Transferase-mediated dUTP-biotin Nick End-labeling Staining Research Articles

Cysteine-rich 61(CYR61) alleviates cyclophosphamide-induced proliferation inhibition in ovarian granulosa cells via suppressing NLRP3/caspase1-mediated pyroptosis.

We investigated the level of Cysteine-rich 61 (CYR61) in premature ovarian failure as well as its regulatory molecular mechanism in this study. Cyclophosphamide (CTX) was used to induce OGCs (rat ovarian granulosa cells) and rats to establish in vivo and in vitro premature ovarian failure models. H&E staining was used to detect the pathological changes of ovarian histopathology. Si-NLRP3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3) and si-CYR61 were transfected into OGCs using lipofectamine 3000. RT-qPCR and western blot were used to detect the expressions of CYR61 in ovarian tissue and OGCs. It showed that the expression of CYR61 was significantly down-regulated in premature ovarian failure model. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling) staining was used to detect the apoptosis. 5-Ethynyl-2'-deoxyuridine (EdU) and SA-β-gal (senescence-associated β-galactosidase) staining were used to assess the proliferation and senescence. The expression of CYR61 in OGCs and ovarian tissues were detected by immunofluorescence and immunohistochemical staining. Overexpression of CYR61 significantly promoted OGCs proliferation and inhibited pyroptosis and apoptosis. Western blot was used to detect the protein expressions of p53 and p21 in OGCs. Flow cytometry was used to detect the pyroptosis. CYR61 overexpression inhibited the expression of NLRP3 and caspase-1 in CTX-induced OGCs according to western blot results. Moreover, we found that CYR61 overexpression down-regulated the protein expressions of p53 and p21 in CTX-induced OGCs. CYR61 inhibited CTX-induced OGCs senescence, and the mechanism may be related to the regulation of caspase-1/NLRP3-induced pyroptosis.

Role of brain and muscle ARNT-like protein 1 in the rat periodontitis-induced liver injury model

Objective: Brain and muscle ARNT-like protein 1 (BMAL1) is a core component of hepatocyte molecular clock and plays an important role in the regulation of other related rhythmic genes in the body through a transcriptional-translational feedback loop in molecular circadian oscillations. Therefore, the aim of this study was to investigate the role of BMAL1 in the rat periodontitis-induced liver injury. Methods: Twelve male Wistar rats were divided into the control group and the periodontitis group according to the random number table method. The rats in the control group were untreated. The periodontitis models were established by ligating the necks of the bilateral maxillary first molars in the periodontitis group rats. After 8 weeks, periodontal clinical indexes of rats in both groups were examined and executed. Micro-CT scans of the maxilla were performed and levels of the alveolar bone resorption were analyzed. Pathological changes in periodontal and liver tissue of rats in two groups were detected by HE and oil red O staining. Biochemical kits were used to detect glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total cholesterol (TC) and triglycerides (TG) in serum. The gene and protein expression levels of BMAL1, nuclear factor kappa-B (NF-κB) and tumor necrosis factor-α (TNF-α) in liver tissue were measured by real time fluorescent quantitative-PCR (qRT-PCR), immunohistochemistry (IHC) and Western blotting (WB) assays. Apoptosis was detected in liver tissues by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit staining. Results: The results of HE staining of maxillary first molars and micro-CT results of maxillary bones showed that alveolar bone resorption was significant in the periodontitis group of rats. The liver histopathology results showed infiltrated inflammatory cells in the liver tissue, disorganized liver cords and a large number of lipid droplets formed in the hepatocytes of the periodontitis group compared with the control group. The results of serum biochemical assay showed that the levels of GOT [(62.77±2.59) U/L], GPT [(47.54±1.04) U/L], TC [(3.19±0.23) mmol/L] and TG [(1.11±0.09) mmol/L] in the serum of rats with periodontitis were significantly higher than that in the control group respectively [GOT: (38.66±2.47) U/L, GPT: (31.48±1.57) U/L, TC: (1.60±0.05) mmol/L and TG: (0.61±0.09) mmol/L](P=0.003, P=0.001, P=0.002, P=0.038). qRT-PCR results showed that the mRNA expression level of BMAL1 was significantly decreased in liver tissue of the periodontitis group [(0.60±0.04)%] compared to the control group [(1.01±0.07)%] (t=4.80, P=0.009), while the mRNA expression levels of NF-κB and TNF-α [(1.62±0.12)%, (2.69±0.16)%] were significantly increased compared to the control group [(1.00±0.03)%, (1.03±0.16)%] (P=0.008, P=0.002); IHC results showed that the protein expression level of BMAL1 in liver tissue of the periodontitis group (averaged optical density, AOD) (11.58±2.15) was down-regulated compared to the control group (AOD) (22.66±1.67) (P=0.015), while NF-κB and TNF-α (AOD) (31.77±2.69, 24.31±2.32) were up-regulated compared to the control group (AOD) (19.40±1.82, 11.92±0.94) (P=0.019, P=0.008). WB results showed that the protein expression level of BMAL1 in liver tissue was down-regulated in the periodontitis group [(0.63±0.10)%] compared to the control group [(1.00±0.06)%] (t=3.19, P=0.033), while NF-κB and TNF-α [(1.61±0.12)%, (2.82±0.23)%] were up-regulated compared to the control group [(1.00±0.12)%, (1.00±0.11)%] (P=0.022, P=0.002). TUNEL staining showed increased apoptotic cells in the liver tissue of the periodontitis group of rats compared to the control group. Conclusions: Periodontitis may induce liver injury by down-regulating the BMAL1 expression levels in liver tissue, which in turn activates NF-κB signaling molecules, leading to the elevated levels of inflammation and apoptosis in rat liver.

