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1.  OnabotulinumtoxinA treatment for chronic migraine: experience in 52 patients treated with the PREEMPT paradigm 
SpringerPlus  2015;4:176.
OnabotulinumtoxinA (OnabotA) was approved for treatment of chronic migraine (CM) after publication of PREEMPT trials. Thus, we set out to evaluate the efficacy of OnabotA in a series of patients with CM treated according to the PREEMPT protocol.
In May 2012 we began to offer OnabotA to patients with CM who did not respond to topiramate and at least one other preventive therapy (beta blocker and/or calcium channel antagonist). We prospectively recorded demographic data and the characteristics of migraine, and we assessed the modifications in monthly headache and migraine days, as well as the number of days of symptomatic medication and triptan intake.
By September 2014 we had treated 52 patients (8 male, 44 female), whose age at treatment onset was 42.8 ± 12.7 years (range: 16–71) and age at migraine onset was 16.8 ± 7.8 years (3–32). In 43 of these patients (82.7%) symptomatic overuse of medication was observed at the onset of treatment. A total of 168 procedures were performed and after the first session, we observed a significant reduction in all the variables considered. Twelve (23.1%) patients failed to perceive a positive effect after the first procedure and it was not repeated in 4 of them. By contrast, there was a significant decreasing in all the variables evaluated compared to the baseline in the 39 patients that received a second series of injections.
The use of OnabotA according to the PREEMPT paradigm is an effective treatment in patients with chronic migraine in a real-life setting.
doi:10.1186/s40064-015-0957-z
PMCID: PMC4398682  PMID: 25897415
Migraine; Chronic migraine; OnabotulinumtoxinA; Topiramate
2.  Preserved micturition after intradetrusor onabotulinumtoxinA injection for treatment of neurogenic bladder dysfunction in Parkinson’s disease 
BMC Urology  2016;16(1):55.
Background
To assess the efficacy and safety of intradetrusor onabotulinumtoxinA (OnabotA) injection treatment in patients with neurogenic lower urinary tract dysfunction (NLUTD), especially for patients with Parkinson disease (PD).
Methods
PD patients refractory to oral antimuscarinic participated in an off-label use study and were evaluated prior and after 200 IU OnabotA injection into detrusor muscle, including trigone. Changes due to treatment were evaluated using bladder diaries, urodynamics, and questionnaires. Statistical analysis comprised Wilcoxon rank-sum test. Values are presented as mean ± standard deviation.
Results
Ten PD patients (4 female and 6 male, mean age: 67.9 ± 5.36 years) with LUTD were enrolled. All patients tolerated the treatment. Bladder diary variables decreased significantly (p ≤ 0.011) after OnabotA injection compared to variables prior injection. Desire to void and maximum bladder capacity increased significantly in urodynamics (p ≤ 0.05). Maximum detrusor pressure during voiding phase normalised from 56.2 to 18.75 cm/H20. Detrusor overactivity was less often detectable. All patients voided spontaneously. Mean post void residual (PVR) volume was 77.0 ± 119.78 mL postoperatively. No urinary retention or side effects have been observed during/after treatment. Mean follow-up time was 4 months (range of 1–12). 4 patients requested repeated injection after a mean period of 10 months between first and second injection.
Conclusions
Our data confirm the efficacy and safety of 200 IU OnabotA injection in patients with neurogenic LUTD due to PD. The risk of urinary retention or high post-urinary residual volumes seems to be minor after OnabotA-injection. More research is needed with larger sample size to confirm the significance of these findings.
Trial registration
Trial Number: ISRCTN 11857462, Registration Date 2016/10/08
doi:10.1186/s12894-016-0174-2
PMCID: PMC5011793  PMID: 27596481
Parkinson’s disease; OnabotulinumtoxinA (OnabotA) injection; Neurogenic lower urinary tract dysfunction; International Consultation and Incontinence Questionnaire-Lower Urinary Tract Symptoms Quality of Life (ICIQ-UI)
3.  Heat shock protein 70 expression is associated with inhibition of renal tubule epithelial cell apoptosis during recovery from low-protein feeding 
Cell Stress & Chaperones  2006;11(4):309-324.
The cellular stress response can mediate cellular protection through expression of heat shock protein (Hsp70), which can interfere with the process of apoptotic cell death. Factors regulating renal epithelial cell apoptosis include angiotensin II. In the present study, we have examined the relationship between the Hsp70 expression and the apoptotic pathway in the kidneys from low-protein–fed rats (8% protein). The possible cytoprotective role of Hsp70 has been evaluated during low-protein feeding and after reincorporation of 24% protein in the diet. The effect of angiotensin II AT1 receptor inhibition has also been studied. Rats were fed with a low-protein (LP) diet (8% protein) for 14 days, and then the animals were recovered by means of a normal protein diet (24% protein) (RP) for 14, 21, and 30 days, and control rats received 24% protein (NP) in the diet. LP and NP rats treated with Losartan (10 mg/kg) were also evaluated. The following methods were performed on the kidneys: terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay for apoptosis, reverse transcriptase-polymerase chain reaction assay for AT1, Bax, and Bcl-2 messenger ribonucleic acid (mRNA) expression, and immunohistochemical and Western blot for Hsp70 and caspase 3 protein expression and activity. In the LP group, the cells of the medullary ducts (MDs) showed increased apoptosis associated with weak immunoreaction for Hsp70 and decreased Hsp70 protein levels. In these animals, enhanced proapoptotic ratio Bax/Bcl-2 linked to decreased procaspase 3 protein levels with increased caspase 3 activation were demonstrated. A cytoprotection attributed to Hsp70 could be noted in the RP rats after 21 days of reincorporation of the normal diet, and in the LP-fed group treated with Losartan. In these cases, the MD cells displayed decreased apoptosis and increased Hsp70 expression in colocalization staining, and high Hsp70 levels in cytosolic fraction. A decreased proapoptotic ratio Bax/Bcl-2, associated with increased Bcl-2 mRNA, was also observed. Our results provide evidence for an antiapoptotic, cytoprotective effect of Hsp70 in kidney MD cells of rats with LP intake, when the animals were recovered with 24% protein in diet and after angiotensin II AT1 receptor inhibition. Angiotensin II seems to play a role in the pathogenesis of tubule epithelial cell apoptosis during LP feeding.
