to the random number table. The mice in chemotherapy group were intraperitoneally injected with DOX (4 mg/kg) once a week for 3 weeks and mice in the control group were injected with the same amount of normal saline. All mice were subjected to 18F-fluorodeoxyglucose (FDG) and 18F-ML-10 PET/CT imaging at day 0, 2, 9, 16, and left ventricular ejection fraction (LVEF) was continuously monitored using cine cardiac MR (cine-CMR) imaging. The region of interest (ROI) was delineated on PET/CT images, and the maximum percentage activity of injection dose per gram of tissue (%ID/g) was calculated. The mice were sacrificed after imaging, and the heart tissue was taken for HE staining and TdT-mediated dUTP nick end labeling (TUNEL) assay. One-way analysis of variance, independent-samples t test and Pearson correlation analysis were used to analyze the data. Results In the chemotherapy group, the myocardial 18F-FDG uptake on day 0, 2, 9, 16 were (63.3±14.5), (93.7±24.0), (153.6±20.6) and (135.8±32.5) %ID/g respectively, and 18F-ML-10 uptake were (0.09±0.02), (0.18±0.03), (0.22±0.04) and (0.55±0.12) %ID/g respectively. Compared with baseline (day 0), 18F-FDG and 18F-ML-10 uptake were significantly increased in the chemotherapy group at each time point after DOX administration(F=6.823, 20.848, both P 0.05). TUNEL and HE staining indicated that the cardiomyocytes in the chemotherapy group showed obvious apoptosis and vacuolization, and the apoptotic index (AI) was positively correlated with the 18F-ML-10 uptake (r=0.950, P<0.01). The cine-CMR imaging results showed that the LVEF in the chemotherapy group continued to decrease after DOX administration (F=4.507, P<0.05), and significant difference was identified at day 16 (t=2.980, P<0.05). There was a significant negative correlation between 18F-ML-10 uptake and LVEF (r=-0.709, P=0.01). Conclusions Both 18F-FDG and 18F-ML-10 PET/CT imaging can early assess DOX-induced cardiotoxicity in vivo. Given the high targeting specificity of 18F-ML-10, it may have a greater clinical transformation advantage over 18F-FDG in early assessment of cardiotoxicity. Key words: Cardiotoxicity; Doxorubicin; Positron-emission tomography; Tomography, X-ray computed; Mice

Protective effect of cycloartenyl ferulate on lipopolysaccharide induced endothelial cell apoptosis and its mechanism

Objective To investigate the effect of cycloartenyl ferulate (CF) on lipopolysaccharide (LPS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC), and to explore its relative mechanism. Methods Human umbilical vein endothelial cells were cultured in vitro, and the experiment was divided into the normal group, CF group, LPS group, and LPS+CF groups at different concentrations. After the corresponding treatment of cells in each group, the cell viability and apoptosis of each group were tested by the cell counter Kit-8 (CCK-8) assay and TdT-mediated dUTP nick end labeling (TUNEL) assay. The protein expression of Bax, Bcl-2, caspase-3 and the effect of CF on the protein expression of Nrf2/HO-1 pathway were determined by Western blot. One-way ANOVA were performed in multigroup mean comparison, LSD-t test was used to compare the mean values of two samples, and P<0.05 was considered statistically significant. Results Compared with the LPS group, the survival rate of HUVECs cells in the CF group was significantly increased with different doses, and the survival rate increased significantly in the 320 μmol/L CF group [(69.85±1.2)% vs ( 100.2±1.824)%, P< 0.01]. TUNEL staining showed that compared with the LPS group, the number of apoptotic positive cells in the 320 μmol/L CF group was significantly reduced [(27.33 ± 3.40) vs (11.67 ± 2.04), P<0.01]. Compared with the LPS group, Bcl-2 protein expression level was significantly increased in the CF group at different doses (P<0.01), caspase-3 and Bax protein expression were significantly decreased (P<0.01). Nrf2 protein expression level increased significantly (P<0.01), and HO-1 protein level increased significantly (P<0.01). Conclusion CF can alleviate LPS-induced apoptosis of HUVEC, which may be related to the increase of bcl-2 expression, the decrease of caspase-3 and Bax protein expression and the activation of Nrf2/ HO-1 signaling pathway. Key words: Cycloartenyl ferulate; Lipopolysaccharide; Vascular endothelial cells; Apoptosis; Nrf2/HO-1 signaling pathway

RepSox Increases Porcine Cloning Efficiency by Improving Pluripotency of Donor Nuclei.

