Cyclophosphamide-treated Rats Research Articles

P-002 Acetate attenuates cyclophosphamide-induced testicular toxicity by modulating steroidogenic and apoptotic signaling via an oxidative stress-dependent pathway

Abstract Study question Will acetate therapy attenuate cyclophosphamide-induced testicular toxicity when used concomitantly? Summary answer Acetate attenuated cyclophosphamide-induced testicular toxicity by modulating steroidogenic and apoptotic signaling via an oxidative stress-dependent pathway. What is known already Cyclophosphamide is an anthracycline that is effective in the management of cancers. Nonetheless, a major drawback of this drug is its testicular toxicity. Available evidence in the literature revealed that cyclophosphamide induces testicular toxicity by targeting steroidogenesis and activating oxidative stress, inflammation, and apoptosis. On the other hand, acetate has been reported to exert antioxidant, anti-inflammatory, and anti-apoptotic activities. However, there is no report on the effect of acetate on cyclophosphamide-induced testicular toxicity. Study design, size, duration This is a prospective experimental study using animal model. Twenty-four sexually mature litter-mate male Wistar rats of comparable weight were used for the study. The study lasted 2 weeks. Participants/materials, setting, methods Animals were acclimatized for two weeks, and then randomized into four groups. The control rats received 0.5mL of distilled water p.o for 14 days, acetate-treated rats received 200 mg/kg/day of acetate p.o for 14 days, cyclophosphamide-treated rats received 150 mg/kg of cyclophosphamide i.p on day 8, while acetate + cyclophosphamide-treated rats received treatment as acetate-treated and cyclophosphamide-treated. The doses of drugs used were the Human Equivalent doses for rats and as earlier reported. Main results and the role of chance Acetate attenuated cyclophosphamide-induced alterations in testicular histoarchitecture and the rise in testicular activities of gamma glutamyl transferase and lactate dehydrogenase activities, and lactate levels. In addition, acetate abrogated cyclophosphamide-induced rise in testicular malondialdehyde, TNF-α, IL-1β, NF-kB, and Bax levels, and myeloperoxidase, caspase 9, and caspase 3 activities, which was accompanied by acetate-driven blockade of cyclophosphamide-induced decline in reduced glutathione, Nrf2, and Bcl-2 levels, and glutathione peroxidase, glutathione-S-transferase, superoxide dismutase and catalase activities. Furthermore, acetate alleviated cyclophosphamide-induced suppression of circulatory levels of FSH, LH, and testosterone, testicular concentrations of 3β-HSD, 17β-HSD, and testosterone, and spermatogenesis and lowered sperm quality. Limitations, reasons for caution This was an experimental study using a rat model; hence, the results of this study should be extrapolated to humans with caution. Clinical trials are recommended to validate these findings. Wider implications of the findings The present study revealed the protective effect of acetate treatment on cyclophosphamide-induced testicular toxicity. These findings provide convincing evidence of the attenuating effect of acetate in cyclophosphamide-induced testicular toxicity. Trial registration number N/A

Modulating effects of heat-killed and live Limosilactobacillus reuteri PSC102 on the immune response and gut microbiota of cyclophosphamide-treated rats.

In the present study, we investigated the potential immunomodulatory effects of heat-killed (hLR) and live Limosilactobacillus reuteri PSC102 (LR; formerly Lactobacillus reuteri PSC102) in RAW264.7 macrophage cells and Sprague-Dawley rats. RAW264.7 murine macrophage cells were stimulated with hLR and LR for 24 h. Cyclophosphamide (CTX)-induced immunosuppressed Sprague-Dawley rats were orally administered with three doses of hLR (L-Low, M-Medium, and H-High) and LR for 3 weeks. The phagocytic capacity, production of nitric oxide (NO), and expression of cytokines in RAW264.7 cells were measured, and the different parameters of immunity in rats were determined. hLR and LR treatments promoted phagocytic activity and induced the production of NO and the expression of iNOS, TNF-α, IL-1β, IL-6, and Cox-2 in macrophage cells. In the in vivo experiment, hLR and LR treatments significantly increased the immune organ indices, alleviated the spleen injury, and ameliorated the number of white blood cells, granulocytes, lymphocytes, and mid-range absolute counts in immunosuppressive rats. hLR and LR increased neutrophil migration and phagocytosis, splenocyte proliferation, and T lymphocyte subsets (CD4+, CD8+, CD45RA+, and CD28+). The levels of immune factors (IL-2, IL-4, IL-6, IL-10, IL-12A, TNF-α, and IFN-γ) in the hLR and LR groups were upregulated compared with those in the CTX-treatment group. hLR and LR treatments could also modulate the gut microbiota composition, thereby increasing the relative abundance of Bacteroidetes and Firmicutes but decreasing the level of Proteobacteria. hLR and LR protected against CTX-induced adverse reactions by modulating the immune response and gut microbiota composition. Therefore, they could be used as potential immunomodulatory agents.