doi:10.1379/CSC-199.1
PMCID: PMC1712679  PMID: 17278880
4.  Apoptosis associated with Wnt/β-catenin pathway leads to steroid-induced avascular necrosis of femoral head 
Background
The objective of the current study was to establish a rat model to investigate apoptosis in steroid-induced femoral head osteonecrosis occurring via the Wnt/β-catenin pathway.
Methods
Male Sprague–Dawley (SD) rats were randomly divided into a control group (group A), model group (group B) and sFRP1 group (group C), each consisting of 24 rats, and the rats were intravenously injected with LPS (10 μg/kg body weight). After 24 h, three injections of MPS (20 mg/kg body weight) were administered intramuscularly at 24-h intervals. The rats in group C were injected intramuscularly with 1 μg/kg sFRP1 protein per day for 30 days, beginning at the time of the first MPS administration. The group A rats were fed and housed under identical conditions but received saline injection. All animals were sacrificed at weeks 2, 4 and 8 from the first MPS injection. Histopathological staining was preformed to evaluated osteonecrosis. Apoptosis was detected via quantitative terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick-end labelling (TUNEL) staining, caspase-3 activity assay, and detection of Bcl-2 and Bax protein expression by immunohistochemistry and Western blotting. Wnt/β-catenin pathway signalling molecules, including activated β-catenin and c-Myc, were detected by immunohistochemistry and Western blotting.
Results
Typical osteonecrosis was observed in groups B and C. Apoptosis gradually increased with increasing time in both groups B and C. More severe osteonecrosis and apoptosis were observed in group C compared with group B. The expression levels of caspase-3 and Bax were higher while that of Bcl-2 was lower in group C compared with group B. The expression levels of activated β-catenin and c-Myc gradually decreased with increasing time in both groups B and C, and they were lower in group C compared with group B.
Conclusions
The Wnt/β-catenin pathway is involved in the pathogenesis of early stage SANFH, as we have demonstrated in an SANFH rat model, and it may act through the regulation of c-Myc, which affects the cell cycle and cell apoptosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12891-015-0606-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12891-015-0606-2
PMCID: PMC4453221  PMID: 26037065
Steroid-induced avascular necrosis of femoral head; Apoptosis; β-catenin; c-Myc; Rat
5.  Role of Bax in quercetin-induced apoptosis in human prostate cancer cells 
Biochemical Pharmacology  2008;75(12):2345-2355.
The aim of this study was to investigate the effect of quercetin, a flavonoid, on the apoptotic pathway in a human prostate cell line (LNCaP). We observed that treatment of cells for 24 h with quercetin induced cell death in a dose-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in quercetin-treated cells. Treatment of LNCaP cells with an apoptosis inducing concentration of quercetin (100 μM) resulted in a rapid decrease in the inhibitory Ser(473) phosphorylation of Akt leading to inhibition of its kinase activity. Quercetin treatment (100 μM) also caused a decrease in Ser(136) phosphorylation of Bad, which is a downstream target of Akt. Protein interaction assay revealed that during treatment with quercetin, Bcl-xL dissociated from Bax and then associated with Bad. Our results also show that quercetin decreases the Bcl-xL:Bax ratio and increases translocation and multimerization of Bax to the mitochondrial membrane. The translocation is accompanied by cytochrome c release, and procaspases-3, -8 and -9 cleavage and increased poly (ADP-ribose) polymerase (PARP) cleavage. Similar results were observed in human colon cancer HCT116Bax+/+ cell line, but not HCT116Bax−/− cell line. Interestingly, at similar concentrations (100 μM), quercetin treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cell line (PrEC) and rat prostate epithelial cell line (YPEN-1). Our results indicate that the apoptotic processes caused by quercetin are mediated by the dissociation of Bax from Bcl-xL and the activation of caspase families in human prostate cancer cells.
doi:10.1016/j.bcp.2008.03.013
PMCID: PMC3266687  PMID: 18455702
Quercetin; apoptosis; caspase; PI3K/Akt pathway; cytochrome c; Bax; Bad; Bcl-xL
6.  Neurotoxic effect of lead on rats: Relationship to Apoptosis 
Background
Lead toxicity has been subjected to intensive research work, but some aspects of its mechanism needs to be elucidated.
Objectives
In the current study we aim to investigate the impact of lead toxicity on some different intermediates of apoptotic signaling pathway in experimental rats.