Accumulating evidence suggests that a low pluripotency of donor nuclei might lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). To improve the pluripotency of SCNT embryo, RepSox, a defined small-molecule compound that functions in inhibiting the transforming growth factor β signaling pathway, was used to treat nuclear-transferred porcine oocytes after activation. We found that the developmental ability of porcine SCNT embryos (defined as the blastocyst rate) was significantly increased (13.5% vs. 21.8%) when 25 μm RepSox was added to the porcine embryo culturing system for 14-16 hours after activation. Of note, RepSox treatment significantly increased the transcriptional expression of the pluripotency gene NANOG at the four- and eight-cell stages. Furthermore, according to the TUNEL (TdT-mediated dUTP nick end labeling) staining and expression levels of the apoptosis-regulated gene Caspase 3 and proapoptotic gene Bax, the percentage of apoptotic cells in blastocyst cells was not affected after RepSox treatment. These results indicated that treatment with RepSox enhanced the developmental competence of porcine SCNT embryos through improvements in nuclear pluripotency.

The therapeutic potential of epigallocatechin‑3‑gallate against human oral squamous cell carcinoma through inhibition of cell proliferation and induction of apoptosis: Invitro and invivo murine xenograft study.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in the oral region. Despite current therapeutic strategies, the survival rate has not been improved for several decades. Thus, it is important to develop a novel approach for the treatment of OSCC. Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea and has previously been demonstrated to inhibit the growth of several types of cancer cells. However, few studies have investigated the effect of EGCG on human OSCC cells, especially in experimental animal models. The aim of the present study was to evaluate the therapeutic potential of EGCG for targeting human OSCC in vitro and in vivo. In the in vitro experiments, EGCG suppressed HSC-3 cell viability in a time- and dose-dependent manner. Cell cycle analysis revealed that EGCG induced G1 phase arrest of the tumor cells. Apoptosis was examined by Annexin V and propidium iodide staining, assays of caspase-3 and −7 activity and TdT-mediated dUTP nick end labeling (TUNEL) staining. Treatment with EGCG significantly increased caspase-3 and −7 activities, and the percentage of apoptotic cells when compared with control cells. In the in vivo xenograft experiment on mice, EGCG treatment resulted in a 45.2% reduction in tumor size as compared with the control group without weight loss. In vivo cell proliferation and apoptosis were assessed by immunohistochemical Ki-67 staining and the TUNEL staining. There were significant differences in Ki-67 expression between the EGCG treatment group and control group, and the percentage of apoptotic cells in the EGCG treatment group was significantly greater than that in the control group. These results indicated that EGCG significantly inhibited cell proliferation by affecting the cell cycle progression and apoptosis in vitro and in vivo. These findings suggest that EGCG may have clinical applications as a novel approach to oral-cancer therapy.

LncRNA PVT1 Mediates Antiapoptosis and 5-Fluorouracil Resistance via Increasing Bcl2 Expression in Gastric Cancer.

Purpose Plasmacytoma variant translocation 1 (PVT1) is a long noncoding RNA encoded by the human PVT1 gene, which has been verified to mediate tumorigenesis in gastric cancer. However, the underlying molecular mechanisms of PVT1 in gastric cancer (GC) remain largely unknown. Methods The tumorigenic ability of PVT1 was verified by subcutaneous and orthotopic mouse models. Flow cytometry assay and TdT-mediated dUTP Nick-End Labeling staining were conducted to explore the effects of PVT1 on gastric cancer cell apoptosis. We investigated the relative gene and protein that are involved in apoptosis in real-time PCR and western blot assay. The resistance to 5- Fluorouracil (5-Fu) caused by PVT1 was evaluated using cell viability assay. Then, to confirm the effects of PVT1 on 5-Fu resistance, we conducted the Kaplan-Meier analysis based on three public databases. Results We confirmed that PVT1 can promote the progression of gastric cancer. PVT1 inhibited the apoptosis of GC cells, which may account for its promotion on GC. We confirmed that PVT1 can regulate the expression of Bcl2 and enhance drug-resistance of gastric cancer to 5-Fu. Kaplan-Meier analysis showed that patients with high PVT1 expression do not experience survival related benefits from 5-Fu based chemotherapy; instead, therapy containing no 5-Fu chemotherapy can improve the first progression survival and overall survival of high PVT1 expression GC patients significantly. Conclusion Our results showed that PVT1 can inhibit the apoptosis and enhance the 5-Fu resistance of gastric cancer through the activation of Bcl2. PVT1 has the potential to serve as an indicator to predict 5-Fu treatment resistance.

