Transferase dUTP Nick-end Labeling Staining Research Articles

MMP13 is a critical target gene during the progression of osteoarthritis

IntroductionOsteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.MethodsTo investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13fx/fx (Mmp13Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.ResultsThe OA progression was decelerated in Mmp13Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.ConclusionsMmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.

WNT AGONIST ATTENUATES LIVER INJURY AND IMPROVES SURVIVAL AFTER HEPATIC ISCHEMIA/REPERFUSION

The Wnt/β-catenin signaling pathway is well characterized in stem cell biology and plays a critical role in liver development, regeneration, and homeostasis. We hypothesized that pharmacologic activation of Wnt signaling protects against hepatic ischemia/reperfusion (I/R) injury through its known proliferative and antiapoptotic properties. Sprague-Dawley rats underwent 70% hepatic ischemia by microvascular clamping of the hilum of the left and median lobes of the liver for 90 min, followed by reperfusion. Wnt agonist (2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine, 5 mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) in 0.5 mL was injected i.p. 1 h before ischemia or infused i.v. over 30 min right after ischemia. Blood and tissue samples from the pretreated groups were collected 24 h after reperfusion, and a survival study was performed. Hepatic expression of β-catenin and its downstream target gene Axin2 were decreased after I/R, whereas Wnt agonist restored their expression to sham levels. Wnt agonist blunted I/R-induced elevations of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase and significantly improved the microarchitecture of the liver. The cell proliferation determined by Ki67 immunostaining significantly increased with Wnt agonist treatment, and inflammatory cascades were dampened in Wnt agonist-treated animals, as demonstrated by attenuations in interleukin 6, myeloperoxidase, inducible nitric oxide synthase, and nitrotyrosine. Wnt agonist also significantly decreased the amount of apoptosis, as evidenced by decreases in both TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining as well as caspase 3 activity levels. Finally, the 10-day survival rate was increased from 27% in the vehicle group to 73% in the pretreated Wnt agonist group and 55% in the Wnt agonist postischemia treatment group. Thus, we propose that direct Wnt/β-catenin stimulation may represent a novel therapeutic approach in the treatment of hepatic I/R.

Conditional deletion of histone deacetylase‐4 in the central nervous system has no major effect on brain architecture or neuronal viability

Evidence from different laboratories using cell culture and in vivo model systems indicates that histone deacetylase-4 (HDAC4) plays an essential role in maintaining neuronal survival. Indeed, HDAC4 null knockout mice, which die within 2 weeks of birth, display cerebellar degeneration, whereas RNAi-mediated knockdown of HDAC4 expression in the retina of normal mice leads to apoptosis of retinal neurons. As a step toward analyzing the role of HDAC4 in the regulation of neuronal survival in more detail, we generated two separate lines of conditional knockout mice by breeding HDAC4-flox mice with mice expressing Cre recombinase through a Thy1 or nestin promoter. Surprisingly, both Thy1-Cre/HDAC4(-/-) mice, in which HDAC4 is ablated in neurons of the cortex and hippocampus, as well as Nes-Cre/HDAC4(-/-) mice, in which HDAC4 is ablated in neural progenitor cells of the CNS, appear normal at birth, have normal body weight, are fertile, and perform normally in locomotor activity assays. Histological analysis of the brains of Nes-Cre/HDAC4(-/-) mice revealed no obvious abnormalities in cytoarchitecture. Immunohistological analysis of tyrosine hydroxylase and calbindin also showed no discernible defects. Terminal deoxynucleotidyl transferase dUTP nick end-labeling staining showed no difference in the level of neuronal death in the cortex and cerebellum of Nes-Cre/HDAC4(-/-) mice compared with controls. These results indicate that neurons are less dependent on HDAC4 expression for their survival than previously believed and suggest that neuronal death observed in HDAC4 null knockout mice and after RNAi injection may result from HDAC4 deficiency in nonneural cells.