Design and methods
We measured caspase-8 and caspase-9 [by chemilumenescence], Bax and Bcl-2 [by ELISA] in Experimental rats, injected intraperitoneally with lead acetate for 7days at the dosage of 25, 50 and l00 mg/kg body weight and compared to control rats injected with deionized distilled water instead. instead.
Results
Lead acetate significantly increased the levels of caspase 8, caspase 9 and Bax in liver, kidney and brain of experimental animals especially those with high doses. Meanwhile, caspase 8 and Bax significantly increased in brain tissue at low dose of lead, while Bcl-2 significantly increased only with advanced toxicity. Furthermore, Bax/bcl2 ratio was significantly high in kidney (p<0.05), liver (p<0.01) and brain (p<0.01) at higher doses of lead toxicity. However, brain tissues showed significant Bax/Bcl2 ratio (p<0.05) at low lead dose. A significant positive correlation was noticed between the blood level of lead and enzymatic level of caspase 8, caspase 9 and Bax in different tissues.
Conclusion
: we concluded that lead might have toxic effect through intrinsic and extrinsic induction of apoptotic pathway with prominent effect on brain tissue even at low dose.
PMCID: PMC3883608  PMID: 24421747
Lead toxicity; Rats; Apoptosis; Bcl-2; Bax; Caspase 8 and Caspase 9
7.  The Effects of Erdosteine and N-Acetylcysteine on Apoptotic and Antiapoptotic Markers in Pulmonary Epithelial Cells in Sepsis 
The Eurasian Journal of Medicine  2013;45(3):167-175.
Objective:
This study investigated the frequency of apoptosis in rat pulmonary epithelial cells after the injection of an intraperitoneal endotoxin lipopolysaccharide (LPS), the effects of LPS on apoptotic (bax, caspase-3) and antiapoptotic (bcl-2) markers during lung damage, and the protective effects of two known antioxidant agents, erdosteine and N-acetylcysteine (NAC).
Materials and Methods:
Male Wistar rats were divided into the following six groups, which included nine rats each: two control groups, two LPS-treated groups, one erdosteine-treated group (150 mg/kg), and one NAC-treated group (150 mg/kg). LPS was injected intraperitoneally at a dosage of 20 mg/kg. Following LPS injection, the antioxidants were orally administered. The rats were sacrificed at 24 h after LPS administration. The levels of apoptosis in bronchiolar and alveolar cells were determined using the TUNEL-staining method. Immunohistochemical staining of cytoplasmic bax, caspase-3, and bcl-2 in the epithelial cells was performed.
Results:
Erdosteine and NAC significantly reduced the rate of LPS-induced pulmonary epithelial cell apoptosis. The effect of NAC on regulating apoptosis was weaker than that of erdosteine. Erdosteine and NAC significantly reduced the local induction of bax and caspase 3 and significantly increased the reduced local production of bcl-2.
Conclusion:
These findings suggest that erdosteine and NΑC can effectively protect the lungs from the damaging effects of LPS.
doi:10.5152/eajm.2013.35
PMCID: PMC4261429  PMID: 25610275
Apoptosis; epithelial cells; erdosteine; N-acetylcysteine; pathogenesis; sepsis
8.  Hydrogen-Rich Saline Promotes the Recovery of Renal Function after Ischemia/Reperfusion Injury in Rats via Anti-apoptosis and Anti-inflammation 
Purpose: Hydrogen is a proven novel antioxidant that selectively reduces hydroxyl radicals. In this study, we investigated the effects of hydrogen-rich saline solution on the prevention of renal injury induced by ischemia/reperfusion (I/R) and on renal function recovery.
Methods: A rat model of renal I/R injury was induced by 45 min occlusion of the left renal pedicle, followed by 108 h reperfusion. The right kidney was surgically removed. Then, 0.9% NaCl solution (1 ml/kg) or hydrogen-rich saline solution (HRSS; 1 ml/kg) was injected into the abdominal cavity at 4 h intervals. We assessed the influence of HRSS or control saline solution on the recovery of renal function after I/R injury. Kidney tissues were taken at different time points (24, 36, 48, 72, and 108 h after reperfusion) and frozen (-80°C). Kidney cell apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive staining. Additionally, the apoptotic factors (Bcl-2, Bax, caspase-3, caspase-9, and caspase-8) and the pro-inflammatory cytokines (IL-6 and TNF-α) were measured in the kidney tissues. Finally, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were measured.
Results: Histological analyses revealed a marked reduction of interstitial congestion, edema and hemorrhage in renal tissue after HRSS treatment compared to saline treatment. After I/R injury, BUN, Cr, Bcl-2, caspase-3, caspase-9, caspase-8, IL-6, and TNF-α were all significantly increased, while Bax expression was decreased. HRSS remarkably reversed these changes. Moreover, BUN and Cr decreased more rapidly in the rats treated with HRSS compared to the rats treated with control saline solution.
Conclusions: HRSS showed a protective effect in the prevention of renal injury and could promote renal function recovery after I/R injury in rats. HRSS might partially exert its role through an anti-apoptotic and anti-inflammatory action in kidney cells.
doi:10.3389/fphar.2016.00106
PMCID: PMC4840252  PMID: 27148060
hydrogen; recovery of function; ischemia/reperfusion; kidney; apoptosis
9.  Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia 
British Journal of Pharmacology  2012;167(1):95-108.
BACKGROUND AND PURPOSE
Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5-lipoxygenase (5-LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid–based ‘dual inhibitor’ of the COX and 5-LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH.