Ranferon-12 tonic mitigates haematological abnormalities in cyclophosphamide-treated rats

Cyclophosphamide (CPA) has anticancer property with many side-effects including haematotoxicity while ranferon-12 tonic (RFT) possesses haematinic effect. This study investigated the mitigating effects of RFT on haematotoxicity in CPA-treated rats. Twenty-four animals were grouped into four (n=6) and treated orally as indicated: Group A: (Control) received 0.4 mL of physiological saline (PS) for 7 consecutive days; Group B: (CPA) received 0.4 mL of PS for 7 days followed by a single intraperitoneal dose of CPA (200 mg/kg) on the 7th day; Group C: (RFT) received RFT at 0.029 mL/kg for 7 days; Group D: (RFT+CPA) received RFT at 0.029 mL/kg for 7 days followed by a single intraperitoneal dose of CPA on the 7th day. Twenty-four hours after the final treatment, the animals were weighed, anaesthetised and sacrificed. Blood was obtained via cardiac puncture and transferred to EDTA and plain tubes for further analysis. Result showed that a single dose of CPA (200 mg/kg) significantly (p < 0.05) reduced the level of white blood cells, haemoglobin, red blood cells, packed cell volume, and platelet (values >36%). Additionally, the body temperature was significantly (p<0.05) elevated by 5.1 %. Furthermore, decreased levels of superoxide dismutase and catalase with a concomitant rise in MDA content were observed. All these alterations were mitigated; to some extent, in animals pre-treated with RFT prior to CPA-administration. The study suggests that RFT could mitigate haematotoxicity in CPA-administered rats by suppressing oxidative stress via modulation of haematopoietic factors, which in turn promotes haematopoiesis.

Acetate attenuates cyclophosphamide-induced cardiac injury via inhibition of NF-kB signaling and suppression of caspase 3-dependent apoptosis in Wistar rats

AimThe goal of the current study was to examine the potential therapeutic effects of sodium acetate on cardiac toxicities caused by cyclophosphamide in Wistar rats. The possible involvement of NF-kB/caspase 3 signaling was also explored. Main methodsThirty-two male Wistar rats were divided into four groups at random. (n = 8). The control animals received 0.5 mL of distilled water orally for 14 days, the acetate-treated group received 200 mg/kg/day of sodium acetate orally for 14 consecutive days, and cyclophosphamide-treated rats received 150 mg/kg /day of cyclophosphamide i.p. on day 8, while cyclophosphamide + acetate group received sodium acetate and cyclophosphamide as earlier stated. Key findingsResults showed that cyclophosphamide-induced cardiotoxicity, which manifested as a marked drop in body and cardiac weights as well as cardiac weight/tibial length, increased levels of troponin, C-reactive protein, lactate, and creatinine kinase, and lactate dehydrogenase activities in the plasma and cardiac tissue. Histopathological examination also revealed toxic cardiac histopathological changes. These alterations were associated with a significant increase in xanthine oxidase and myeloperoxidase activities, uric acid, malondialdehyde, TNF-α, IL-1β, NFkB, DNA fragmentation, and caspase 3 and caspase 9 activities in addition to a marked decline in Nrf2 and GSH levels, and SOD and catalase activities in the cardiac tissue. Acetate co-administration significantly attenuated cyclophosphamide cardiotoxicity by its antioxidant effect, preventing NFkB activation and caspase 9/caspase 3 signalings. SignificanceThis study shows that acetate co-administration may have cardio-protective effects against cyclophosphamide-induced cardiotoxicity by inhibiting NF-kB signaling and suppressing caspase-3-dependent apoptosis.