Effects of zerumbone on proliferation and apoptosis of esophageal cancer cells and on P53 and Bcl-2 expression levels.

The effects of zerumbone on the proliferation and apoptosis of esophagus cancer cells and on the P53 and Bcl-2 expression levels were studied. The esophagus cancer EC-109 cells were cultured and inoculated. The effect of zerumbone on proliferation of EC-109 cells was detected via the Cell Counting Kit-8 (CCK-8) method. Cell apoptosis was detected via TdT-mediated dUTP nick end-labeling (TUNEL) staining. Moreover, the mRNA expression levels of P53 and Bcl-2 were detected via reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression levels of P53 and Bcl-2 were evaluated via western blotting. CCK-8 detection results showed that compared with control group, zerumbone in different concentrations could inhibit the activity of EC-109, and the proliferation inhibition rate was significantly increased in a concentration-dependent manner with the increase of concentration. TUNEL staining showed that cell apoptosis gradually occurred in administration group, and the number of apoptotic cells was increased in a concentration-dependent manner with the increase of concentration. RT-PCR detection results showed that the mRNA expression level of P53 in administration group was significantly increased compared with that in control group, but that of Bcl-2 was significantly decreased. Western blotting showed that the protein expression level of Bcl-2 in administration group in different concentrations was significantly increased with the increase of zerumbone concentration, but that of Bcl-2 was significantly decreased in a concentration-dependent manner. Zerumbone can inhibit the proliferation and induce apoptosis of esophageal cancer EC-109 cells, and its induction of apoptosis may be realized through upregulating the mRNA expression of P53 and downregulating the mRNA expression of Bcl-2, and upregulating the protein expression of P53 and downregulating the protein expression of Bcl-2.

Cyanidin Protects SH-SY5Y Human Neuroblastoma Cells from 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

Cyanidin is an anthocyanidin extracted from a variety of fruits and vegetables. Cyanidin showed benefits against diabetes, cancer, and atherosclerosis. However, the potential neuroprotective effects of cyanidin against Parkinson’s disease (PD) have not been examined. Indicated concentrations of cyanidin (1, 3, 10, and 30 μmol/L) were incubated together with 0.5 mmol/L 1-methyl-4-phenylpyridinium (MPP+) to human neuroblastoma SH-SY5Y cells. We found cyanidin prevented MPP+-induced cell demise in a concentration-dependent manner. Cyanidin significantly reduced MPP+-induced apoptosis, this is reflected by decreased TdT-mediated dUTP nick-end labeling staining and caspase-3 expressions. Further, MPP+ increased the Bax/Bcl-2 ratio, which was partly reversed by cyanidin. We also found cyanidin attenuated the MPP+-induced mitochondrial oxidative stress as revealed by decreased MitoSOX staining. Taken together, these data for the first time indicated the ­neuroprotective effects of cyanidin against MPP+-induced ­SH-SY5Y cell death. These findings shed light on the potential implications of cyanidin for PD treatment.

Soluble Flt-1 Has Cytotoxic Effects on BeWo Choriocarcinoma Cells

Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and is overexpressed in many kinds of malignant tumors. Soluble VEGF receptor 1 (sVEGFR-1/ soluble fms-like tyrosine kinase 1 [sFlt-1]) plays a role as an inhibitor of VEGF, and an antitumor effect has been shown in several studies using sFlt-1. Recently, in addition to its antiangiogenic effect, it was reported that sFlt-1 has direct cytotoxicity. Transfection of sFlt-1 plasmid DNA was performed in the BeWo choriocarcinoma cell line. Overexpression of sFlt-1 in BeWo cells was confirmed by ELISA. In order to evaluate cell proliferation, cell counting and BrdU uptake assay were performed. Cytotoxicity was tested by LDH assay. TdT-Mediated dUTP Nick end Labeling (TUNEL) staining and quantitative analysis of caspase-cleaved keratin 18 (ccK18) level were done to evaluate cell apoptosis. The cell number was significantly less, and the ratio of cytotoxicity was significantly higher in sFlt-1 group compared to the control group. TUNEL staining and ccK18 level suggested nonapoptotic cell death. Soluble Flt-1 showed a cytotoxic effect on BeWo cells. Our results suggest that sFLT-1 could be therapeutic for malignant tumors.

Effects of Periostracum Cicadae on Cytokines and Apoptosis Regulatory Proteins in an IgA Nephropathy Rat Model.