EXPERIMENTAL APPROACH
Rats were treated daily with testosterone propionate (3 mg·kg−1 s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg−1, i.p.) or flavocoxid (20 mg·kg−1, i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells.
KEY RESULTS
Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE2 and leukotriene B4 (LTB4), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth.
CONCLUSION AND IMPLICATIONS
Our results show that a ‘dual inhibitor’ of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism.
doi:10.1111/j.1476-5381.2012.01969.x
PMCID: PMC3448916  PMID: 22471974
apoptosis; BPH; COX; flavocoxid; growth factors; inflammation; 5-LOX
10.  BCL-2 AND BAX EXPRESSION AND PROSTATE CANCER OUTCOME IN MEN TREATED WITH RADIOTHERAPY IN RADIATION THERAPY ONCOLOGY GROUP PROTOCOL 86–10 
Purpose
Bcl-2 and bax are proteins with opposing roles in apoptosis regulation; yet abnormal expression of either has been associated with failure after radiotherapy (RT). In this study we examined bcl-2 and bax expression as predictive markers in men treated with radiotherapy ± androgen deprivation on Radiation Therapy Oncology Group (RTOG) protocol 86-10.
Experimental Design
Suitable archival diagnostic tissue was obtained from 119 (26%) patients for bcl-2 analysis and 104 (23%) patients for bax analysis. Cox proportional hazards multivariate analysis was used to determine the relationship of abnormal bcl-2 and bax expression to the end points of local failure, distant metastasis, cause-specific mortality, and overall mortality. Bcl-2 overexpression was classified as any tumor cell cytoplasmic staining and altered bax expression was classified as greater or lesser cytoplasmic staining intensity of tumor cells as compared with adjacent normal prostate epithelium.
Results
The study cohort exhibited bcl-2 overexpression in 26% (n = 30) of cases and abnormal bax expression in 47% (n = 49) of cases. A borderline significant relationship was observed between abnormal bax expression and higher Gleason score (p = 0.08). In univariate and multivariate analyses, there was no statistically significant relationship seen between abnormal bcl-2 or bax expression and outcome.
Conclusions
Abnormal bcl-2 and bax expression were not related to any of the end points tested. The cohort examined was comprised of patients with locally advanced disease and it is possible that these markers may be of greater value in men with earlier-stage prostate cancer.
doi:10.1016/j.ijrobp.2006.03.056
PMCID: PMC1950741  PMID: 16814949
Bcl-2; Bax; Androgen Deprivation; Radiation Therapy
11.  Quercetin postconditioning attenuates myocardial ischemia/reperfusion injury in rats through the PI3K/Akt pathway 
Quercetin (Que), a plant-derived flavonoid, has multiple benefical actions on the cardiovascular system. The current study investigated whether Que postconditioning has any protective effects on myocardial ischemia/reperfusion (I/R) injury in vivo and its potential cardioprotective mechanisms. Male Sprague-Dawley rats were randomly allocated to 5 groups (20 animals/group): sham, I/R, Que postconditioning, Que+LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor], and LY294002+I/R. I/R was produced by 30-min coronary occlusion followed by 2-h reperfusion. At the end of reperfusion, myocardial infarct size and biochemical changes were compared. Apoptosis was evaluated by both TUNEL staining and measurement of activated caspase-3 immunoreactivity. The phosphorylation of Akt and protein expression of Bcl-2 and Bax were determined by Western blotting. Que postconditioning significantly reduced infarct size and serum levels of creatine kinase and lactate dehydrogenase compared with the I/R group (all P<0.05). Apoptotic cardiomyocytes and caspase-3 immunoreactivity were also suppressed in the Que postconditioning group compared with the I/R group (both P<0.05). Akt phosphorylation and Bcl-2 expression increased after Que postconditioning, but Bax expression decreased. These effects were inhibited by LY294002. The data indicate that Que postconditioning can induce cardioprotection by activating the PI3K/Akt signaling pathway and modulating the expression of Bcl-2 and Bax proteins.
doi:10.1590/1414-431X20133036
PMCID: PMC3854307  PMID: 24068165
Ischemia and reperfusion; Quercetin; Postconditioning; PI3K/Akt
12.  Long-Lasting Effect of Infant Rats Endotoxemia on Heat Shock Protein 60 in the Pancreatic Acinar Cells: Involvement of Toll-Like Receptor 4 
Introduction. Lipopolysaccharide endotoxin (LPS) is responsible for septic shock and multiorgan failure, but pretreatment of rats with low doses of LPS reduced pancreatic acute damage. Aim. We investigated the effects of the endotoxemia induced in the early period of life on Toll-like receptor 4 (TLR4), heat shock protein 60 (HSP60) and proapoptotic Bax, caspase-9 and -3 or antiapoptotic Bcl-2 protein expression in the pancreatic acinar cells of adult animals. Material and Methods. Newborn rats (25 g) were injected with endotoxin (Escherichia coli) for 5 consecutive days. Two months later, pancreatic acinar cells were isolated from all groups of animals and subjected to caerulein stimulation (10−8 M). Protein expression was assessed employing Western blot. For detection of apoptosis we have employed DNA fragmentation ladder assay. Results. Preconditioning of newborn rats with LPS increased TLR4, Caspase-9 and -3 levels, but failed to affect basal expression of HSP60, Bax, and Bcl-2. Subsequent caerulein stimulation increased TLR4, Bcl-2, and caspases, but diminished HSP60 and Bax proteins in pancreatic acinar cells. Endotoxemia dose-dependently increased TLR4, Bax, HSP60, and both caspases protein signals in the pancreatic acini, further inhibiting antiapoptotic Bcl-2. Conclusions. Endotoxemia promoted the induction of HSP60 via TLR4 in the infant rats and participated in the LPS-dependent pancreatic tissue protection against acute damage.