Efficacy of Carob Leaves and Pods in Reducing Cyclophosphamide-Induced Toxicity on Bone Marrow and Peripheral Blood Leukocytes

Cyclophosphamide is one of the chemotherapeutics for cancer, however this drug has severe side effects in multiple organs and is a well-known mutagenic agent. In this work the protective activity of antioxidant-rich Carob (Ceratonia siliqua L.) pods and leaves extracts against cyclophosphamide induced genotoxicity was studied. Safety of these extracts was evaluated in acute and subacute toxicity tests at a dose of 1500 mg/kg/day in albino rats. Both extracts exhibited no mortality or significant changes in rats' general behavior. Moreover, organ weights and blood chemistry revealed no toxic effects of these extracts (p>0.05).The anti-mutagenic potential of carob pods and leaves extracts were evaluated using in vivo bone marrow chromosomal aberration and micronucleus assays and in vitro comet assay for peripheral blood white cells. Results showed that rats that were treated with cyclophosphamide along with carob pods had a significant decrease in chromosomal aberrations when compared with cyclophosphamide-treated rats (p<0.05). Pods extract also showed significant reduction in cyclophosphamide-induced DNA migration (p<0.05). There were also highly statistically significant differences in the means of micronuclei (MNs) in immature red blood cells between cyclophosphamide group and carob leaves group (p<0.01).It could be concluded that Carob extracts had protective effect against cyclophosphamide- induced genotoxicity and may be considered as anti-mutagenic agent after conducting more studies.

Influence of 5-HT2A receptor function on anxiety-like behavior induced by a combination treatment with doxorubicin and cyclophosphamide in rats.

Anxiety-like behavior induced by a combination of doxorubicin and cyclophosphamide may be mediated by serotonin (5-HT)2A receptor hyperactivity. The anxiolytic effects of fluoxetine may be inhibited by this combination. The present study examined the mechanisms underlying anxiety-like behavior induced by the combination doxorubicin and cyclophosphamide in rats. Anxiety-like behavior was induced during a light-dark test by the doxorubicin and cyclophosphamide treatment (once a week for 2weeks). 5-HT2A receptor and 5-HT2A receptor-mediated extracellular signal-related kinase (ERK)1/2 levels were measured using Western blotting. 5-HT reuptake activity in fluoxetine-treated rats was also examined using microdialysis. ( ±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane, a 5-HT2A receptor agonist, induced anxiety-like behavior. The fluoxetine treatment increased extracellular 5-HT concentrations in the hippocampus of vehicle- and doxorubicin and cyclophosphamide-treated rats. 5-HT transporter levels in the hippocampus were not affected by chemotherapy. The doxorubicin and cyclophosphamide treatment did not alter 5-HT2A receptor levels in the frontal cortex. However, chemotherapy increased 5-HT2A receptor-mediated ERK1/2 phosphorylation levels significantly more than the vehicle treatment. The present results suggest that anxiety-like behavior induced by the combination of doxorubicin and cyclophosphamide is mediated by 5-HT2A receptor hyperactivity without an increase in 5-HT2A receptor levels in rats.

N-Acetylcysteine Attenuates the Anxiety-Like Behavior and Spatial Cognition Impairment Induced by Doxorubicin and Cyclophosphamide Combination Treatment in Rats

