Terminal Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate-biotin Research Articles

Targeting intestinal epithelial cell–programmed necrosis alleviates tissue injury after intestinal ischemia/reperfusion in rats

BackgroundIntestinal dysfunction, especially acute pathologies linked to intestinal ischemia/reperfusion (I/R) injury, is profoundly affected by inflammation and improper execution of cell death. Few studies have examined the efficacy of combined strategies in regulated intestinal epithelial necrosis after intestinal I/R. Here, we evaluated the functional interaction between poly (adenosine diphosphate-ribose) polymerase 1 (PARP-1)-induced parthanatos and receptor-interacting protein 1/3 (RIP1/3) kinase-induced necroptosis in the pathophysiological course of acute ischemic intestinal injury. MethodsAnesthetized adult male Sprague–Dawley rats were subjected to superior mesenteric artery occlusion consisting of 1.5 h of ischemia and 6 h of reperfusion. The PARP-1-specific inhibitor PJ34 (10 mg/kg) and the RIP1-specific inhibitor Necrostatin-1 (1 mg/kg) were intraperitoneally administered 30 min before the induction of ischemia. ResultsIntestinal I/R was found to result in PARP-1 activation and RIP1/3-mediated necrosome formation. PJ34 or Necrostatin-1 treatment significantly improved the mucosal injury, while the combined inhibition of PARP-1 and RIP1/3 conferred optimal protection of the intestine. Meanwhile, results from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay showed a decrease in intestinal epithelial cell death. Interestingly, we further showed that PARP-1 might act as a downstream signaling molecule of RIP1 in the process of I/R-induced intestinal injury and that the RIP1/PARP-1-dependent cell death signaling pathway functioned independently of caspase 3 inhibition. ConclusionsThe results of our study provide a molecular basis for combination therapy that targets both pathways of regulated necrosis (parthanatos and necroptosis), to treat acute intestinal I/R-induced intestinal epithelial barrier disruption.

Granulocyte Colony-Stimulating Factor Improves Motor Function in Rats Developing Compression Myelopathy.

Basic animal research. The effects of granulocyte colony-stimulating factor (G-CSF) were assessed in a rat chronic spinal cord compression model to explore the potential of G-CSF as a pharmacological treatment for cervical spondylotic myelopathy. G-CSF is a hematopoietic cytokine used clinically to treat neutropenia. Recently, neuroprotective effects of G-CSF have been reported in spinal cord disorders. To introduce the chronic cervical cord compression, thin polyurethane sheets were implanted under C5-C6 laminae of rats and gradually expanded by absorbing water. This model reproduces delayed compressive myelopathy of the cervical spine. In sham operations, the sheets were immediately removed. G-CSF (15 μg/kg) or normal saline (NS) was administered subcutaneously 5 days a week. Experimental groups were sham operation given NS; cord compression given NS; and cord compression given G-CSF. To assess motor functions, rotarod performance, and grip strength were measured. Twenty-six weeks after surgery, cervical spinal cords were examined histopathologically. In the prevention experiment, G-CSF or NS administration was started immediately after surgery. In the treatment experiment, their administration was started 8 weeks after surgery. In another experiment, in three groups in the prevention experiment, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining was performed to assess apoptotic cell death at 8 weeks after surgery. In the prevention experiment, administration of G-CSF preserved the motor functions and motor neurons throughout the 26 weeks, and significantly decreased the number of apoptotic cells at 8 weeks. In the treatment experiment, G-CSF administration from 8 weeks after surgery markedly restored the motor function temporarily to a level equal to the sham group. G-CSF prevents the decline in motor functions and preserves motor neurons in the rat chronic cord compression model. G-CSF also improves motor function in the progressive phase of compression myelopathy. N/A.

The Effect of Intravitreal Azithromycin on the Albino Newborn Rabbit Retina.