doi:10.1155/2012/354904
PMCID: PMC3364569  PMID: 22685683
13.  Ceftriaxone, a Beta-Lactam Antibiotic, Modulates Apoptosis Pathways and Oxidative Stress in a Rat Model of Neuropathic Pain 
BioMed Research International  2014;2014:937568.
Purpose. In our previous study, ceftriaxone, a beta-lactam antibiotic, elicited antinociceptive effects in the chronic constriction injury (CCI) of neuropathic pain. In this study, we assessed apoptosis and oxidative stress in the spinal cord of neuropathic rats treated with ceftriaxone. Methods. 45 male Wistar rats were divided as naïve, sham, normal saline-treated CCI rats, and CCI animals treated with the effective dose of ceftriaxone. Involvement of Bax, Bcl2, and caspases 3 and 9, important contributors of programmed cell death (apoptosis), was determined using western blotting at days 3 and 7. The markers of oxidative stress including malondialdehyde (MDA) and reduced glutathione (GSH) were measured on days 3 and 7. Results. Increased Bax/Bcl2 ratio and cleaved active forms of caspases 3 and 9 were observed in the spinal cord of CCI rats on day 3. Ceftriaxone attenuated the increased levels of Bax and cleaved forms of caspases 3 and 9, while it increased Bcl2 levels. Bax and active forms of caspases declined by day 7. Consequently, comparison among groups showed no difference at this time. CCI enhanced MDA and decreased GSH on days 3 and 7, while ceftriaxone protected against the CCI-induced oxidative stress. Conclusion. Our results suggest that ceftriaxone, an upregulator/activator of GLT1, could concomitantly reduce oxidative stress and apoptosis and producing its new analogs lacking antimicrobial activity may represent a novel approach for neuropathic pain treatment.
doi:10.1155/2014/937568
PMCID: PMC4084648  PMID: 25028668
14.  Protective effect of Curcumin on chemotherapy-induced intestinal dysfunction 
Objective: Chemotherapy is one of most important treatments for human cancers. However, side effects such as intestine dysfunction significantly impaired its clinical efficacy. This study aimed to investigate the protective effect of Curcumin on chemotherapy-induced intestinal dysfunction in rats. Methods: Sixty healthy Wistar rats were randomly divided into control group (normal saline), 5-FU group and 5-FU+Curcumin group. The weight, serum level of endotoxin, DAO and D-lactate were determined. The pathological change of intestinal mucosa structure was studied under light microscopy and electron microscopy. The expression of Bax, Bcl-2 and Caspase-3 were assessed by immunohistochemical staining. Results: The Curcumin intragastrically administrated obviously reduced 5-FU-induced weight-loss. 5-FU induced dramatic increase of serum endotoxin, D-lactate and D-Amino-Acid Oxidase (DAO) that were significantly reversed by Curcumin treatment. Meanwhile, 5-FU-induced-damage to intestinal mucosa structure was markedly recovered by Curcumin. The expression of Bax and Caspase-3 were dramatically increased after 5-FU treatment (p<0.01) and Curcumin treatment significantly reduced Bax expression (p<0.05) but had only a moderate effect on reducing caspase-3 expression (p>0.05). Interestingly, Bcl-2 expression was low in control group but increased after 5-FU treatment (p>0.05) and Curcumin treatment further stimulated Bcl-2 expression (p<0.05). Conclusions: Curcumin can significantly reverse chemotherapy-induced weight-loss, increase of serum endotoxin, D-lactate and DAO and damage to intestinal mucosa structure. Curcumin also reduced the expression of pro-apoptotic Bax but stimulated anti-apoptotic Bcl-2 to attenuate 5-FU-induced apoptosis of intestinal epithelial cells. The clinical administration of Curcumin may improve chemotherapy-induced intestinal dysfunction, thus increasing the clinical efficacy of chemotherapy.
PMCID: PMC3816802  PMID: 24228095
Curcumin; chemotherapy-induced; intestinal dysfunction; Bax; Bcl-2; 5-FU; ultrastructures
15.  An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats 
Background
Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.
Methods
We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.
Results
The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.
Conclusion
These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.
doi:10.1186/1477-7827-7-68
PMCID: PMC2713246  PMID: 19570211
16.  Evaluation of Bcl-2 Family Gene Expression and Caspase-3 Activity in Hippocampus STZ-Induced Diabetic Rats 
Experimental Diabetes Research  2008;2008:638467.