Oxidative Stress Is Associated With Cell Death, Wall Degradation, and Increased Risk of Rupture of the Intracranial Aneurysm Wall

The cause of rupture of intracranial aneurysms (IA) is not well understood. We previously demonstrated that loss of cells from the IA wall is associated with wall degeneration and rupture. To investigate the mechanisms mediating cell death in the IA wall. Snap-frozen tissue samples from aneurysm fundi were studied with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunostaining (14 unruptured and 20 ruptured), as well as with Western blot (12 unruptured and 12 ruptured). : Ruptured IA walls had more TUNEL-positive cells than unruptured walls (P < .001). Few cells positive for cleaved caspase-3 were detected. Cleaved caspase-9 (intrinsic activation of apoptosis) was significantly increased in ruptured IA walls, whereas cleaved caspase-8 (extrinsic activation of apoptosis) was not detected. Increased expression of hemeoxygenase-1, a marker for oxidative stress, was associated with IA wall degeneration and rupture. Our results show that programmed cell death is activated in the IA wall via the intrinsic pathway. High oxidative stress in the IA wall is probably a significant cause of the intrinsic activation of cell death.

The Salvia miltiorrhiza monomer IH764-3 induces apoptosis of hepatic stellate cells in vivo in a bile duct ligation-induced model of liver fibrosis

During the process of liver fibrosis, hepatic stellate cells (HSCs) play a critical role in the excessive production of extracellular matrix (ECM). Previous studies have indicated that the monomer IH764-3, one of the major bioactive components of Salvia miltiorrhiza, is able to inhibit HSC proliferation and induce the apoptosis of activated HSCs invitro. In the current study, we used a rat model of liver fibrosis induced by bile duct ligation (BDL) to investigate the effect of the monomer IH764-3 on the induction of apoptosis in HSCs in vivo. The rat model of liver fibrosis was established by BDL. Immunohistochemical staining of α-smooth muscle actin (α-SMA) was performed to detect HSC activation and proliferation and HSC apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and α-SMA immunohistochemical double staining. In addition, the protein expression levels of focal adhesion kinase (FAK), p-FAK (Tyr397), extracellular signal-regulated kinase (ERK) and p-ERK and the mRNA expression levels of FAK and ERK were measured by western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The monomer IH764-3 was associated with a significant decrease in intrahepatic fibrogenesis and collagen deposition and attenuated the liver fibrosis induced by BDL. Immunohistochemical staining revealed that the expression of α-SMA in the IH764-3 group was significantly decreased compared with that in the model group (12.92±2.45 vs. 22.65±2.16%, P<0.01). TUNEL and α-SMA immunohistochemical double staining also confirmed that IH764-3 increased the apoptotic rate of the activated HSCs (34.8±4.5 vs. 4.72±0.37%, P<0.01). Moreover, the results revealed that IH764-3 downregulated the expression levels of FAK, p-FAK (Tyr397), ERK and p-ERK in the liver tissue of rats with liver fibrosis. The monomer IH764-3 ameliorates experimental liver fibrosis by inhibiting HSC proliferation and inducing HSC apoptosis, warranting its use as a potential therapeutic agent in the treatment of liver fibrosis.

Interleukin-1β is internalised by viable Aggregatibacter actinomycetemcomitans biofilm and locates to the outer edges of nucleoids

The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1β, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1β; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1β when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1β and caused IL-1β leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1β localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1β interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1βin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1β. The interaction of IL-1β with conserved HU provides a potential mechanism for shaping bacterial gene expression.

Abstract 342: Increased Mitochondrial Permeability Transition Pore Opening Dominates Ischemia-Reperfusion Injury in the Aged Female Rat Heart