Itaconate Attenuates Neuroinflammation and Exerts Dopamine Neuroprotection in Parkinson's Disease through Inhibiting NLRP3 Inflammasome.

Parkinson’s disease (PD) is a common age-associated neurodegenerative motor disorder, which is mainly caused by dopaminergic neuron loss in the substantia nigra. This study aimed to evaluate the function and the underlying molecular mechanism of itaconate in PD. PD models were established in vivo and in vitro using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+), respectively. Pole and rotarod tests were applied to evaluate the motor coordination of mice. The expression of tyrosine hydroxylase (TH) in MPTP-induced mice and the MPP+ revulsive PD cell model were detected using Western blotting and immunofluorescence. The inflammatory factors level was detected by quantitative real-time polymerase chain reaction. The content of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) in substantia nigra, striatum, and SH-SY5Y cells were analyzed. Moreover, the apoptosis of MPP+ revulsive SH-SY5Y cells was determined using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining and flow cytometry. The expression of apoptosis- and Nod-like receptor family protein 3 (NLRP3) inflammasome-associated proteins was measured using Western blotting and immunofluorescence. Itaconate attenuated motor deficits of MPTP-induced PD mice. Itaconate inhibited dopamine neuronal damage, inflammatory response, oxidative stress, and neuronal apoptosis in MPTP-caused PD mice and the MPP+ revulsive PD cell model. Additionally, itaconate notably repressed the activation of NLRP3 inflammasome. This research demonstrated that itaconate could attenuate neuroinflammation and exert dopamine neuroprotection in PD through inhibiting NLRP3 inflammasome.

Effect of mistletoe extract on tumor response in neoadjuvant chemoradiotherapy for rectal cancer: a cohort study

BackgroundMistletoe extract, used as a complementary chemotherapeutic agent for cancer patients, has anticancer effects against various malignancies. The aim of the present study was to evaluate the effect of mistletoe extract (Abnoba Viscum Q®) on tumor responses to neoadjuvant chemoradiotherapy (NCRT) for locally advanced rectal cancer.MethodsThis study included patients with rectal cancer who underwent NCRT between January 2018 and July 2020. In the mistletoe group (MG), the patients were administered Abnoba Viscum Q® subcutaneously during chemoradiotherapy—maintained just before surgery. Patient demographics, clinical outcomes, histopathological outcomes, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay results were compared between the MG and non-mistletoe group (NMG). Two rectal cancer cell lines (SNU-503 and SNU-503R80Gy) were treated with Abnoba Viscum Q® to assess its mechanistic effects in vivo.ResultsOverall, the study included 52 patients (MG: n = 15; NMG: n = 37). Baseline demographics between the two groups were similar, except carbohydrate antigen 19-9 levels and tumor location from the anal verge. There was no difference in the clinical stage between the two groups. A better tumor response in the MG, relative to the NMG, was observed with respect to tumor regression grade (TRG), T stage, and overall tumor–node–metastasis stage. Tumor response was significantly better in the MG than in the NMG in terms of pathologic complete response rate (53.3% vs. 21.6%, P = 0.044), good TRG response (66.7% vs. 32.4%, P = 0.024), T downstaging (86.7% vs. 43.2%, P = 0.004), and overall downstaging (86.7% vs. 56.8%, P = 0.040). The toxicities during NCRT were minimal in both groups. More apoptotic cells were noted in MG samples than in the NMG samples on TUNEL staining. Cleaved caspase-3 level following treatment with Abnoba Viscum Q® was higher in SNU-503R80Gy cells than in SNU-503 cells.ConclusionPatients treated with chemoradiation combined with mistletoe extract showed better outcomes than patients not treated with mistletoe extract in terms of tumor responses. This diversity in treatment may improve the efficacy of NCRT, leading to better oncologic outcomes. Prospective and randomized studies with long-term follow-up are warranted to confirm and extend these results.