We assessed the expression of Bcl-2 family members at both mRNA and protein levels as well as the Caspase-3 activity, in order to investigate the occurrence of apoptosis in hippocampus of STZ-induced diabetic rats. We selected twenty-four Wistar rats; half of them were made diabetic by intraperitoneal injection of a single 60 mg/kg dose of streptozotocin (STZ, IP), while the others received normal saline and served as controls. The expressions of Bcl-2, Bcl-xL, and Bax mRNA and proteins were measured using RT-PCR and western blotting, respectively. Caspases-3 activity was determined by using the Caspase-3/CPP32 Fluorometric Assay Kit. The result showed that mRNA and protein levels of Bcl-2 and Bcl-xL were lower in hippocampus of diabetic group than that of the control group, whereas expressions of Bax in hippocampus of diabetic rats were higher than that of controls at both mRNA and protein levels (P < .01). Hyperglycemia was found to raise 6.9-fold hippocampal caspase-3 activity in diabetic group compared with control group (P < .001). Therefore, the induction of diabetes is associated with increased ratios of Bax/Bcl-2, Bax/Bcl-xL, and increased caspase-3 activity in hippocampus which shows that apoptosis is favored in hippocampal region.
doi:10.1155/2008/638467
PMCID: PMC2566751  PMID: 18923682
17.  Efficient Elimination of Cancer Cells by Deoxyglucose-ABT-263/737 Combination Therapy 
PLoS ONE  2011;6(9):e24102.
As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre-treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies.
doi:10.1371/journal.pone.0024102
PMCID: PMC3176271  PMID: 21949692
18.  Changes in apoptotic factors in hypothalamus and hippocampus after acute and subchronic stress induction during conditioned place preference paradigm  
EXCLI Journal  2013;12:1001-1016.
The hypothalamus (HYP) and hippocampus (HIP) are important regions involved in stress responses. These areas are also associated with reward processing. In this study, the effects of acute and subchronic stress on the changes in apoptotic factors (Bax/Bcl-2 ratio, caspase-3 activation and PARP degradation) in the HYP and HIP during conditioned place preference (CPP) paradigm were evaluated. Male Wistar rats were divided into two saline- and morphine-treated supergroups. Each supergroup contained control, acute stress (AS) and subchronic stress (SS) groups. In all groups, CPP paradigm was done and thereinafter alterations of apoptotic factors were measured by western blot. The results revealed that in the HYP, all mentioned factors increased significantly in saline- or morphine-treated animals during AS and SS. On the other hand, in the HIP, Bax/Bcl-2 ratio in saline-treated animals increased significantly during AS and SS, while in morphine-treated animals this ratio did not have any significant alteration during AS and was decreased during SS compared with morphine-control group. Caspase-3 and PARP increased during AS and SS in saline- or morphine-treated animals. For example, caspase-3 increased during AS and SS in morphine-treated animals by 2.4 folds and PARP (89 KDa) increased by 3.1 and 3.5 folds, respectively. Interestingly, the increase of apoptotic factors in morphine-treated animals was more considerable than that of saline-treated animals. These results strongly implied that AS and SS trigger apoptotic events in the HYP and HIP of saline- and/or morphine-treated animals as well as morphine reinforces the effect of stress on the induction of apoptosis.
PMCID: PMC4932884  PMID: 27385959
apoptosis; hypothalamus; hippocampus; forced swim stress; conditioned place preference; rat
19.  Liguzinediol improved the heart function and inhibited myocardial cell apoptosis in rats with heart failure 
Acta Pharmacologica Sinica  2014;35(10):1257-1264.
Aim:
Liguzinediol is a novel derivative of ligustrazine isolated from the traditional Chinese medicine Chuanxiong (Ligusticum wallichii Franch), and produces significant positive inotropic effect in isolated rat hearts. In this study we investigated the effects of liguzinediol on a rat model of heart failure.
Methods:
To induce heart failure, male SD rats were injected with doxorubicin (DOX, 2 mg/kg, ip) once a week for 4 weeks. Then the rats were administered with liguzinediol (5, 10, 20 mg·kg−1·d−1, po) for 2 weeks. Hemodynamic examination was conducted to evaluate heart function. Myocardial cell apoptosis was examined morphologically. The expression of related genes and proteins were analyzed using immunohistochemical staining and Western blot assays, respectively.
Results:
Oral administration of liguzinediol dose-dependently improved the heart function in DOX-treated rats. Electron microscopy revealed that liguzinediol (10 mg·kg−1·d−1) markedly attenuated DOX-induced injury of cardiomyocytes, and decreased the number of apoptotic bodies in cardiomyocytes. Furthermore, liguzinediol significantly decreased Bax protein level, and increased Bcl-2 protein level in cardiomyocytes of DOX-treated rats, led to an increase in the ratio of Bcl-2/Bax. Moreover, liguzinediol significantly decreased the expression of both cleaved caspase-3 and NF-κB in cardiomyocytes of DOX-treated rats. Administration of digitalis (0.0225 mg·kg−1·d−1) also markedly improved the heart function and the morphology of cardiomyocytes in DOX-treated rats.
Conclusion:
Liguzinediol improves the heart function and inhibits myocardial cell apoptosis in the rat model of heart failure, which is associated with regulating Bcl-2, Bax, caspase-3 and NF-κB expression.
doi:10.1038/aps.2014.75
PMCID: PMC4186991  PMID: 25220638
heart failure; cardiomyocyte; liguzinediol; apoptosis; caspases; Bcl-2; NF-κB; doxorubicin; Ligusticum wallichii
20.  Role of Bax in Death of Uninfected Retinal Cells During Murine Cytomegalovirus Retinitis 
Purpose.
Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3–dependent and –independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells.
Methods.
BALB/c mice, Bax−/− mice, or Bax+/+ mice were immunosuppressed with methylprednisolone and infected with 5 × 103 plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3.