Background: Cardiovascular disease remains the leading cause of death in older post-menopausal women. Ischemia/Reperfusion (I/R) injury triggers mitochondrial calcium (Ca 2+ ) overload inducing mitochondrial permeability transition pore (MPTP) opening, mitochondrial dysfunction, and cell death potentially by necrosis, apoptosis, and/or autophagy. Purpose: We sought to determine if age-associated estrogen deficiency increases mitochondrial Ca 2+ sensitivity, providing a possible mechanism for increased vulnerability to I/R injury in older women. Methods: Mitochondrial respiration (MR) was assessed in isolated mitochondria from ventricles of adult (6 mo; n=15) and aged (24 mo; n=18) ovary-intact or ovariectomized (OVX) female F344 rats. MR at complexes I and II was compared in the absence (State 2) and presence (State 3) of ADP to calculate respiratory control index (RCI; state3/state 2). Reduced RCI following Ca 2+ addition was used to assess Ca 2+ sensitivity, while mitochondrial Ca 2+ retention capacity was measured to quantify MPTP opening (CRC; n=4-5/group) prior to and following coronary artery ligation (55 min I and 6 hr R). Apoptosis was examined using DNA laddering and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Markers of autophagy were evaluated by western blotting and mitochondrial morphology through electron microscopy (EM). Results: Significant age-dependent decreases in RCI for complex I (12%) and complex II (8%) were observed in the absence of Ca 2+ , and correlated with increased necrosis in aged hearts revealed by triphenyltetrazolium chloride (TTC) staining (p &lt; 0.05). Ca 2+ exposure decreased MR (18-30%; p &lt; 0.05) in Complex I of aged and OVX mitochondria vs adults. Furthermore, CRC worsened with age requiring less Ca 2+ to open the MPTP. Reduced DNA laddering and TUNEL staining combined with increased beclin-1 and cathepsinD expression in aged vs. adult further support a dominant role for necrosis over apoptosis underlying cell death in aged females (n=4-5/group). EM revealed morphological alterations with age and OVX. Conclusion: Decreased MR and increased MPTP opening with aging are likely causal in necrotic cell death mechanisms associated with I/R injury observed in post-menopausal women.

Effect of Fasudil, a Selective Inhibitor of Rho Kinase Activity, in the Secondary Injury Associated with the Experimental Model of Spinal Cord Trauma

Rho kinase (ROK) may play an important role in regulating the biological events of cells, including proliferation, differentiation, and survival/death. Blockade of ROK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. The aim of this experimental study was to determine the role of ROK signaling pathways in the inflammatory response, in particular in the secondary injury associated with the experimental model of spinal cord trauma. The injury was induced by application of vascular clips to the dura via a four-level T5 to T8 laminectomy in mice. Fasudil was administered in mice (10 mg/kg i.p.) 1 and 6 h after the trauma. The treatment with fasudil significantly decreased 1) histological damage; 2) motor recovery; 3) nuclear factor-κB (NF-κB) expression; 4) ROK activity; 5) inflammasome activation (caspase-1 and NOD-like receptor family, pyrin domain-containing 3 expression); 6) production of proinflammatory cytokine such as tumor necrosis factor and interleukin-1β (IL-1β); 7) neutrophil infiltration; 8) nitrotyrosine and poly-ADP-ribose formation; 9) glial fibrillary acidic protein expression; 10) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, FAS ligand expression, and Bax and Bcl-2 expression); and 11) mitogen-activated protein kinase activation (phospho-extracellular signal-regulated kinase and phospho-c-Jun NH(2)-terminal kinase expression). Our results indicate that inhibition of ROK by fasudil may represent a useful therapeutic perspective in the treatment of inflammation associated with spinal cord trauma.

Protective effect of NecroX, a novel necroptosis inhibitor, on gentamicin-induced ototoxicity

IntroductionNecroX is a novel necrosis and necroptosis inhibitor that shows scavenger activity against mitochondrial reactive oxygen species (ROS) and cytoprotective activity against various insults. These findings raise the possibility of its protective effect in ototoxicity. This study was performed to investigate the protective effect of NecroX on gentamicin (GM)-induced hair cell loss in neonatal mouse cochlea cultures. Materials and methodsThe protective effects of NecroX were measured by phalloidin staining of cultures from postnatal day 2–3 mice with GM-induced hair cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptosis. The radical-scavenging activity of NecroX was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. ResultsNecroX showed a significant and concentration-dependent protective effect against GM-induced hair cell loss, and hair cells retained their stereocilia well. NecroX decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. Additionally, NecroX showed direct radical scavenging activity in the DPPH assay. ConclusionsIn this study, we demonstrated the protective effect of NecroX on GM-induced hair cell loss in neonatal cochlea cultures, and suggest that it may be of therapeutic use in the treatment of drug-induced ototoxicity.