Purpose: To evaluate the effect of intravitreal azithromycin on the retina in a newborn rabbit model.Methods: Twelve, two-week old New Zealand albino rabbits were divided into two groups (six in each). The right eyes of six rabbits received 0.75 mg (0.05 mL) azithromycin and the right eyes of the remaining six rabbits 1.5 mg (0.1 mL) azithromycin intravitreally. Left eyes were served as the control and received the same volume of saline. All eyes were enucleated at the third postinjection week. Retinal histology was examined by light microscopy. Apoptosis of the retinal cells was further evaluated by immunohistochemical staining for caspase-3 and in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) of DNA fragments. Results: Light microscopy demonstrated no retinal abnormalities in all eyes. However, retinal nuclear DNA fragmentation was evident in both study groups (33.6% with 1.5 mg and 21.4% with 0.75 mg azithromycin) with the TUNEL method. TUNEL staining ratio was statistically higher only in the second group treated with 1.5 mg azithromycin when compared to the control group (p=0.01 Mann Whitney U test). The ratio of caspase-3 positive cells in the two study groups was 21.5% and 20.2%, respectively. Caspase-3 staining ratio was statistically higher in both study groups when compared to the control eyes (p=0.00, p=0.00 respectively). The difference of TUNEL staining ratio between the two study groups was statistically significant (p=0.028), but there were no statistically significant differences in the two study groups by caspase-3 staining (p=0.247). Conclusion: In newborn rabbits, intravitreal azithromycin injection resulted in an apoptotic activity in the photoreceptor, bipolar and ganglion cells. Immunohistochemical analysis suggested that doses of 0.75 mg and 1.5 mg azithromycin, administered intravitreally might be toxic to the newborn rabbit retina.

Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

Treatment of near-infrared photodynamic therapy using a liposomally formulated indocyanine green derivative for squamous cell carcinoma.

IntroductionPhotodynamic therapy (PDT) is a less invasive option for cancer treatment that has evolved through recent developments in nanotechnology. We have designed and synthesized a novel liposome system that includes an indocyanine green (ICG) derivative, ICG-C18, in its bilayer. In addition to its use as an optical imager to visualize blood, lymphatic, and bile flow, ICG has also been used as an optical sensitizer. In the present report, we evaluate the use of our novel liposome system, LP-ICG-C18, in PDT for squamous cell carcinoma in an autologous murine model.Materials and MethodsAn excitation pulse beam (300 μJ/pulse) of a single band (800 nm) was used for sensitization. The cytotoxicity of the photodynamic therapy was evaluated in terms of cellular morphology changes, methyl thiazolyl tetrazolium (MTT) assay results, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. We tested the enhanced permeability and retention effect of LP-ICG-C18 in tumor-bearing C3H/He mice using a near-infrared fluorescence imaging system and fluorescence microscopy. We also examined the antitumor effect of PDT by measuring tumor volume in tumor-bearing mice.ResultsCell death and apoptosis were only observed in the PDT group receiving LP-ICG-C18. LP-ICG-C18 itself had no cytotoxic activity and showed good biocompatibility. LP-ICG-C18 accumulated on the tumor 24 hours after injection and was retained for approximately 3 weeks. Tumor cell apoptosis following PDT with LP-ICG-C18 was also observed under optical microscopy, MTT assay, and TUNEL staining.ConclusionThese findings suggest that LP-ICG-C18 may be an effective intervening material in PDT for malignant disease.

Expression of caspase-3 predicts prognosis in advanced noncardia gastric cancer.

There is strong evidence that tumor growth is not only a result of uncontrolled cell proliferation but also of decreased apoptosis. Caspase-3 is a member of interleukin-1 beta-converting enzyme which is involved in the induction of apoptosis. Data on the expression of caspase-3 in patients with gastric cancer and its association with patient outcome are somewhat contradictory. We aimed to investigate the potential relation of the expression of caspase-3 protein with response to therapy and overall survival in patients with advanced noncardia gastric cancer. Tumor tissue samples collected from 359 consecutive patients with gastric cancer stage IV were retrospectively analyzed for the expression of caspase-3 in the primary tumor. The DNA apoptotic index assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling method. All patients were followed up until death. Caspase-3 was expressed in 43.5% of tumors. Caspase-3 expression compared to no expression was related with a higher DNA apoptotic index (p<0.05). In multivariate analysis, tumor expression of caspase-3 was found to be an independent predictor of poor treatment response and survival (p<0.05). Expression of caspase-3 in advanced gastric cancer is a predictor of poor response to treatment and survival. Further studies are needed to fully elucidate the prognostic value of caspase-3 expression in these patients.