Results.
Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax−/− eyes than in MCMV-infected Bax+/+ eyes. However, more caspase 3–independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax−/− than in Bax+/+ eyes.
Conclusions.
During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3–dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3–independent pathway.
Bax is activated during murine cytomegalovirus retinitis and plays an important role in the death of uninfected bystander retinal cells by caspase 3–dependent apoptosis.
doi:10.1167/iovs.14-15404
PMCID: PMC4224579  PMID: 25298417
murine cytomegalovirus; retinitis; apoptosis; Bax
21.  Bufothionine induced the mitochondria-mediated apoptosis in H22 liver tumor and acute liver injury 
Chinese Medicine  2015;10:5.
Background
Bufothionine is an alkaloid in Cinobufacini (Huachansu). This study aims to investigate the effects of bufothionine on liver tumors and acute liver injury.
Methods
In the hepatoprotective experiment, fifty rats were randomly divided into five groups (n = 10): normal saline group, model group, compound glycyrrhizin injection (9.14 mL/kg); cinobufacini injection (3.42 mL/kg) (InjA) and bufothionine (9.77 mL/kg) (BufoA) group. Liver weight indices were recorded to judge the degree of liver swelling, hematoxylin and eosin (H&E) staining of liver tissues was carried out to observe liver histological morphology injury and biochemical indicators including aspartate aminotransferase (AST); alanine aminotransferase (ALT); alkaline phosphatase (ALP); and total bilirubin (TBIL) were determined by modular auto-analyzer. In anti-tumor experiment, H22-tumor-bearing mice were randomly divided into five groups (n = 10): normal saline group, model group, cinobufacini injection (InjB) (5.14 mL/kg), bufothionine (8.02 mL/kg) (BufoB) and 5-fluorouracil (5-Fu) (3.42 mL/kg). Tumors were picked out and determined with vernier calipers. Histological morphology of tumors was observed by H&E staining. In SMMC-7721 cells, expressions of proteins related to mitochondria-mediated apoptosis pathway including Bcl-2, Bax, caspase-3, caspase-9, cyto-c, Bid, and p53 were analyzed by western blotting at low, medium, high concentrations of bufothione (3.62 μg/mL, 18.12 μg/mL,90.62 μg/mL).
Results
Butothionine relieved CCl4-induced liver morphology, decreased the level of ALT (P =2.46 × 10-2) and expressed tendency to decrease other biochemical markers including AST, ALP and TBIL. Butothionine could also promote necrosis of tumor tissue in H22-tumor-bearing mice and restrained tumor growth with 65.16% inhibition rate. Its mechanism might relate to up-regulation of p53 (at low, mediate and high concentration, corresponding P values were 0.142, 0.0257, 0.0162), caspase-3 (P = 0.246, 0.0267 and 0.0236), cyto-c (P = 0.276, 0.0343 and 0.0429), Bid (P = 0.0125, 0.0395 and 0.0132) and Bax (P = 0.563, 0.0492 and 0.0357) in a dose-dependent manner, down-regulation of Bcl-2 expression (P = 0.0232, 0.0178 and 0.0464), but had no significant effects on caspase-9 (P = 0.253, 0.147 and 0.287).
Conclusion
Bufothionine induced the proteins for the mitochondria-mediated apoptosis that inhibits liver tumors and protects the liver against acute injury.
doi:10.1186/s13020-015-0033-1
PMCID: PMC4371615  PMID: 25806084
22.  A Novel Angiogenesis Inhibitor Bevacizumab Induces Apoptosis in the Rat Endometriosis Model 
Our aim was to investigate the effects of anti-vascular endothelial growth factor (anti-VEGF) antibody Bevacizumab on endometrial explants and on apoptotic gene expression levels in the rat endometriosis model. Endometriotic implants were surgically formed, and rats treated with (i) 1 mg/kg single subcutaneous injection of depot leuprolide acetate; (ii) 2.5 mg/kg of single intaperitoneal injection of bevacizumab; (iii) intraperitoneal injection of saline. Histopathologic scores and adhesion scores of endometriotic foci and levels of Bcl-2-associated X protein (Bax), Cytochrome c (Cyt-c), B-cell lymphoma/leukemia 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) mRNA gene expressions of endometriotic foci. Bevacizumab treatment decreased the endometriotic explant size compared with control. Bevacizumab-treated rats had lower total adhesion scores when compared with the control group. Semi-quantitative evaluation of the persistence of endometrial epithelial cells in the explants showed a lower score in gonadotropin-releasing hormone (GnRH) agonist-treated rats compared with control rats. In Bevacizumab increased expression of Bax 3.1-fold, Cyt-c 1.3-fold and decreased expression of Bcl-2 0.4-fold, Bcl-xl 0.8-fold compared with the control group. The GnRH agonist increased expression of Bax 3.0 fold, Cyt-c 1.3 fold and decreased expression of Bcl-2 0.4-fold, Bcl-xl 0.8-fold, compared with the control group. This study suggests that a novel angiogenesis inhibitor, anti-VEGF antibody bevacizumab is as effective as GnRH agonist in the regression of the endometriotic lesions in rat endometriosis model. One possible mechanism of this effect is the induction of apoptosis.
doi:10.2478/bjmg-2014-0077
PMCID: PMC4413445  PMID: 25937801
Endometriosis; Apoptosis; Bevacizumab; Angiogenesis
23.  Glutamine Treatment Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis 
PLoS ONE  2012;7(11):e50407.