Background: Cancer patients can suffer from psychological and cognitive disorders after chemotherapy, which influence quality of life. Objective: Oxidative stress may contribute to the psychological and cognitive disorders induced in rats by chemotherapy. In the present study, we examined the effects of N-acetylcysteine, an anti-oxidant, on anxiety-like behavior and cognitive impairment in rats treated with a combination of doxorubicin and cyclophosphamide. Methods: Rats were intraperitoneally injected with doxorubicin and cyclophosphamide once a week for 2 weeks. The light-dark test and the novel location recognition test were used to assess anxiety-like behavior and spatial cognition, respectively. The rats’ hippocampal levels of glutathione (GSH) and glutathione disulfide (GSSG) were measured using a GSSG/GSH quantification kit. Results: Combined treatment with doxorubicin and cyclophosphamide produced anxiety-like behavior and cognitive impairment in rats. N-acetylcysteine reversed the anxiety-like behavior and inhibition of novel location recognition induced by the combination treatment. Furthermore, the combination of doxorubicin and cyclophosphamide significantly reduced the rats’ hippocampal GSH/GSSG ratios. N-acetylcysteine reversed the reduction in the GSH/GSSG ratio seen in the doxorubicin and cyclophosphamide-treated rats. Conclusion: These results suggest that N-acetylcysteine inhibits doxorubicin and cyclophosphamide-induced anxiety-like behavior and cognitive impairment by reducing oxidative stress in the hippocampus.

Effects of Two Different Doses of Vitamin B2 and a Single Dose of Vitamin B12 Against Cyclophosphamide Induced Nephrotoxicity

Cyclophosphamide, a medication that is used for the treatment of different types of cancer; however its’ use associated with numerous adverse effects. Vitamin B2 and vitamin B12 suggested having nephroprotective effect. This work is designed to investigate the nephroprotective effect of both vitamins against cyclophosphamide induced nephrotoxicity. One hundred adult rats of both sexes were used in this study. The animals were randomly enrolled into ten groups of 10 rats each. On day eight, animals were sacrificed and blood collected for the measurement of serum superoxide dismutase 1, glutathione peroxidase; and kidney extracted for histological examination. Vitamin B2 and vitamin B12 significantly (P<0.05) increase superoxide dismutase 1, glutathione peroxidase; and the combination of vitamins produce significant (P<0.05) increase in superoxide dismutase 1 and glutathione peroxidase compared to the corresponding levels in other groups; and improve histopathological changes compared to cyclophosphamide-treated rats. In conclusion both vitamins may have nephroprotective effects against cyclophosphamide-induced nephrotoxicity.

Curcumin Administration Mitigates Cyclophosphamide-Induced Oxidative Damage and Restores Alteration of Enzymes Associated with Cognitive Function in Rats' Brain.

The use of chemotherapeutic drugs is associated with oxidative damage, cognitive dysfunction, and brain damage. This study sought to investigate the neuroprotective effect of curcumin against cognitive problems associated with treatment with cyclophosphamide via assessment of biomolecules associated with cognitive function in rats' brain homogenates. Rats were divided in to five groups: Control (vehicle), CUR (curcumin [20mg/kg]), CPA (cyclophosphamide [150mg/kg]), CUR1 + CPA (curcumin [20mg/kg] and cyclophosphamide [150mg/kg]), and CPA + CUR2 (cyclophosphamide [150mg/kg] and curcumin [20mg/kg]). After the treatment, cognitive behavior was assessed and enzymes [cholinesterases, purinergic enzymes, arginase, and angiotensin I-converting enzyme] associated with cognitive function were examined. Oxidative stress parameters [total thiol, non-protein thiol, malondialdehyde, and nitric oxide] including the expression of caspase-3 were also assessed in rats' brain. Our results showed that curcumin improved cognitive behavior, attenuated cholinergic deficit as revealed by the inhibition of cholinesterases, and improved purinergic signaling in cyclophosphamide-treated rats. Furthermore, curcumin reduced angiotensin-I-converting enzyme and arginase activities before and after treatment with cyclophosphamide. Curcumin also improved redox balance and showed protection against cyclophosphamide-induced oxidative damage to rats' brain via an increase in protein and non-protein thiols and nitric oxide levels as well as a significant reduction in malondialdehyde levels. Curcumin also prevented neuronal degeneration in different brain regions and reduced caspase-3 expression. Hence this study suggests that pre and post-treatment with curcumin improved neurobehavior, modulates some biomarkers associated with cognitive function and exhibit neuroprotection against cyclophosphamide-induced neurotoxicity in rats.

Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats.

Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20kDa- or a branched 40kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10days to 5 or 6days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

Effects of intravesical administration of sensory neuron-specific receptor agonist on voiding function in rats with cyclophosphamide-induced cystitis

IntroductionThe aim of this study was to investigate the urodynamic effects of intravesical administration of bovine adrenal medulla 8-22 (BAM8-22), a selective rat sensory neuron-specific receptor 1 agonist, on the micturition reflex in normal rats and rats with cyclophosphamide-induced bladder overactivity.Material and methodsContinuous cystometrograms (0.04 ml/min) were performed in urethane-anesthetized rats. After stable micturition cycles were established, vehicle (saline) or BAM8-22 was instilled intravesically and changes in bladder activity were monitored. The experiments using BAM8-22 were also performed in capsaicin-pretreated rats. In another experiment, vehicle (saline) or BAM8-22 was instilled intravesically and changes in bladder activity were monitored in cyclophosphamide-treated rats. Continuous cystometrograms were performed 48 hours after cyclophosphamide injection. Cystometric parameters were recorded and compared before and after intravesical drug administration.ResultsIntravesical administration of BAM8-22 significantly increased the intercontraction interval and threshold pressure in urethane-anesthetized rats, but did not affect the basal pressure or maximum pressure at any doses tested. The inhibitory effects of intravesical administration of BAM8-22 were not inhibited by capsaicin pretreatment. Intravesical administration of BAM8-22 also significantly increased intercontraction interval in the cyclophosphamide-treated rats.ConclusionsThe current results indicate that intravesical administration of a selective rat sensory neuron-specific receptor 1 agonist can inhibit the micturition reflex and can ameliorate cyclophosphamide-induced bladder overactivity in rats.

Extracorporeal shockwave against inflammation mediated by GPR120 receptor in cyclophosphamide-induced rat cystitis model

BackgroundWe tested the hypothesis that extracorporeal shockwave treatment (ESWT) can abolish inflammation and restore urothelial barrier integrity in acute interstitial cystitis by upregulating the fatty acid receptor GPR120.MethodsA total of 30 female Sprague-Dawley rats were categorized into five groups: (1) sham-operated rats (SC); (2) rats treated with ESWT (SC + ESWT); (3) rats with bladder irritation using 150 mg/kg cyclophosphamide through intraperitoneal injection; (4) cyclophosphamide rats treated with ESWT (cyclophosphamide+ESWT); (5) cyclophosphamide rats treated with GPR120 agonist (cyclophosphamide+GW9508).ResultsOn Day 3, urine and bladder specimens were collected for biochemical, histopathological, immunological, and immunoblotting analysis. Following stimulation with cyclophosphamide, the inhibition of the elevated levels of TAK1/NF-κB and phospho-TAK1/NF-κB by ESWT and GPR120 agonists in RT4 cells was associated with a suppression of NF-κB translocation from the cytosol to the nucleus. Accordingly, this anti-inflammatory effect was abolished by GPR120 antagonist and knockdown of GPR120. Histologically, bladder inflammation in cyclophosphamide-treated rats was suppressed by GW9508 or ESWT. Masson’s trichrome and Sirius red staining revealed that cyclophosphamide treatment enhanced synthesis of extracellular matrix in rats that was reversed by GW9508 or ESWT. Upregulated pro-inflammatory mediators and cytokines in the cyclophosphamide-treated rats were also suppressed in the GW9508- or ESWT-treated rats. The significantly increased inflammatory cell infiltration as well as the impaired urothelial integrity of the bladder after cyclophosphamide treatment were reversed by treatment with GW9508 or ESWT.ConclusionsThese findings suggest that GPR120, the sensing receptor for ESWT, may be useful in the treatment of interstitial cystitis by inhibiting inflammatory response in bladder cells.

Rho Kinase and Protein Kinase C Pathways are Responsible for Enhanced Carbachol Contraction in Permeabilized Detrusor in a Rat Model of Cystitis.