Stearic Acid Accumulation in Macrophages Induces Toll-Like Receptor 4/2-Independent Inflammation Leading to Endoplasmic Reticulum Stress–Mediated Apoptosis

Elevated serum free fatty acid levels are associated with an increased risk of cardiovascular disease and type 2 diabetes mellitus. Macrophages are recruited to atherosclerotic plaques and metabolic tissues during obesity and accumulate lipids, including free fatty acids. We investigated the molecular consequences of intracellular saturated free fatty acid accumulation in macrophages. Previously, we demonstrated that cotreatment of mouse peritoneal macrophages (MPMs) with stearic acid and triacsin C (an inhibitor of long-chain acyl coenzyme A synthetases) results in intracellular free fatty acid accumulation and apoptosis. Here, we used Western blotting analysis, real-time reverse transcription polymerase chain reaction, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining to assess endoplasmic reticulum (ER) stress, inflammation, and apoptosis in MPMs. Intracellular stearic acid accumulation induces Toll-like receptor 4/2-independent inflammation that results in ER stress-mediated apoptosis of MPMs. Polarization of MPMs to a proinflammatory M1 phenotype increases their susceptibility to inflammation and ER stress, but not apoptosis, in response to cotreatment with stearic acid and triacsin C. Intracellular accumulation of stearic acid in MPMs activates inflammatory signaling, leading to ER stress-mediated apoptosis. M1 macrophages are more prone to stearic acid-induced inflammation and ER stress. These same pathways may be activated in macrophages residing in atherosclerotic plaques and metabolic tissues during conditions of obesity and hyperlipidemia.

Histopathologic and apoptotic effect of nanosilver in liver of broiler chickens

Silver nanoparticles can destroy bacteria, viruses and fungi; therefore, it is recommended as a disinfectant and can be used as a drug in the treatment of some non-curable viral disease in livestock. The aim of the present investigation was to study the effects of nanosilver induced toxicity on the liver of broiler chickens. This study was carried out on 240 one-day-old male broiler chickens (Ross 308) in acompletely randomized design (CRD) in four treatments at 0 (control), 4, 8 and 12 ppm levels of silver nanoparticles that was added daily to the drinking water with four repetitions within 16 separate cages and 15 birds in each pen. At the end of theexperiment (day 42), 32 chicks were randomly selected and slaughtered thereafter,H&E, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and light microscope was used to examine livers. Liver histopathology and H&E staining showed dose depended changes such as fatty degeneration, sinusoidal congestion, central vein dilatation, necrosis in hepatocytes and fibrosis. TUNEL staining showed significant (p<0.01) increase of apoptotic cells in groups 3 (8 ppm) and 4 (12 ppm). Data were statistically analyzed using ANOVA. Lesions and apoptotic cells in groups 3 (8 ppm) and 4 (12 ppm) were more severe than in group 2 (4 ppm). It can be concluded that higher concentrations of nanosilver (8 and 12 ppm) can induce sever lesions in chickens liver. Key words: Broiler chicks, liver, histopathology, immunohistochemistry, TUNEL.

Ischemic Postconditioning Protects Liver From Ischemia-Reperfusion Injury by Modulating Mitochondrial Permeability Transition

We tested the effectiveness of ischemic postconditioning (iPoC) in mitigating ischemia-reperfusion (I/R) injury of liver and the mechanism involves inhibiting the opening of the mitochondrial permeability transition pore (mPTP). iPoC, performed by three cycles of 1 min I/R of the liver, was tested on a partial liver I/R model on rats. The serum alanine transaminase levels, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, cytochrome c release, the formation of 4-hydroxy-2-nonnenal-modified proteins, and mitochondrial membrane potential (Δψm) were measured. Atractyloside (ATR) and NIM811, which modify the opening of mPTP, were administered in selected groups. iPoC, and NIM811, diminished the elevation of serum alanine transaminase level after I/R injury (174.0±28.3 U/L for iPoC; 94.3±25.4 U/L for control+NIM811) when compared with others (416.3±16.7 U/L for control, 557.0±86.7 U/L for iPoC+ATR, P<0.05). The expressions of cytosolic cytochrome c after I/R injury were decreased in iPoC and control+NIM811 groups when compared with others. After I/R, the apoptosis and the 4-hydroxy-2-nonnenal-modified proteins were attenuated in iPoC group when compared (apoptotic counts/50 HPF: 723.3±98.7 for iPoC, 1274±201.2 for control, 1057.6±39 for iPoC+ATR, P<0.05). The Δψm measured by flow cytometry was better preserved in iPoC and NIM811 groups. iPoC attenuated cell deaths after I/R injury of liver. The protective effects were negated by the addition of ATR--a mPTP opener--and mimicked by injection of NIM811--a mPTP opening inhibitor. The study indicated iPoC conferred protection by modulating mPTP.