The protective effects of dexmedetomidine against apoptosis in retinal ischemia/reperfusion injury in rats

Objective: Dexmedetomidine is an alpha 2 adrenoceptor agonist and can be used for postoperative sedation, analgesia and anesthesia-sparing properties. Furthermore, the neuroprotective effects against ischemia/reperfusion (I/R) injury in the central nervous system have been shown in experimental studies. This study aimed to investigate the protective effects of dexmedetomidine against apoptosis in retinal I/R injury in the rat. Materials and methods: Retinal I/R injury was induced by transient elevation of intraocular pressure. Eighteen animals were divided into three groups (n = 6): sham, I/R and treatment. The I/R injury and protective effects of the dexmedetomidine were evaluated by retinal thickness determined by histological sections, terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) and immunohistochemistry of caspases 3. Results: A decrease in the retinal thickness and an increase in the apoptotic cells were found to be statistically significant in I/R and treatment groups when compared with the control group. However, in comparison with the I/R group we realized that the administration of dexmedetomidine reduced the thinning of retinal thickness and also decreased the number of caspases 3 and TUNEL-positive cells. Conclusion: Dexmedetomidine is protective against apoptosis in retinal I/R injury in rats.

Chitosan-zinc chelate improves intestinal structure and mucosal function and decreases apoptosis in ileal mucosal epithelial cells in weaned pigs

The present study was conducted to investigate the effects of chitosan (CS)-Zn on intestinal morphology, mucosal epithelial cell apoptosis and mucosal immune function in weanling pigs. A total of 150 weanling barrows with a body weight of 7.2 kg were randomly allocated into five groups. A basal diet without Zn supplementation was used as the control and other four groups were fed the control diet supplemented with 50 or 100 mg/kg of Zn as CS-Zn, 100 mg/kg of Zn as ZnSO4 and 3000 mg/kg of Zn as ZnO, respectively. The feeding trial lasted for 28 d. The results showed that serum diamine oxidase activities, d-lactate levels and endotoxin contents were lower in pigs fed dietary 100 mg/kg of Zn as CS-Zn or 3000 mg/kg of Zn as ZnO than in pigs fed the control or 100 mg Zn/kg as ZnSO4 diet. The ratios of the villus height:crypt depth of the duodenum, jejunum and ileum were higher in pigs that received 100 mg/kg of Zn as CS-Zn or a high level of Zn as ZnO than in pigs fed the control diet. Moreover, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL)-stained ileal epithelial cells were found in the control group, and apoptotic cells did not appear prominently in pigs that received the 100 mg/kg of CS-Zn or ZnO diet. Secretory IgA concentration in ileal mucus was increased in the dietary group that received 100 mg/kg of CS-Zn or ZnO. These results indicated that dietary 100 mg CS-Zn/kg had similar biological effects to dietary 3000 mg ZnO/kg on intestinal morphology, mucosal epithelial cell apoptosis and mucosal immune function.

Thiamine Deficiency Induces Massive Cell Death in the Olfactory Bulbs of Mice

Thiamine (vitamin B1) deficiency (TD) leads to focal brain necrosis in particular brain regions in humans and in experimental animal models. The precise mechanism of the selective topographic vulnerability triggered by TD still remains unclear. We examined the distribution pattern of cell death in the brains of mice in an experimental model of TD using anti-single-strand DNA immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling methods. We found that interneurons in the olfactory bulb were sensitive to TD. The morphologic aspects of cell death in the olfactory bulb resembled those of cell death in thalamic neurons, which have previously been examined in detail. Furthermore, cell death in the olfactory bulb was partly relieved by the administration of an N-methyl-d-aspartate receptor antagonist, as was the case in thalamic lesions by TD. The superficial part of the olfactory granule cell layer seemed to be the most sensitive to TD, suggesting that differences in the afferents between superficial and deep granule cells may influence the sensitivity of these cells to TD. Our results indicate that the olfactory bulb should be considered as one of the vulnerable regions to TD.