The Ameliorating Effects of Bushen Huatan Granules and Kunling Wan on Polycystic Ovary Syndrome Induced by Dehydroepiandrosterone in Rats.

AimTo investigate the effects of Bushen Huatan Granules (BHG) and Kunling Wan (KW), the two Chinese medicines, on the regulation of polycystic ovary syndrome (PCOS) and their underlying mechanisms.Materials and MethodsPCOS rat model was established by subcutaneous injection of dehydroepiandrosterone (DHEA) (6 mg/100 g/day) for 20 days, followed by treatment with BHG (0.75, 1.49, and 2.99 g/kg) or KW (0.46, 0.91, and 1.82 g/kg) by gavage for 4 weeks. Estrous cycle was detected by vaginal smears. Follicles development was assessed by histology. Levels of testosterone and insulin in serum were tested by ELISA. Apoptosis of Granulosa cells (GCs) was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining. Pathways associated with apoptosis were detected with western blot. Pregnancy outcome was also assessed. GCs were pre-treated with 10–5 M testosterone in vitro for 24 h, then incubated with serum from rats receiving BHG (1.49 g/kg) or KW (1.82 g/kg). The parameters concerning apoptosis, mitochondrial function and endoplasmic reticulum stress were assessed.ResultsPost-treatment with either BHG or KW ameliorated DHEA-induced irregular estrous cycles, follicles development abnormalities, increase of testosterone and insulin in serum, and the apoptosis of GCs. Post-treatment with BHG decreased the expression of cleaved caspase-9/caspase 9, release of cytochrome C from mitochondria, and mitochondria reactive oxygen species production, increased activities of complex I, II, IV of ovarian tissue. Post-treatment with KW decreased the levels of caspase-12, GRP78, C/EBP homologous protein, phosphorylation of IRE-I, x-box-binding protein 1s, as well as phosphorylation of proline-rich receptor-like protein kinase, phosphorylation of eukaryotic translation initiation factor 2α and ATF4 of ovarian tissue and GCs. Both BHG and KW ameliorated pregnancy outcome.ConclusionThis study demonstrated BHG or KW as a potential strategy for treatment of PCOS induced by DHEA, and suggested that the beneficial role of the two medicines were mediated by different pathway with the effect of BHG being correlated with the regulation of mitochondria, while the effect of KW being attributable to protection of endoplasmic reticulum stress.

TT01001 attenuates oxidative stress and neuronal apoptosis by preventing mitoNEET-mediated mitochondrial dysfunction after subarachnoid hemorrhage in rats.

Oxidative stress and neuronal apoptosis are considered crucial therapeutic targets against early brain injury (EBI) after subarachnoid hemorrhage (SAH). Emerging evidence indicates that mitochondrial dysfunction is the main reason for oxidative stress and neuronal apoptosis. MitoNEET, an outer mitochondrial membrane protein, has been shown to regulate mitochondrial function. However, whether mitoNEET activation attenuates oxidative stress and neuronal apoptosis after SAH remains unknown. This study was therefore conducted to verify the neuroprotective role of mitoNEET in EBI after SAH in rats. A total of 93 rats were subjected to an endovascular perforation model of SAH. TT01001, a selective agonist of mitoNEET, was administered intraperitoneally 1 h after SAH induction. Neurological tests, immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining, dihydroergotamine (DHE) staining, and western blot experiments were performed. The results showed that MitoNEET is expressed in neurons, but significantly decreased at 24 h after SAH induction. Activating mitoNEET with TT01001 significantly improved the neurological deficits, and reduced oxidative stress and neuronal apoptosis as measured by DHE and TUNEL staining, when compared with the SAH+vehicle group. Furthermore, TT01001 treatment decreased the expression of the proapoptotic marker, Bax, while increasing the expression of the antiapoptotic marker, Bcl-2. Together, our results suggested that mitoNEET activation with TT01001 reduced oxidative stress injury and neuronal apoptosis by improving mitochondrial dysfunction in EBI after SAH.