Interstitial cystitis is a syndrome characterized by detrusor overactivity and chronic inflammation of the bladder. The mechanisms responsible for the altered smooth muscle contractility remain poorly understood. The aim of the study was to investigate the role of intracellular signalling pathways in carbachol-induced detrusor contraction in a rat model of interstitial cystitis. Cyclophosphamide (150 mg/kg, dissolved in saline) was injected to rats (Sprague-Dawley, female, 200-250 g) intraperitoneally once a day on days 1, 4 and 7 to induce interstitial cystitis. Control groups were injected with saline (0.9% NaCl). Detrusor smooth muscle strips were mounted in 1-ml organ baths containing HEPES-buffered modified Krebs' solution and permeabilized with 40 μM β-escin for 30 min. Carbachol-induced contractions were significantly increased from 21.2 ± 1.6% (saline-treated) to 44 ± 4.4% in cyclophosphamide-treated group. The Rho kinase inhibitor Y-27632 (8.8 ± 2%) and the protein kinase C inhibitor GF-109203X (11.7 ± 2.8%) inhibited the increased contractile response (44 ± 4.4%) in rats with cystitis. The increased carbachol-induced contraction (44 ± 4.4%) was also significantly inhibited by the sarcoplasmic reticulum ryanodine channel blocker ryanodine (25.8 ± 3.2%) and the sarcoplasmic reticulum IP3 receptor blocker heparin (17.2 ± 2.2%) in cystitis. RhoA protein levels in the bladder of cyclophosphamide-treated rats were significantly increased while pan-protein kinase C (α, β and γ isoforms) protein expression was unaltered between experimental groups. Carbachol-induced calcium sensitization at constant and clamped calcium (pCa 6) was also increased in cystitis (from 15.8 ± 2.2% to 24.7 ± 2.8%). This increased response (24.7 ± 2.8%) was significantly inhibited by both Y-27632 (7.9 ± 0.7%) and GF-109203X (4.4 ± 1.5%). We conclude that interstitial cystitis is characterized by an enhanced carbachol contractile response as well as by calcium sensitization of the detrusor smooth muscle. Activation of Rho kinase and protein kinase C pathways may be the molecular culprits responsible for the augmented muscarinic response observed in cystitis.

Effect of Transplantation of Bone Marrow Stromal Cell- Conditioned Medium on Ovarian Function, Morphology and Cell Death in Cyclophosphamide-Treated Rats.

Objective Although stem cell transplantation has beneficial effects on tissue regeneration, but there are still problems such as high cost and safety issues. Since stem cell therapy is largely dependent on paracrine activity, in this study, utilization of transplantation of bone marrow stromal cells (BMSCs)-secretome instead of the cells, into damaged ovaries was evaluated to overcome the limitations of stem cell transplantation. Materials and Methods In this experimental study, BMSCs were cultured and 25-fold concentrated conditioned medium (CM) from BMSCs was prepared. Female rats were injected intraperitoneally with cyclophosphamide (CTX) for 14 days. Then, BMSCs and CM were individually transplanted into bilateral ovaries, and the ovaries were excised after four weeks of treatment. The follicle count was performed using hematoxylin and eosin (H&E) staining and the apoptotic cells were counted using TUNEL assay. Ovarian function was evaluated by monitoring the ability of ovulation and the levels of serum estradiol (E2) and follicle-stimulating hormone (FSH). Results Evaluation of the ovarian function and structure showed that results of secretome transplantation were almost similar to those of BMSCs transplantation and there was no significant differences between them. ConclusionBMSCs-secretome is likely responsible for the therapeutic paracrine effect of BMSCs. Stem cell- secretome is expected to overcome the limitations of stem cell transplantation and become the basis of a novel therapy for ovarian damage.