Periostracum cicadae, the cast-off shell of the cicada Cryptotympana pustulata Fabricius, is used in traditional Chinese medicine for its diaphoretic, anticonvulsive, sedative, antipyretic, and antiallergic effects. However, the exact pathogenesis of immunoglobulin A nephropathy (IgAN) remains unclear, thereby hindering investigations to identify novel therapeutic agents. A rat IgAN model was established by administration of bovine serum albumin, lipopolysaccharide, and carbon tetrachloride, which simultaneously established blood stasis and a heat syndrome model. The animals were sacrificed to detect changes in protein levels in urine and blood. Immunofluorescence was performed to assess IgA deposition in the glomeruli. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6) levels were measured in bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay. Hematology and eosin, periodic acid-Schiff, TUNEL (TdT-mediated dUTP Nick-End Labeling), and immunohistochemical staining were performed to evaluate histopathological changes in kidney tissues. Additionally, target-related proteins were measured by Western blotting. Periostracum cicadae resulted in a reduction in blood and urine protein levels. Serum TNF-α, IL-1β, and IL-6 levels significantly decreased in the periostracum cicadae-treated groups compared to the IgAN group. Furthermore, a reduction in MCP-1 (Monocyte Chemotactic Protein-1), TLR4 ((Toll-Like Receptor 4)), and IgA expression levels and a dose-dependent increase in caspase 3 expression were observed in response to periostracum cicadae treatment. TGF-β1(Transforming Growth Factor-β) levels decreased, whereas that of Fas increased in the kidney tissues of the periostracum cicadae-treated groups. The findings of the present study indicate that periostracum cicadae induces apoptosis and improves kidney inflammation and fibrosis in IgA nephropathy rat models.

Lutein Inhibits Cell Growth and Activates Apoptosis via the PI3K/AKT/mTOR Signaling Pathway in A549 Human Non-Small-Cell Lung Cancer Cells.

Cancer, the uncontrolled growth of cells, is a major disease that threatens the worldwide population. Among all cancer types, lung cancer has the highest morbidity rate, with a survival rate of less than 5%. Various studies have focused on discovering a potent anticancer drug that will increase the survival rate of lung cancer patients. Lutein (3,3'-dihydroxy-β, ε-carotene), a carotenoid present in fruits and vegetables, is one such compound that possesses excellent antioxidant properties. The present study was designed to determine the anticancer effect of lutein against A549, a non-small-cell lung cancer cell line. The cytotoxic effect of lutein against lung cancer cells (A549 and HCC827) and normal cells (BEAS-2B) was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The Transwell assay was performed to detect the inhibitory potential of lutein against cell invasion and migration of A549 cells. The induction of apoptosis by lutein in A549 was analyzed by a double-staining technique using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling) and DAPI (4',6-diamidino-2-phenylindole) staining assays to confirm the molecular mechanism exhibited by lutein to induce apoptosis through regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling molecules that are often deregulated in cancerous condition. The results show that lutein inhibits the PI3K/AKT signaling pathway and induces apoptosis in A549, which may therefore be used as a potent natural anticancer drug with no side effects to treat lung cancer.

Protective effects of atorvastatin on cerebral vessel autoregulation in an experimental rabbit model of subarachnoid hemorrhage.

The aim of the present study was to assess the therapeutic effects of atorvastatin on cerebral vessel autoregulation and to explore the underlying mechanisms in a rabbit model of subarachnoid hemorrhage (SAH). A total of 48 healthy male New Zealand rabbits (weight, 2–2.5 kg) were randomly allocated into SAH, Sham or SAH + atorvastatin groups (n=16/group). The Sham group received 20 mg/kg/d saline solution, whereas 20 mg/kg/d atorvastatin was administered to rabbits in the SAH + atorvastatin group following SAH induction. Changes in diameter, perimeter and basilar artery (BA) area were assessed and expression levels of the vasoactive molecules endothelin-1 (ET-1), von Willebrand factor (vWF) and thrombomodulin (TM) were measured. Neuronal apoptosis was analyzed 72 h following SAH by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) staining. The mortality rate in the SAH group was 18.75, 25% in the SAH + atorvastatin treated group and 0% in the Sham group (n=16/group). The neurological score in the SAH + atorvastatin group was 1.75±0.68, which was significantly higher compared with the Sham group (0.38±0.49; P<0.05). The BA area in the SAH + atorvastatin group (89.6±9.11) was significantly lower compared with the SAH group (115.4±11.0; P<0.01). The present study demonstrated that SAH induction resulted in a significant increase in the diameter, perimeter and cross-sectional area of the BA in the SAH + atorvastatin group. Administration of atorvastatin may significantly downregulate the expression levels of ET-1, vWF and TM (all P<0.01) vs. sham and SAH groups. TUNEL staining demonstrated that neuronal apoptosis was remarkably reduced in the hippocampus of SAH rabbits following treatment with atorvastatin (P<0.05). Atorvastatin treatment may alleviate cerebral vasospasm and mediate structural and functional remodeling of vascular endothelial cells, in addition to promoting anti-apoptotic signaling. These results provided supporting evidence for the use of atorvastatin as an effective and well-tolerated treatment for SAH in various clinical settings and may protect the autoregulation of cerebral vessels.