Manganese–Fluorouracil Metallodrug Nanotheranostic for MRI-Correlated Drug Release and Enhanced Chemoradiotherapy

For cancer therapy, drug delivery systems are often limited by insufficient drug loading capacity, which usually results in systemic toxicity and heavy metabolic burden to excrete the carriers. Her...

3-Bromopyruvate alleviates the development of monocrotaline-induced rat pulmonary arterial hypertension by decreasing aerobic glycolysis, inducing apoptosis, and suppressing inflammation.

Background:Pulmonary arterial hypertension (PH) is a progressive disease with limited therapeutic options, ultimately leading to right heart failure and death. Recent findings indicate the role of the Warburg effect (aerobic glycolysis) in the development of PH. However, the effect of the glycolysis inhibitor 3-bromopyruvate (3-BrPA) on the pathogenesis of PH has not been well investigated. This study aimed to determine whether 3-BrPA inhibits PH and its possible mechanism.Methods:PH was induced in adult Sprague-Dawley rats by a single intraperitoneal injection of monocrotaline (MCT). 3-BrPA, or phosphate-buffered saline (PBS) was administered via intraperitoneal injection every other day from the first day of MCT-injection to 4 weeks of follow-up, and indices such as right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), pulmonary arteriolar remodeling indicated by percent media thickness (% MT), lactate levels and glucose consumption, were evaluated. Pulmonary arteriolar remodeling and right ventricular hypertrophy were observed in hematoxylin-eosin-stained lung sections. Western blotting, immunohistochemistry, and/or immunofluorescence analyses were used to measure the expression of relevant proteins. A cytochrome C release apoptosis assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining were used to measure cell apoptosis.Results:MCT-induced PH showed a significant increase in glucose consumption (0 vs. 4 weeks: 0.87 ± 0.23 vs. 2.94 ± 0.47, P = 0.0042) and lactate production (0 vs. 4 weeks: 4.19 ± 0.34 vs. 8.06 ± 0.67, P = 0.0004). Treatment with 3-BrPA resulted in a concomitant reduction in glucose consumption (1.10 ± 0.35 vs. 3.25 ± 0.47, P = 0.0063), lactate production (5.09 ± 0.55 vs. 8.06 ± 0.67, P = 0.0065), MCT-induced increase in RVSP (39.70 ± 2.94 vs. 58.85 ± 2.32, P = 0.0004), pulmonary vascular remodeling (% MT, 43.45% ± 1.41% vs. 63.66% ± 1.78%, P < 0.0001), and right ventricular hypertrophy (RVHI, 38.57% ± 2.69% vs. 62.61% ± 1.57%, P < 0.0001) when compared with those of the PBS-treated group. 3-BrPA, a hexokinase 2 inhibitor, exerted its beneficial effect on PH by decreasing aerobic glycolysis and was also associated with inhibiting the expression of glucose transporter protein-1, inducing apoptosis, and suppressing inflammation.Conclusions:3-BrPA might have a potential beneficial effect on the PH treatment.

Intraperitoneal injection of ketamine enhances apoptosis in urothelium via autophagy in rats

Ketamine abusing is associated with ulcerative cystitis, but the mechanisms remain unclear. This study aimed to investigate the existence of ketamine-induced symptom in a rat model and evaluate the underlining mechanisms. Sprague-Dawley rats were chosen and randomly divided into 12 groups (n = 8), such as the control group, low dose of ketamine (10 mg/kg/day), middle dose of ketamine (30 mg/kg/day) and high dose of ketamine (50 mg/kg/day) groups. The experimental groups were administrated ketamine i.p. daily, whereas the control groups were administrated with saline. After 1, 3, and 6 months of treatment, the bladder tissues were collected. Haematoxylin and eosin (HE) staining and a transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to evaluate the bladder epithelium pathology and urothelial apoptosis, respectively. The protein expression levels of LC3, p62, Beclin1 were assessed by Western blotting. HE staining results of the experimental rats showed the bladder tissue denudation of the urothelial epithelium with edema and congestion compared with the control groups. TUNEL staining showed a significantly higher number of apoptotic cells in experimental groups than in the control groups. The protein LC3 and Beclin1 had significantly higher levels compared with control groups. The protein p62 had lower levels compared with control groups. The expression levels correlated with contraction of ketamine and treatment time. HE staining, TUNEL staining and Western blot results showed dose-dependent, time-dependent autophage in ketamine-treated rats. All the results suggested that autophagy proteins might be involved in inflammatory response in rats.