Overexpression of extracellular superoxide dismutase has a protective role against hyperoxia‐induced brain injury in neonatal mice

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P< 0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P < 0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.

Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells

Ethnopharmacological relevanceZiziphus extracts have been used in Traditional Chinese Medicine for the treatment of cancer. Aim of the studyIn the present study we have investigated the effects of Ziziphus jujube extracts (ZEs) on breast cancer. Materials and methodsWe evaluated the effects of increasing concentrations of ZEs on ERα positive MCF-7 and ERα negative SKBR3 breast cancer cell proliferation using MTT assays. Apoptosis was analyzed by evaluating the involvement of some pro-apoptotic proteins, including Bax, Bad, Bid and PARP cleavage by immunoblotting analysis. Moreover, the effects of ZEs treatment on apoptosis were tested by both DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. By using chromatographic techniques, we identified the constituents of the effective extracts. ResultsZE1, ZE2, and ZE4 exerted significant antiproliferative effects on estrogen receptor alpha (ERα) positive MCF-7 (IC50 values of 14.42, 7.64, 1.69μg/mL) and ERα negative SKBR3 (IC50 values of 14.06, 6.21, 3.70μg/mL) human breast cancer cells. Remarkably, ZEs did not affect cell viability of both normal human fibroblasts BJ1-hTERT and nonmalignant breast epithelial MCF-10A cells. Treatment with ZEs induced cell death by apoptosis in both malignant breast cells. We found that the most effective extracts ZE2 and ZE4 shared a number of triterpenic acids, already known for their anticancer activities. ConclusionsOur data provide a rational base for the use of Ziziphus extracts in the treatment of breast cancer in Traditional Chinese Medicine.

Transforming Growth Factor- Beta and its Signaling Mediator Smad3 Enhance Cell Survival After Vascular Injury

Introduction: It is well established that Transforming Growth Factor-beta (TGF-β) promotes intimal hyperplasia (IH); levels of this cytokine are elevated after vascular injury and blocking TGF-β results in inhibition of the hyperplastic response. IH is usually accompanied by inhibition of apoptosis/increased cell survival, which contributes to the increased cellularity that accompanies hyperplastic plaque. Paradoxically, TGF-β traditionally is thought to be pro-apoptotic. We have previously shown injured smooth muscle cells (SMCs) contain elevated levels of Smad3 and may respond to TGF-β differently compared to normal SMCs. in the current study, we tested a hypothesis that TGF-β in the presence of elevated Smad3 may be anti-apoptotic. Methods: Cultured aortic SMCs were infected with adenovirus expressing Smad3 or GFP and then stimulated with TGF-β for 24 hours. Apoptosis was induced with 5 minutes of ultraviolet light. Cleaved Caspase-3 immunofluorescence and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with adenovirus expressing Smad3; or GFP control and arteries harvested at 3 days were subject to TUNEL staining and immunohistochemistry for cleaved Caspase-3. Akt activation was measured with phospho-Ser473 Akt (p-Akt). SIS3 was used to inhibit Smad3 activation. Results: in vitro, stimulation of SMCs withTGF-β reduced the number of TUNEL positive cells (Control = 35.0 vs TGF-β = 22.6%, p < 0.05) and diminished activation of Caspase-3 (31.4 vs 21.8%, p < 0.01) suggesting that TGF-β has an anti-apoptotic or protective effect on vascular SMCs. These protective effects were reversed by knockdown (siRNA) of TGF-β’s primary signaling protein, Smad3 (cleaved Caspase-3, p < 0.05). Moreover, overexpression of Smad3 in SMCs further enhanced TGF-β’s antiapototic effect (cleaved Caspase-3, p < 0.05). TGF-β phosphorylation of Akt was time-dependent and significantly enhanced when Smad3 was overexpressed. Both chemical inhibition and knockdown of Smad3 prevented phosphorylation of Akt (n = 3, p < 0.01). in injured carotid arteries, overexpression of Smad3 resulted in a dramatic inhibition of apoptosis but increased phosphorylation of Akt in the medial layer of the arterial wall: cleaved Caspase-3 (GFP = 35.2 vs Smad3 = 4.9, p < 0.01), TUNEL (32.1 vs 8.5, p < 0.01) and p-Akt (6.2 vs 22.4 p < 0.05). Conclusions: Our data show that TGF-β promotes SMC survival both in vitro and in vivo. This anti-apoptotic effect may well be one of the primary mechanisms through which TGF-β enhances intimal hyperplasia. Knockout and overexpression studies suggest that TGF-β’s inhibitory effect on apoptosis is mediated through the signaling protein Smad3. the correlation with Akt activation suggests that this may be mediated by the Akt-cell survival pathway.