Mitochondrial quality control in alveolar epithelial cells damaged by S. aureus pneumonia in mice.

Mitochondrial damage is often overlooked in acute lung injury (ALI), yet most of the lung's physiological processes, such as airway tone, mucociliary clearance, ventilation-perfusion (Va/Q) matching, and immune surveillance require aerobic energy provision. Because the cell's mitochondrial quality control (QC) process regulates the elimination and replacement of damaged mitochondria to maintain cell survival, we serially evaluated mitochondrial biogenesis and mitophagy in the alveolar regions of mice in a validated Staphylococcus aureus pneumonia model. We report that apart from cell lysis by direct contact with microbes, modest epithelial cell death was detected despite significant mitochondrial damage. Cell death by TdT-mediated dUTP nick-end labeling staining occurred on days 1 and 2 postinoculation: apoptosis shown by caspase-3 cleavage was present on days 1 and 2, while necroptosis shown by increased levels of phospho- mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was present on day 1 Cell death in alveolar type I (AT1) cells assessed by bronchoalveolar lavage fluid receptor for advanced glycation end points (RAGE) levels was high, yet AT2 cell death was limited while both mitochondrial biogenesis and mitophagy were induced. These mitochondrial QC mechanisms were evaluated mainly in AT2 cells by localizing increases in citrate synthase content, increases in nuclear mitochondrial biogenesis regulators nuclear respiratory factor-1 (NRF-1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and increases in light chain 3B protein (LC3-I)/LC3II ratios. Concomitant changes in p62, Pink 1, and Parkin protein levels indicated activation of mitophagy. By confocal microscopy, mitochondrial biogenesis and mitophagy were often observed on day 1 within the same AT2 cells. These findings imply that mitochondrial QC activation in pneumonia-damaged AT2 cells promotes cell survival in support of alveolar function.

Effect of L-Deprenyl on the Putrescine Level and Neuronal Damage after Transient Global Cerebral Ischemia in Gerbils

L-Deprenyl is selective and irreversible monoamine oxidase B inhibitor, known to have neuroprotective properties. Putrescine, one of polyamine, is thought to be important in the neuronal cell damage associated with various type of excitatory neurotoxicity. We examined the effects of L-deprenyl on the changes in putrescine level and neuronal damage after transient global ischemia in ger-bils. Male Mongolian gerbils weighing 65 - 75 g were used in the experiment. Global ischemia was induced by occlusion of common carotid arteries for 3 min to observe neuronal injury in hippocampal pyramidal cells. L-Deprenyl group was given 10 mg/kg of L-deprenyl intraperitoneally immediately after, 3 h and 6 h after global ischemia. Treated animals were processed in parallel with ischemic animals receiving saline as a vehicle and with sham- operated controls. Hippocampal putrescine level was increased by global ischemia and inhibited by L-deprenyl treatment. In histological findings, counts of viable neurons were made in the pyramidal cell layer of the hippocampal CA1 area 3 days after ischemic insult. The number of viable neurons in the pyramidal cell layer of CA1 area was significantly increased in animals treated with L-deprenyl compared to vehicle-treated ischemic animals (p < 0.05). In terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick endlabeling (TUNEL) assay, semiquantitative analysis of dark-brown neuronal cells was made in the hippocampal CA1 area. There was also a significant difference in the degree of TUNEL staining in the hippocampal CA1 area between vehi-cle-treated and L-deprenyl-treated animals (p < 0.05). These data show L-deprenyl is effective as a prophylactic treatment for neuronal injury when it is administrated before ischemia but a further study need to know the effects of administration of L-deprenyl after ischemia and at given times after reper-fusion.