The effect of di-n-butyl phthalate on testis and the potential protective effects of resveratrol

This study aimed to observe the possible protective effects of resveratrol (RSV) against the damage of di-n-butyl phthalate (DBP) on the testis. The study was conducted in 6 groups of rats with 6 animals in each group aged 20 days. The groups include group 1: control group; group 2: solvent (carboxymethylcellulose (CMC), 10 ml/kg); group 3: 500 mg/kg/day DBP; group 4: 500 mg/kg/day DBP + 20 mg/kg/day RSV; group 5: 1000 mg/kg/day DBP; and group 6: 1000 mg/kg/day DBP + 20 mg/kg/day RSV. Groups were treated by gavage for 30 days. Indirect immunohistochemical staining was performed with c-kit, AT1, and ER-α antibodies. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method was used for apoptosis. It was found in the DBP-applied groups the C-kit immunostaining, which is parallel to increasing dose, decreased in comparison with the control. C-kit reactivity was similar to that of the control group in the group applied with 500 mg/kg/day + RSV; however, the reactivity was not same in the 1000 mg/kg/day DBP-applied group. It was observed that the reactivity of AT1 increased in the DBP-applied groups. RSV reversed these changes with its protective effects. While there was not much difference between the groups in terms of estrogen receptor reactivity, it was observed that the high dose of DBP reduced the level of estrogen receptor and the resveratrol was not at enough levels in all doses. In TUNEL analysis, high doses of DBP increased the apoptosis in all types of cells; nevertheless, the resveratrol application decreased the apoptosis in the low-level DBP dose. In the statistical analysis, while the length of epithelium and the diameter of seminiferous tubules decreased for all the other groups, it reverted to its original state in the RSV-applied groups. In conclusion, DBP (with increasing dose) administration caused cycle and hormonal changes in testis, resveratrol were recovered the cyclic changes but in hormonal changes, RSV is efficient too but inadequate.

Evaluation of Sperm Deoxyribonucleic Acid (DNA) Damage and Effects on Embryo Development Using a Mouse Cryptorchidism Model

To investigate the effects of sperm deoxyribonucleic acid (DNA) damage on fertilization and embryo development using a mouse cryptorchidism model of sperm DNA damage induction. Male ICR mice (aged 5-6 weeks) underwent cryptorchidism on their left testicles and sham operations on their right testicles. Spermatogenesis and sperm DNA fragmentation were assessed after 1, 2, and 4 weeks using hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays. Intracytoplasmic sperm injection into the oocytes of BDF1 females (aged 4-6 weeks) was performed using DNA-damaged sperm and normal sperm, and the fertilization rates and embryonic development were compared. The testicular weight and size gradually decreased after induction of cryptorchidism, with progressive reduction of spermatogenesis and increased DNA damage after 1, 2, and 4 weeks. After intracytoplasmic sperm injection, the fertilization and blastocyst development rates were significantly lower in the cryptorchidism group; however, about one quarter of the embryos arising from DNA-damaged sperm continued to develop. This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism. This model could be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development.

Mechanism of CCAAT/enhancer-binding protein-homologous protein mediated endoplasmic reticulum stress involving in the epileptic brain damage

Objective To explore the neuronal apoptosis, endoplasmic reticulum stress(ERS)-related factors glucose-regulated protein 78(GRP78) and CCAAT/enhancer-binding protein-homologous protein(CHOP) expressions in hippocampus and possible mechanism of brain protection by 2-deoxy-D-glucose(2-DG) on their expressions in epileptic brain damage rats induced by pentetrazole. Methods Rats aged 21 d (n=120) were randomly divided into 3 groups: control group, status epilepticus (SE) group and SE+ DG group, with 40 rats in each group.On a SE rat mo-del, the neuronal apoptosis in the hippocampus was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling method.And the expression of GRP78 and CHOP were detected by Western blot and Real-time PCR. Results Apoptotic cells in the hippocampus were increased remarkably at 12 h, 24 h, and 48 h after the seizures, as compared with those of control rats.In SE group, chromatin condensation and breakdown of the nucleus in neurons in hippocampus were also easily observed by Hoechst staining.Meanwhile, the ERS was induced by SE, as witness by up-regulating GRP78 expression at both protein and mRNA level.The expression of GRP78 protein in the hippocampus was elevated at 3 h, 6 h and 12 h, with the maximum elevation noted at 6 h after SE.The GRP78 transcript expression was increased at 3 h, 6 h, and 12 h after SE.DG treatment enhanced the changes of GRP78 protein and transcript at 3 h, and abrogated the increase of GRP78 at 6 h and 12 h, as compared with those in SE group.CHOP activation occurred at 12 h, 24 h and 48 h in SE group.And the CHOP-mRNA expression was the same as the CHOP protein expression.The expression of CHOP protein and mRNA was decreased at 12 h, 24 h, and 48 h after using DG. Conclusion The endoplasmic reticulum stress response mediated by CHOP seems to be involved in the neuronal apoptosis caused by status epilepticus. Key words: Status epilepticus; CCAAT/enhancer-binding protein-homologous protein; Glucose-regulated protein 78; 2-deoxy-D-glucose; Apoptosis