The Evaluation of TUNEL, PCNA and SOX2 Expressions in Lens Epithelial Cells of Cataract Patients with Pseudoexfoliation Syndrome

ABSTRACT Purpose This study aims to determine the expression patterns of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), proliferating cell nuclear antigen (PCNA) and SOX2 in lens epithelial cells (LEC) of cataract patients with pseudoexfoliation syndrome (PEX), and to determine the effect of apoptosis, proliferative activity and stem/progenitor cells on cataract formation in patients with PEX. This is a prospective, randomized clinical trial. Materials and Methods Setting: institutional. 50 eyes of 50 patients were included. Anterior capsule samples were obtained during phacoemulsification surgery. The specimens of LEC were also examined using the TUNEL, PCNA and SOX2 immunohistochemical staining method. To detect the number of immunopositive cells, the total number of cells in a 3 mm2 area was counted using a microscope under x20 magnification and the percentage of cells stained positive was determined. Results In Group 1, increased expression was observed with TUNEL, while decreased expression was detected with PCNA (p = .008, p = .015). The average percentage of TUNEL immunopositive cells was significantly higher in Group 1 than in Group 2, but there was no statistically meaningful SOX2 expression in Group 1 and Group 2 (P = .44). Apoptosis rates were 61.75 ± 14.5% and 36.91 ± 14.6% in Groups 1 and 2, respectively. Proliferation rates were 40.96 ± 16.8% and 65.45 ± 16.9% in Groups 1 and 2, respectively. Conclusion We found increased apoptosis and decreased proliferation of LECs in cataract patients with PEX. We suspected that this could be related to oxidative stress.

Inhibition of long noncoding RNA growth arrest–specific 5 attenuates cerebral injury induced by deep hypothermic circulatory arrest in rats

ObjectiveWe sought to investigate cerebroprotection by targeting long noncoding RNA growth arrest–specific 5 in a rat model of prolonged deep hypothermic circulatory arrest. MethodsDeep hypothermic circulatory arrest was conducted for 60 minutes when the pericranial temperature was cooled to 18°C in rats. Dual luciferase assay was used to detect the binding relationship between growth arrest–specific 5 and putative target microRNAs. Adeno-associated viral vectors containing growth arrest–specific 5 small interfering RNA or negative control small interfering RNA were administered by intracerebroventricular injection 14 days before deep hypothermic circulatory arrest. Expressions of growth arrest–specific 5, microRNA-23a, phosphate and tension homology, Bcl-2–associated X protein, Bcl-2, phospho-protein kinase B, protein kinase B, and cleaved caspase-3 in the hippocampus were measured by quantitative reverse transcription polymerase chain reaction and Western blot. Spatial learning and memory functions were evaluated by the Morris water maze test. The hippocampus was harvested for histologic examinations and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining. ResultsLuciferase assay showed that growth arrest–specific 5 targeted and inhibited microRNA-23a expression. After deep hypothermic circulatory arrest, hippocampal growth arrest–specific 5 expression was significantly enhanced with a robust decrease of hippocampal microRNA-23a expression. Small interfering RNA growth arrest–specific 5 significantly inhibited growth arrest–specific 5 expression and enhanced microRNA-23a expression in the hippocampus, accompanied with decreases of phosphate and tension homology and Bcl-2–associated X protein expression, and increases of Bcl-2 expression and phospho-protein kinase B/protein kinase B ratio. Growth arrest–specific 5 knockdown inhibited neuronal apoptosis, attenuated histologic damages, and increased the number of surviving neurons in the hippocampus. Spatial learning and memory functions after deep hypothermic circulatory arrest were also markedly improved by growth arrest–specific 5 inhibition. ConclusionsInhibition of large noncoding RNA growth arrest–specific 5 can provide a powerful cerebroprotection against deep hypothermic circulatory arrest, which may be mediated through microRNA-23a/phosphate and tension homology pathway.