Oral phenytoin protects against experimental cyclophosphamide-chemotherapy induced hair loss

In the current study, effects of oral phenytoin on hair growth in cyclophosphamide-treated rats were assessed with the goal of evaluating the ability of phenytoin to suppress chemotherapy-induced hair loss. Thirty-six rats were randomly assigned to six groups (1k6) of six each (n=6). In all groups, anagen was induced in flank skin of rats by depilation. On day 9 (anagen VI), rats were injected once with either distilled water (groups 1–3) or cyclophoshamide (groups 4–6). From day 10, rats in group 1 and 4 received oral vehicle (distilled water), groups 2 and 5 received oral phenytoin (50mg/kg), while groups 3 and 6 also received oral phenytoin (100mg/kg). Drug or vehicle was administered daily for a period of 28days. The flank area was serially photographed. At the end of the experimental period, rats were sacrificed to correlate visible hair growth with a histological profile of follicle response and recovery. Glutathione (GSH), glutathione peroxidase (GPX) and lipid peroxidation status were assessed. Cyclophosphamide (CYP) treatment was associated with gross morphologic and histological evidence of hair loss in the flanks, microscopic evidence of hair-shaft thinning, increased skin lipid peroxidation, decreased GSH level, and reduction in GPX activities. Phenytoin co-administration was associated with evidence of improved hair growth, increased hair-shaft thickness, reduced skin lipid peroxidation, increased GSH level, and increased GPX activities. This study showed that oral phenytoin can suppress hair loss due to CYP therapy in rats; however, further studies are needed to evaluate its potential application in chemotherapy-induced alopecia.

Protective effect of catechin on humoral and cell mediated immunity in rat model

The present study was focused on examining the effect of catechin on the cellular and humoral immunity in rat model. Immunomodulatory effect of catechin was determined by delayed-type hypersensitivity (DTH) response, carbon clearance assay, leucocyte mobilization test and cyclophosphamide-induced myelosuppression and hemagglutinating antibody (HA) titer assay. Catechin in experimental dose (25, 50 and 100mg/kg, p.o.) elevated a significant increase in antibody titer in the hemagglutination test with increased levels of immunoglobulin. There was an enhancement in the delayed-type hypersensitivity reaction produced by sheep red blood cells. There was also restoration in the functioning of leucocytes in cyclophosphamide-treated rats with an increased clearance of carbon particles. The results of the present study signify that catechin possesses sufficient potential for modulating immune activity by cellular and humoral mechanisms.

THERAPEUTIC EFFECT OF TRIDHAM AND 1,2,3,4,6-PENTA-O-GALLOYL-Β-D-GLUCOSE ON ALTERED ENERGY METABOLISM IN MAMMARY CARCINOMA BEARING RATS

ABSTRACT Objective: Emerging evidence indicates that cancer is primarily a metabolic disease involving disturbances in energy production, and hence, reprogramming of cellular energy metabolism is deemed to be one of the principal hallmarks of cancer. Tridham (TD) has been used by traditional practitioners of Siddha medicine against various ailments. Hence, the present study has been designed to evaluate the therapeutic effect of TD on altered energy metabolism in mammary carcinoma-bearing rats. Methods: Adult female albino rats of Sprague-Dawley strain weighing 170-190 g were used and 7,12-dimethylbenzeneanthracene (DMBA) was used for induction of mammary carcinoma and rats were divided into seven groups. Group I - control rats, Group II - DMBA-induced rats, Group III - DMBA- and TD-treated rats, Group IV - DMBA- and 1,2,3,4,6-Penta-O-galloyl-β-d-glucose (PGG)-treated rats, Group V - DMBA- and cyclophosphamide-treated rats, Groups VI and VII are TD and PGG control rats. Results: Significant (p<0.05) increase in the glycolytic enzymes, hexokinase, phosphoglucoisomerase and aldolase, was observed in tumor-bearing rats whereas gluconeogenic enzymes, glucose-6-phosphatase and fructose-1,6-biphosphatase, were significantly decreased. The activities of the mitochondrial Krebs cycle enzymes, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase, and respiratory chain enzymes, nicotinamide dinucleotide dehydrogenase and cytochrome c oxidase, were significantly reduced in tumor tissue of the mammary carcinoma-bearing rats. These biochemical disturbances were effectively counteracted by TD and PGG which restore the activities of all these enzymes to the respective control levels. Conclusion: TD and PGG effectively ameliorated the altered glucose metabolism resulting in the regression of breast cancer. Keywords: Breast cancer, Cancer cachexia, Carbohydrate-metabolizing enzymes, Krebs cycle.