Endoplasmic reticulum (ER) stress and apoptotic cell death play an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). We aimed to explore the potential of glutamine to reduce ER stress and apoptosis in a rat model of experimental IBD. Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/dL) was given by rectal route daily for 2 d or 7 d. Both oxidative stress (TBARS concentration and oxidised/reduced glutathione ratio) and ER stress markers (CHOP, BiP, calpain-1 and caspase-12 expression) increased significantly within 48 h of TNBS instillation, and glutamine attenuated the extent of the changes. Glutamine also inhibited the significant increases of ATF6, ATF4 and spliced XBP-1 mRNA levels induced by TNBS instillation. TNBS-colitis resulted in a significant increase in p53 and cytochrome c expression, and a reduced Bcl-xL expression and Bax/Bcl-2 ratio. These effects were significantly inhibited by glutamine. Treatment with the amino acid also resulted in significant decreases of caspase-9, caspase-8 and caspase-3 activities. Double immunofluorescence staining showed co-localization of CHOP and cleaved caspase-3 in colon sections. Phospho-JNK and PARP-1 expression was also significantly higher in TNBS-treated rats, and treatment with glutamine significantly decreased JNK phosphorylation and PARP-1 proteolysis. To directly address the effect of glutamine on ER stress and apoptosis in epithelial cells, the ER stress inducers brefeldin A and tunicamycin were added to Caco-2 cells that were treated with glutamine (5 mM and 10 mM). The significant enhancement in PERK, ATF6 phosphorylated IRE1, BiP and cleaved caspase-3 expression induced by brefeldin A and tunicamycin was partly prevented by glutamine. Data obtained indicated that modulation of ER stress signalling and anti-apoptotic effects contribute to protection by glutamine against damage in TNBS-induced colitis.
doi:10.1371/journal.pone.0050407
PMCID: PMC3508929  PMID: 23209735
24.  The anticancer drug cisplatin induces an intrinsic apoptotic pathway inside the inner ear 
British Journal of Pharmacology  2007;152(7):1012-1020.
Background and purpose:
Ototoxicity is a known adverse effect of cisplatin (CDDP). Since apoptosis is involved in the development of some pathological conditions associated with the administration of anticancer drugs, we examined, using immunohistochemical and electrophysiological techniques, the apoptotic changes in the cochlea of Sprague-Dawley (SD) rats after an injection of CDDP (5 mgkg-1 body weight).
Experimental approach:
Luciferase assays were used to determine the different caspase activities and ATP levels in protein extracts of whole cochleae. The expression of several apoptotic-related proteins was measured by means of Western blotting. These analyses were performed 2, 7 and 30 days after the CDDP injection. The auditory brain stem response was obtained before and at the different times after the injection of CDDP, before the animals were killed.
Key results:
CDDP significantly increased the levels of caspase-3/7 activity and active caspase-3 protein expression and caspase-3 immunofluorescence staining, caspase-9 activity, and Bax protein expression but decreased Bcl-2 protein expression within the rat cochleae. Threshold shifts were significantly elevated 2 days after CDDP treatment.
Conclusions and implications:
These findings support the hypothesis that cisplatin-related apoptosis evokes an intrinsic pathway of pro-apoptotic signalling within the rat cochleae. Thus, selective inhibition of the sequence of events involved in the intrinsic apoptotic pathway could provide a strategy to minimize cisplatin-induced ototoxicity.
doi:10.1038/sj.bjp.0707405
PMCID: PMC2095105  PMID: 17906689
cisplatin; ototoxicity; apoptosis; caspase-3; caspase-9
25.  The prognostic significance of apoptosis-associated proteins BCL-2, BAX and BCL-X in clinical nephroblastoma 
British Journal of Cancer  2001;85(10):1557-1563.
Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers in normal tissues. Various apoptosis-associated regulatory proteins, such as Bcl-2, Bax and Bcl-X, may contribute to the rate of apoptosis in neoplasia. The present study was performed to evaluate the prognostic value of these molecules in a group of 61 Wilms' tumours of chemotherapeutically pre-treated patients using an immunohistochemical approach. Generally, Bcl-2, Bax and for Bcl-X S/L were expressed in the blastemal and epithelial components of Wilms' tumour. Immunoreactive blastema cells were found in 53%, 41% and 38% of tumours for Bcl-2, Bax and for Bcl-X S/L, respectively. An increased expression of Bcl-2 was observed in the blastemal component of increasing pathological stages. In contrast, a gradual decline of Bax expression was observed in the blastemal component of tumours with increasing pathological stages. Also blastemal Bcl-X S/L expression decreased with stage. Univariate analysis showed that blastemal Bcl-2 expression and the Bcl-2/Bax ratio were indicative for clinical progression, whereas epithelial staining was of no prognostic value. Multivariate analysis showed that blastemal Bcl-2 expression is an independent prognostic marker for clinical progression besides stage. These findings demonstrate that alterations of the Bcl-2/Bax balance may influence the clinical outcome of Wilms' tumour patients by deregulation of programmed cell death.   http://www.bjcancer.com © 2001 Cancer Research Campaign
doi:10.1054/bjoc.2001.2146
PMCID: PMC2363928  PMID: 11720445
Wilms’; tumour; Bcl-2; Bax; Bcl-X; prognosis

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