Inhibition of MicroRNA-204 Conducts Neuroprotection Against Spinal Cord Ischemia

MicroRNA(miR)-204 is an autophagy- and apoptosis-related gene. Neuroprotection by the inhibition of miR-204 against spinal cord ischemia was evaluated, and the roles of neuronal autophagy and apoptosis were investigated. Spinal cord ischemia was conducted in rats by cross-clamping the descending aorta for 14 minutes. Inhibition of miR-204 was induced by intrathecal injection of lentivirus vectors containing antagomiR-204. Hind-limb motor function was assessed with the motor deficit index. Lumbar spinal cords were harvested for histologic examinations and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining. Autophagy was evaluated by the LC3-II/LC3-I ratio and beclin-1 expression. Expressions of LC3-I, LC3-II, beclin-1, B-cell lymphoma-2 (BCL-2), caspase-3, and miR-204 were measured by Western blot and quantitative real-time polymerase chain reaction. Autophagy was blocked by 3-methyladenine. Transient ischemia enhanced miR-204 expression and the LC3-II/LC3-I ratio and downregulated BCL-2expression in spinal cords in a time-dependent manner. AntagomiR-204 significantly reduced expressions of miR-204 and caspase-3, dramatically upregulated expressions of beclin-1 and BCL-2 and the LC3-II/LC3-I ratio in spinal cords after reperfusion. Compared with controls, inhibition of miR-204 markedly decreased the motor deficit index scores at 6, 12, 24, and 48 hours after reperfusion; increased the number of viable motor neurons; and decreased the number ofapoptotic neurons. 3-Methyladenine completely abolished enhancements of the LC3-II/LC3-I ratio and beclin-1 expression induced byantagomiR-204 and inhibited the protective effect on hind-limb motor function. Inhibition of miR-204 exerts spinal cordprotection against ischemia-reperfusion injury, possibly via promotion of autophagy and antiapoptotic effects.

Amplifying Apoptosis Homing Nanoplatform for Tumor Theranostics.

Nanomedicine has significantly impacted cancer theranostics. However, its efficiency is restricted by the limited enhanced permeability and retention effect of nanomaterials and insufficient density/specificity of receptors of tumor cells. Herein, an apoptosis-homing nanoplatform based on zinc(II) dipicolylamine (ZnDPA) conjugated Fe/Fe3 O4 nanoparticles (MNPs/ZnDPA), which demonstrates amplified magnetic resonance signal and photothermal therapy, is developed. In an apoptotic xenograft model constructed by doxorubicin, due to the high affinity between ZnDPA and the upregulated level of phosphatidylserine on the outer surface of apoptotic cancer cells, the accumulation value of MNPs/ZnDPA is enhanced two-fold and the tumor/muscle ratio of T2 values is decreased to 50% compared to that in the normal xenograft model. In the apoptotic xenograft model, the amplifying photothermal therapy is confirmed by the changes of the relative tumor volume and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining. This nanoplatform provides a promising strategy to improve the targeting efficiency of nanoparticles and the enhancement of tumor-targeting theranostics.

Acquired inhibition of microRNA-124 protects against spinal cord ischemia–reperfusion injury partially through a mitophagy-dependent pathway

ObjectiveMitophagy results in selective clearance of damaged mitochondria. We investigated whether mitophagy was involved in the neuroprotection by inhibiting microRNA (miRNA)-124 on ischemic spinal cords. MethodsInhibition of miRNA-124 was conducted by intrathecal injection of lentivirus vectors containing antagomiR-124. Spinal cord ischemia was induced in rats by crossclamping the descending aorta just distal to the left subclavian artery for 14 minutes. Hind-limb motor function was assessed with the motor deficit index (MDI). Lumbar spinal cords were harvested for ultrastructural, histologic examinations, and terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling staining. Mitophagy was evaluated by expressions of beclin-1 and LC3-II in mitochondria. Expressions of inhibitory member of the apoptosis-stimulating proteins of p53 family, p53, beclin-1, LC3-II, and miRNA-124 were measured by Western blot and quantitative real-time polymerase chain reaction. Mitophagy was inhibited by the antagonist of 3-methyladenine. ResultsCompared with control animals, antagomiR-124 significantly inhibited expressions of miRNA-124 (P < .01) and p53 (P < .05) and enhanced expressions of inhibitory member of the apoptosis-stimulating proteins of p53 family, becline-1 and LC3-II (P < .01, respectively) in spinal cords. MDI at 6, 12, 24, and 48 hours after reperfusion were markedly lower in antagomiR-124 group (P < .01, vs control group, respectively). More motor neurons and less apoptotic cells were detected in lumbar spinal cords of antagomiR-124 group (P < .01 vs control group). Administration of 3-methyladenine completely abolished enhancements of mitochondrial becline-1 and LC3-II by antagomiR-124 (P < .01 vs antagomiR-124 group) and partially inhibited effects of antagomiR-124 on MDI, number of motor neurons, and apoptotic cells (P < .01 or < .05 vs control group and antagomiR-124 group, respectively). ConclusionsInhibition of miRNA-124 exerts neuroprotection on spinal cords against ischemia–reperfusion injury, possibly by induction of mitophagy and antiapoptotic effects.