Transforming Growth Factor- Beta and its Signaling Mediator Smad3 Enhance Cell Survival After Vascular Injury

Introduction: It is well established that Transforming Growth Factor-beta (TGF-β) promotes intimal hyperplasia (IH); levels of this cytokine are elevated after vascular injury and blocking TGF-β results in inhibition of the hyperplastic response. IH is usually accompanied by inhibition of apoptosis/increased cell survival, which contributes to the increased cellularity that accompanies hyperplastic plaque. Paradoxically, TGF-β traditionally is thought to be pro-apoptotic. We have previously shown injured smooth muscle cells (SMCs) contain elevated levels of Smad3 and may respond to TGF-β differently compared to normal SMCs. In the current study, we tested a hypothesis that TGF-β in the presence of elevated Smad3 may be anti-apoptotic. Methods: Cultured aortic SMCs were infected with adenovirus expressing Smad3 or GFP and then stimulated with TGF-β for 24 hours. Apoptosis was induced with 5 minutes of ultraviolet light. Cleaved Caspase-3 immunofluorescence and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with adenovirus expressing Smad3; or GFP control and arteries harvested at 3 days were subject to TUNEL staining and immunohistochemistry for cleaved Caspase-3. Akt activation was measured with phospho-Ser473 Akt (p-Akt). SIS3 was used to inhibit Smad3 activation. Results: in vitro, stimulation of SMCs withTGF-β reduced the number of TUNEL positive cells (Control = 35.0 vs TGF-β = 22.6%, p < 0.05) and diminished activation of Caspase-3 (31.4 vs 21.8%, p < 0.01) suggesting that TGF-β has an anti-apoptotic or protective effect on vascular SMCs. These protective effects were reversed by knockdown (siRNA) of TGF-β’s primary signaling protein, Smad3 (cleaved Caspase-3, p < 0.05). Moreover, overexpression of Smad3 in SMCs further enhanced TGF-β’s antiapototic effect (cleaved Caspase-3, p < 0.05). TGF-β phosphorylation of Akt was time-dependent and significantly enhanced when Smad3 was overexpressed. Both chemical inhibition and knockdown of Smad3 prevented phosphorylation of Akt (n = 3, p < 0.01). in injured carotid arteries, overexpression of Smad3 resulted in a dramatic inhibition of apoptosis but increased phosphorylation of Akt in the medial layer of the arterial wall: cleaved Caspase-3 (GFP = 35.2 vs Smad3 = 4.9, p < 0.01), TUNEL (32.1 vs 8.5, p < 0.01) and p-Akt (6.2 vs 22.4 p < 0.05). Conclusions: Our data show that TGF-β promotes SMC survival both in vitro and in vivo. This anti-apoptotic effect may well be one of the primary mechanisms through which TGF-β enhances intimal hyperplasia. Knockout and overexpression studies suggest that TGF-β’s inhibitory effect on apoptosis is mediated through the signaling protein Smad3. the correlation with Akt activation suggests that this may be mediated by the Akt-cell survival pathway.