Protective role of human insulin against the cytotoxicity associated with human mutant S20G islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a main component of islet amyloid in type 2 diabetes and cosecreted from β-cell with insulin. Clinical evidence from the patients with S20G mutation of the IAPP gene, as well as experimental evidence that insulin could inhibit amyloid formation of IAPP, suggests that a gradual reduction of insulin could be related to the cytotoxicity associated with S20G-IAPP through long-term deterioration of β-cells in type 2 diabetes. Our objective was to show an effect of human insulin on S20G-IAPP associated cytotoxicity. We analyzed the cytotoxicity associated with S20G-IAPP by controlling human insulin expression using adenovirus vectors with micro ribonucleic acid specifically against human insulin in endocrine AtT-20ins cells, which express human insulin permanently. Additionally, we carried out a follow-up study of circulating IAPP and insulin in type 2 diabetic patients. S20G-IAPP expression was associated with a decrease in viability and an increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-positive cells in AtT-20ins cells. Furthermore, downregulation of human insulin enhanced the cytotoxicity associated with S20G-IAPP, and induced the cytotoxicity associated with wild-type (WT)-IAPP. Reduction of ubiquitin carboxy-terminal hydrolase L1 activity enhanced cytotoxicity under the downregulation of human insulin expression in both S20G- and WT-IAPP transduced cells. A 5-year follow up of type 2 diabetic patients showed a disproportionate increase of serum fasting IAPP-to-insulin ratio from baseline. Human insulin plays a protective role against the cytotoxicity associated with S20G-IAPP, as well as WT-IAPP. The findings could suggest long-term deterioration of insulin secretion associates with IAPP linked cytotoxicity in type 2 diabetes.

Neuroprotective effect of penehyclidine hydrochloride on focal cerebral ischemia-reperfusion injury.

Penehyclidine hydrochloride can promote microcirculation and reduce vascular permeability. However, the role of penehyclidine hydrochloride in cerebral ischemia-reperfusion injury remains unclear. In this study, in vivo middle cerebral artery occlusion models were established in experimental rats, and penehyclidine hydrochloride pretreatment was given via intravenous injection prior to model establishment. Tetrazolium chloride, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and immunohistochemical staining showed that, penehyclidine hydrochloride pretreatment markedly attenuated neuronal histopathological changes in the cortex, hippocampus and striatum, reduced infarction size, increased the expression level of Bcl-2, decreased the expression level of caspase-3, and inhibited neuronal apoptosis in rats with cerebral ischemia-reperfusion injury. Xanthine oxidase and thiobarbituric acid chromogenic results showed that penehyclidine hydrochloride upregulated the activity of superoxide dismutase and downregulated the concentration of malondialdehyde in the ischemic cerebral cortex and hippocampus, as well as reduced the concentration of extracellular excitatory amino acids in rats with cerebral ischemia-reperfusion injury. In addition, penehyclidine hydrochloride inhibited the expression level of the NR1 subunit in hippocampal nerve cells in vitro following oxygen-glucose deprivation, as detected by PCR. Experimental findings indicate that penehyclidine hydrochloride attenuates neuronal apoptosis and oxidative stress injury after focal cerebral ischemia-reperfusion, thus exerting a neuroprotective effect.

Expression of Rap1 During Germ Cell Development in the Rat and Its Functional Implications in 2-Methoxyacetic Acid-induced Spermatocyte Apoptosis

To investigate the expression pattern of Ras-related protein 1 (Rap1) during testicular development and to clarify whether its expression is developmentally regulated and whether this expression is involved in the process of germ cell apoptosis. The expression pattern of Rap1 in the adult rat testicle was compared with that in the developing rat testicle using immunoblotting and immunohistochemical analyses. After the adult rats were treated with methoxyacetic acid (MAA), which selectively depletes primary spermatocytes, we correlated Rap1 expression with apoptotic dynamics using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), double immunofluorescent staining, and coimmunoprecipitation assays. During testicular development, Rap1 was expressed in the nucleus of gonocytes and in the Golgi apparatus of spermatocytes. The expression pattern of Rap1 during spermatogenesis was also shown to be stage specific. After 12 hours of MAA treatment, we found that Rap1 was translocated into the nucleus of some spermatocytes and the overlapping rate of Rap1 and NF-κB was much greater than that of Rap1 and TUNEL staining. In addition, Rap1 protein could be co-immunoprecipitated with NF-κB protein. In cooperation with NF-κB, Rap1 might be involved in the early stage of the apoptotic process occurring in the MAA-treated rat testicle. Additional characterization of this small guanosine triphosphatase in the apoptotic dynamics should provide information on the early diagnosis of MAA-induced male infertility.