The Relationship Between Apoptotic Activity and Prognostic Factors in Neuroblastomas.

Prognostic parameters in determining risk groups for treatment in neuroblastoma are cellular differentiation, mitosis karyorrhexis index (MKI), N-myc amplification and age. However, additional prognosticators are still needed because patients can show unpredictable biological behavior. We aimed to study the prognostic significance of apoptotic activity in neuroblastomas. Thirty-five primary neuroblastoma were evaluated. The data including stage, treatment protocol, metastatic disease, survival, N-myc status, age and prognostic categorization were obtained from the clinical records. The differentiation and MKI were evaluated in hematoxylin and eosin stained slides. Apoptotic activity was assessed by both bcl-2 immunohistochemical staining and the transferase-mediated d-UTP-biotin nick end labeling (TUNEL) method. Data were correlated with established prognostic factors and clinical outcome. Twenty-five (71.4%) cases were located in the adrenal. Sixteen cases showed low and 19 high MKI. Thirty-three (%94) were immunopositive for bcl-2. TUNEL staining was negative in 2 cases. Of the remaining positive 33 cases, 14 had an apoptotic index of ≤2%, 11 of 2-4% and 8 of ≥4%. Cases located in the adrenal showed higher scores of bcl-2 positivity than extra-adrenal tumors. There was no statistical significance for both bcl-2 staining and apoptotic index in correlation with cellular differentiation, MKI, N-myc amplification, age and other clinical parameters. Statistical significance was observed between bcl-2 scoring and tumor localization. According to our results, apoptotic activity is unlikely to be a prognostic parameter in neuroblastomas. Some studies showing significant correlations between clinical outcome and both bcl-2 immunoscoring and apoptotic index, as assessed by the TUNEL method, differences in case numbers and methodology can explain these conflicting results. Larger series and different methodologies are needed to evaluate the prognostic value of apoptotic activity in neuroblastomas.

Effects of salidroside on myocardial cell apoptosis in acute myocardial ischemia rats and its mechanism

Background: To observe the effects of salidroside on myocardial cell apoptosis in acute myocardial ischemia (AMI) rats and explore its possible molecular mechanisms. Methods: Sprague-Dawley (SD) rat models of AMI were established and randomized into salidroside high-dose (40 mg/kg) and low-dose (10 mg/kg) groups, AMI group, and sham group. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was applied to analyze the myocardial cell apoptosis. Western blotting was used to determine the protein expressions of Bcl-2, Bax, Cytochrome c (Cyt-c), cleaved caspase-3, and cleaved caspase-9. Results: TUNEL staining showed that salidroside inhibited AMI-induced myocardial cell apoptosis in a dose-dependent manner. Moreover, compared with AMI group, the salidroside treatment groups had significantly increased protein expression of Bcl-2 and significantly decreased protein expression of Bax, Cyt-c, cleaved caspase-3, and cleaved caspase-9. Conclusions: Salidroside inhibits myocardial cell apoptosis during AMI, which may be achieved by inactivating the mitochondria-dependent pathway. This finding may provide labarotary evidence for the clinical application of salidroside in treating ischemic heart disease.

The canonical Notch signaling was involved in the regulation of intestinal epithelial cells apoptosis after intestinal ischemia/reperfusion injury.

Notch signaling plays a critical role in the maintenance of intestinal homeostasis. The aim of the present study was to investigate the role of Notch signaling in the apoptosis of intestinal epithelial cells after intestinal ischemia reperfusion (I/R) injury. Male C57BL/6 mice were subjected to sham operation or I/R injury. Intestinal tissue samples were collected at 12 h after reperfusion. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) staining showed that intestinal I/R injury induced significantly increased apoptosis of intestinal epithelial cells. Meanwhile, the mRNA expression of Jagged1, DLL1, Notch2, and Hes5, and protein expression of NICD2 and Hes5 were increased significantly after I/R injury in intestinal epithelial cells. In an in vitro IEC-6 culture model, flow cytometry analyses showed that inhibition of Notch signaling by γ-secretase inhibitor DAPT and the suppression of Hes5 expression using siRNA both significantly increased the apoptosis of IEC-6 cells under the condition of hypoxia/reoxygenation (H/R). In conclusion, the Notch2/Hes5 signaling pathway was activated and involved in the regulation of intestinal epithelial cells apoptosis in intestinal I/R injury.

Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivo

To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA (shRNA) on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism. Human ovarian cancer cell line HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer. Tumor-bearing nude mice were assigned randomizely to three groups: Lenti-RhoA-sh group, Lenti- negative control (NC) group and phosphate buffered saline (PBS) group.lentivirus mediated RhoA shRNA, negative control lentivirus and PBS were respectively injected in the three groups. Effects of treatment were observed by tumor growth curve, tumor volume, tumor weight, and tumor inhibition rate. Xenograft tissues and liver, spleen, lung, and renal tissues were examined by hematoxylin and eosin (HE) staining or were detected by streptavidin-perosidase(SP)immunochemical method. The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR, immunochemistry and Western blot assay. Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and apoptotic index (AI) were counted. Compared with Lenti-NC group and PBS group, the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days (P < 0.01) . Tumor volume (338 ± 114) mm(3) decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group (1190 ± 332) mm(3) and PBS group (1101 ± 396) mm(3) (P < 0.01) . Tumor weight (0.23 ± 0.11) g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group (0.79 ± 0.19) g and PBS group (0.74 ± 0.17) g (P < 0.01) . Real-time qPCR result shown that the expression of RhoA mRNA (0.30 ± 0.05) decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group (0.95 ± 0.06) and PBS group(1.00 ± 0.11; P < 0.01) .Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P < 0.01). TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) % compared with those in Lenti-NC group (5.2 ± 2.0) % and PBS group (6.0 ± 2.1) % (P < 0.01). Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA, inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.

Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway during Porcine Circovirus Type 2 Infection Facilitates Cell Survival and Viral Replication

Virus infection activates host cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates diverse cellular activities related to cell growth, survival, and apoptosis. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), a major causative agent of postweaning multisystemic wasting syndrome, which is an emerging and important swine disease, can transiently induce the PI3K/Akt pathway in cultured cells at an early step during PCV2 infection. Activation of the PI3K/Akt signal was also induced by UV-irradiated PCV2, indicating that virus replication was not required for this induction. Inhibition of PI3K activation leads to reduced virus yield, which is associated with decreased viral DNA replication and lower virus protein expression. However, inhibition of PI3K activation greatly enhanced apoptotic responses as evidenced by the cleavage of poly-ADP ribose polymerase and caspase-3 as well as DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining during the early stage of PCV2 infection. Furthermore, the pancaspase inhibitor zVAD.fmk alleviated the reduction in Akt phosphorylation levels by inhibiting PI3K activation, indicating that the signaling promotes cell survival and thereby favors viral replication. These results reveal that an antiapoptotic role for the PI3K/Akt pathway induced by PCV2 infection to suppress premature apoptosis for improved virus growth after infection, extending our understanding of the molecular mechanism of PCV2 infection.

Overexpression of CREG attenuates atherosclerotic endothelium apoptosis via VEGF/PI3K/AKT pathway

AimsCellular repressor of E1A-stimulated genes (CREG) is a homeostasis-modulating gene abundantly expressed in adult artery endothelium. Previous studies have demonstrated a protective effect of CREG against atherosclerosis through prevention of vascular smooth muscle cell apoptosis. However, the role of CREG in endothelial cells (ECs) apoptosis and the underlying signaling mechanisms are unknown. Method and resultsWe ascertained that CREG expression was decreased in atherogenesis-prone endothelium in apolipoprotein E-null (apoE−/−) mice compared with their wild-type littermates using in situ immunofluorescent staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining and caspase-3 activity assays determined that treatment of apoE−/− mice arteries with staurosporine (STS) significantly induced endothelial apoptosis associated with a reduction of CREG expression. Gain- and loss-of-function analyses revealed that silencing CREG expression significantly enhanced ECs apoptosis, whereas CREG overexpression abrogated apoptosis stimulated by STS or etoposide (VP-16). Blocking assays using the neutralizing antibody for vascular endothelial growth factor (VEGF) and the specific inhibitor of phosphoinositide 3-kinase (PI3K), such as LY294002 or wortmannin, demonstrated that the protective effect of CREG on ECs apoptosis was mainly mediated by activation of the VEGF/PI3K/AKT signaling pathway. ConclusionsThese data demonstrate that CREG plays a critical role in protecting the vascular endothelium from apoptosis, and the protective effort of CREG against ECs apoptosis is through the activation of the VEGF/PI3K/AKT signaling pathway.