Edaravone inhibits apoptosis caused by ischemia/reperfusion injury in a porcine hepatectomy model

To investigate the effect of E3-methyl-1-phenyl-2-pyrazolin-5-one (Edr) on hepatic ischemia-reperfusion (I/R) injury and liver regeneration in a porcine hepatectomy model. One hour ischemia was induced by occluding the vessels and the bile duct of the right and median lobes. A 40% left hepatectomy was performed after reperfusion. Six animals received Edr (3 mg/kg per hour) intravenously and six control animals received saline just before reperfusion. Remnant liver volume, hemodynamics, aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase and lactic acid, were compared between the groups. The expression of transforming growth factor-β (TGF-β1) and toll-like receptor (TRL) mRNA in hepatic tissues was examined using reverse transcription polymerase chain reaction. Apoptosis was demonstrated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Serum AST (P = 0.029), and toll like receptor 4 level (P = 0.043) were significantly lower after 3 h in animals receiving Edr. In addition, TUNEL staining in Edr-treated pigs showed significantly fewer hepatocytes undergoing apoptosis compared with control pigs. After 1 mo, all factors were non-significantly different between the two groups. Edr is considered to reduce hepatic injury in the early stage of I/R injury in a porcine model.

Polo-like kinase 1, a new therapeutic target in hepatocellular carcinoma

To investigate the role of polo-like kinase 1 (PLK1) as a therapeutic target for hepatocellular carcinoma (HCC). PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA (siRNA) was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) and bromodeoxyuridine (BrdU) assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progression was compared with controls. RT-PCR showed that PLK1 was overexpressed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells. siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLK1-treated mice, but not in controls. Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G pathway.

Role of Oxidative Stress in Retinal Photoreceptor Cell Death in N-Methyl-N-nitrosourea–Treated Mice

This study aimed to investigate whether oxidative stress contributes to retinal cell death in a mouse model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU). We measured in vitro MNU-induced radical production in retinal cell cultures of murine 661W photoreceptor–derived cells; RGC-5, a mouse ganglion cell line; and primary retinal cells. The addition of MNU induced oxidative radical generation in 661W and primary retinal cells, but not in RGC-5 cells. Edaravone, a free radical scavenger, at 1 μM reduced MNU-induced radical production in 661W and primary retinal cells. To induce in vivo retinal photoreceptor degeneration in mice, we administered 60 mg/kg MNU by intraperitoneal injection. We intravenously administered 1 mg/kg edaravone immediately and at 6 h after the MNU injection. Retinal photoreceptor degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and by oxidative stress markers. MNU caused photoreceptor cell loss at 7 days after administration. Edaravone inhibited ONL thinning and reduced TUNEL-positive cells and the oxidative stress markers. These findings indicate that MNU leads to selective photoreceptor degradation via oxidative stress in vitro and in vivo and may help to understand the pathogenic mechanism of retinitis pigmentosa.

Inhibitory effect of endostatin combined with paclitaxel-cisplatin on breast cancer in xenograft-bearing mice

In the present study, we aimed to investigate the tumor-inhibiting effects of recombinant human endostatin (rhES) combined with paclitaxel-cisplatin (TP regimen) on human breast cancer in xenograft-bearing nude mice. A total of 24 mice bearing human breast cancer xenografts were administered both rhES and TP, TP alone, rhES alone or saline. The tumor growth inhibition was observed. Serum vascular endothelial growth factor (VEGF) levels and microvessel density (MVD) were determined by ELISA and immunohistochemistry, respectively. Cell apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. Survival time was observed in another 24 nude mice with the same treatment. MVD expression in the group administered rhES and TP was lower than that in the other groups (P<0.05); serum VEGF levels in the combined drug group were lower compared to the other groups; the apoptotic index increased in the combined drug group. We conclude that the effect of the TP regimen combined with rhES on breast cancer is better than that of the TP regimen alone.