Protective effects of Chinese herbal medicine Naoshuantong on neurovascular unit in rats with cerebral ischemia/reperfusion injury

To investigate the protective effects of Naoshuantong, a compound traditional Chinese herbal medicine, on the main components of neurovascular unit in rats with cerebral ischemia/reperfusion injury. A total of 30 male Sprague-Dawley rats were randomly divided into sham-operated, ischemia/reperfusion, and ischemia/reperfusion plus Naoshuantong groups. The cerebral ischemia was induced by 1.5 h of middle cerebral artery occlusion, followed by unlocking the thread to induce reperfusion injury. After 24 h of reperfusion, neurological functional deficits were assessed, apoptosis of main components of neurovascular unit was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling combined with neuronal nuclei antigen, glial fibrillary acidic protein or CD31 immunofluorescence double staining. Naoshuantong significantly reduced neurological functional deficits, and reduced neuron, astrocyte and vascular endothelium cell apoptosis induced by ischemia/reperfusion injury in the border zone of ischemic cortex. Naoshuantong is effective in protecting the neurovascular unit including neurons, astrocytes and vascular endothelium cells against cerebral ischemia/reperfusion-induced apoptosis.

A clinical study on regional lymphatic chemotherapy using an activated carbon nanoparticle–epirubicin in patients with breast cancer

The purpose of this study was to understand the short-term therapeutic effects of an activated carbon nanoparticle-epirubicin suspension for regional lymphatic chemotherapy in patients with breast cancer. One hundred and twenty patients with stage I, II, or III primary breast cancer were randomly divided into three groups: the lymphatic chemotherapy group using the activated carbon nanoparticle-epirubicin suspension, the epirubicin control group, and the activated carbon control group. Each group of 40 patients was further divided into two subgroups with the drug injected either 24 or 48 h before surgery. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was used to determine cancer cell apoptotic indices in metastatic lymph nodes. The epirubicin concentration in the black-stained lymph nodes in the lymphatic chemotherapy treatment group was 4,144.64 ± 2,426.44 ng/g, which is significantly higher than in the epirubicin control group (335.87 ± 212.82 ng/g, P < 0.001). The plasma epirubicin concentrations at 0.5, 1.5, and 24 h postinjection in the regional lymphatic chemotherapy treatment group were significantly lower than in the epirubicin control group (P < 0.001). Tolerable mild pain was observed at the injection area after administration of the epirubicin-activated carbon nanoparticle suspension. No regional necrosis or adverse effects were found. The TUNEL assay demonstrated that there was no significant difference in the apoptotic indices in the metastatic lymph nodes from the three groups. Performing lymphatic chemotherapy by regionally injecting the epirubicin-activated carbon nanoparticle suspension could significantly enhance the drug concentration in the stained lymph nodes and lower the plasma drug concentration. The epirubicin-activated carbon nanoparticle suspension has the ability to release the drug slowly in the lymph nodes and, as a result, can prolong the chemotherapeutic effects.

Antitumor activity of Xiaoaiping injection on human gastric cancer SGC-7901 cells

Xiaoaiping, extracted from Marsdenia tenacissima (Asclepiadaceae), is a Chinese medicine used to treat cough and asthma. Previous studies have shown that its active ingredients possess anti-inflammatory and anticancer effects. The aim of this study was to investigate the antitumor activities of Xiaoaiping through apoptosis induction in human gastric cancer SGC-7901 cells. The MTT assay was used to assess the cell growth and cell viability. SGC-7901 cells were treated with Xiaoaiping injection [(20–40) mg·mL−1] for 24 and 48 h. The apoptotic cells and the cell cycle distribution were analyzed by flow cytometry. The in vivo activity of Xiaoaiping was determined by the growth inhibition of the established tumor xenografts in nude mice. Caspase-3 activity, Bax and Bcl-2 proteins in tumor tissue were measured by immunohistochemistry and apoptosis was assayed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method. Xiaoaiping inhibited SGC-7901 cell growth in a time and dose dependent-manner and the estimated IC50 was (38.20 ± 0.27) mg·mL−1 after 24 h of treatment. The body weight and the tumor volume were significantly reduced in nude mice bearing human gastric tumor treated with Xiaoaiping. The inhibition rate of tumor growth in the mid-dose (200 mg·kg−1) group and high dose (400 mg·kg−1) group were 61.19% and 69.07%, respectively. Immunohistochemical staining showed an increase in caspase-3 and Bax expression whereas Bcl-2 expression decreased gradually. Xiaoaiping exerted potent antitumor activity in vitro and in vivo against human SGC-7901 cells; induced apoptosis and G1 cell cycle arrest. These results suggest that Xiaoaiping is a promising antitumor agent for the treatment of human gastric cancer.

RETRACTED: Ischemia postconditioning and mesenchymal stem cells engraftment synergistically attenuate ischemia reperfusion-induced lung injury in rats

BackgroundIt has been reported that ischemic postconditioning (IPO) or mesenchymal stem cell (MSC) engraftment could protect organs from ischemia/reperfusion (I/R) injury. We investigated the synergetic effects of combined treatment on lung injury induced by I/R. MethodsAdult Sprague-Dawley rats were randomly assigned to one of the following groups: sham-operated control, I/R, IPO, MSC engraftment, and IPO plus MSC engraftment. Lung injury was assessed by arterial blood gas analysis, the wet/dry lung weight ratio, superoxide dismutase level, malondialdehyde content, myeloperoxidase activity, and tissue histologic changes. Cytokine expression was detected using real-time polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end assay and annexin V staining. ResultsMSC engraftment or IPO alone markedly attenuated the lung wet/dry weight ratio, malondialdehyde and myeloperoxidase production, and lung pathologic injury and enhanced arterial partial oxygen pressure, superoxide dismutase content, inhibited pro-inflammatory cytokine levels, and decreased cell apoptosis in lung tissue, compared with the I/R group. In contrast, IPO pretreatment enhanced the protective effects of MSC on I/R-induced lung injury compared with treatment alone. Moreover, in the combined treatment group, the number of MSC engraftments in the lung tissue was increased, associated with enhanced survival of MSCs compared with MSC treatment alone. Additional investigation showed that IPO treatment increased expression of vascular endothelial growth factor and stromal cell-derived factor-1 in I/R lung tissue. ConclusionsIPO might contribute to the homing and survival of transplanted MSCs and enhance their therapeutic effects through improvement of the microenvironment of I/R injury.

N-acetylcysteine may prevent severe intestinal damage in necrotizing enterocolitis

ObjectiveThe aim of this study was to evaluate the preventive effect of N-acetylcysteine (NAC) on the development of necrotizing enterocolitis (NEC) in an experimental rat model. Material and MethodsThirty newborn Sprague-Dawley rats were randomly divided into 3 groups: NEC, NEC + NAC, and control. Necrotizing enterocolitis was induced by enteral formula feeding, exposure to hypoxia-hyperoxia, and cold stress. Pups in the NEC + NAC group were administered NAC at a dose of 150 mg/kg daily by intraperitoneal route from the first day until the last day of the study. All pups were killed on the fifth day. Proximal colon and ileum were excised for histopathologic, immunohistochemical (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end-labeling and caspase-3, caspase-8, caspase-9), and biochemical evaluation, including xanthine oxidase, total antioxidant status, total oxidant status, malondialdehyde, and myeloperoxidase activities. ResultsThe pups in the NEC + NAC group had better clinical sickness scores compared with those in the NEC group (P < .05). In histopathologic and apoptosis evaluations (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end-labeling and immunohistochemical evaluation for caspase-3 and caspase-9), the severity of bowel damage was significantly less in the NEC + NAC group compared with the NEC group (P < .01). Tissue malondialdehyde, myeloperoxidase, xanthine oxidase levels, and total oxidant status were significantly decreased in the NEC + NAC group, whereas total antioxidant status (TAS) was significantly increased in the NEC + NAC group (P < .01). ConclusionN-acetylcysteine therapy significantly reduced the severity of intestinal damage in NEC.