CARDIOPROTECTIVE EFFECT OF MENTHA LONGIFOLIA AGAINST CYCLOPHOSPHAMIDE INDUCED CARDIOTOXICITY IN RATS: A BIOCHEMICAL, ELECTROCARDIOGRAPHIC AND HISTOPATHOLOGICAL STUDY

&lt;p&gt;&lt;strong&gt;Objective: &lt;/strong&gt;The current research was designed to evaluate the cardioprotective activity of &lt;em&gt;Mentha longifolia&lt;/em&gt; (ML) leaf extract on cyclophosphamide-induced cardiotoxicity in rats.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;Cardiotoxicity was induced in &lt;em&gt;Albino wistar&lt;/em&gt; rats of either sex by administering a single injection of cyclophosphamide (200 mg/kg, i. p.) on the first day of the experimental period. &lt;em&gt;Mentha longifolia&lt;/em&gt; (250 and 500 mg/kg, p. o.) was administered daily for 10 d immediately after administration of cyclophosphamide on the first day. The general observations such as oxidative marker enzyme assays, ECG and histopathology were examined.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;Cyclophosphamide administration significantly (p&amp;lt;0.05) increased lipid peroxidation (LPO) and decreased the levels of antioxidant markers such as superoxide dismutase (SOD) and catalase (CAT). Cyclophosphamide elevated the levels of biomarker enzymes like creatine kinase isoenzyme MB (CK-MB), creatine kinase isoenzyme NAC (CK-NAC) and lactate dehydrogenase (LDH). Further, the cyclophosphamide-treated rats showed changes in electrocardiographic parameters. Treatment with &lt;em&gt;Mentha longifolia&lt;/em&gt; significantly (p&amp;lt;0.05) reversed the status of cardiac biomarkers, ECG and oxidative enzymes in cyclophosphamide-induced cardiotoxicity. Histopathological examination was also supported the potential cardioprotective effect of &lt;em&gt;Mentha longifolia&lt;/em&gt; with reduced damage to the myocardium.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion: &lt;/strong&gt;The biochemical, ECG and histopathology reports support the potential benefits of &lt;em&gt;Mentha longifolia&lt;/em&gt; against myocardial damage which could be attributed to antioxidant activity.&lt;/p&gt;

Immunostimulant activities of the aqueous methanolic extract of Leptadenia pyrotechnica, a plant from Cholistan desert

Ethnopharmacological relevanceLeptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is a well-renowned medicinal shrub, used by herbal practitioners for various ailments including allergic rhinitis, productive cough, abortion, diabetes, stomach disorders, fever, kidney disorders, stones and cancer Aim of the studyOn the basis of folkloric uses, L. pyrotechnica was selected from the wide medicinal flora of the Cholistan desert of Pakistan, for the exploration of immunomodulatory potential. Materials and methodsAqueous methanolic (30:70) extract of aerial parts of L. pyrotechnica (Lp. Cr) was prepared by 3 days thrice maceration and subsequent evaporation under reduced pressure. In-vivo experiments were performed in Wistar albino rats including neutrophil adhesion test, haemagglutinating antibody (HA) titre, delayed-type hypersensitivity response, phagocytic activity and cyclophosphamide-induced myelosuppression. ResultsLp. Cr produced a significant increase in phagocytic index in dose-dependent fashion (3.56, 4.18 and 5.42 at 30, 100 and 300mg/kg, respectively) as well as an augmented response in the delayed-type hypersensitivity reaction and HA titre induced by sheep erythrocytes. Lp. Cr also showed improved adhesion of neutrophils with nylon pellets (15.28, 27.85 and 38.42% at the doses of 30, 100 and 300mg/kg) and prevented myelosuppression in cyclophosphamide-treated rats as evidenced from the hematological parameters. The results of treatment were compared with normal and standard control groups throughout the study and the effects by Lp. Cr (300mg/kg) were found to be comparable with Levamisole. ConclusionsThe results indicated that L. pyrotechnica possesses immunostimulatory activity and justify its traditional use for the control and management of diseases in which the immune system needs to be stimulated like infectious diseases.

Cyclophosphamide-induced vasopressin-independent activation of aquaporin-2 in the rat kidney.

Because cyclophosphamide-induced hyponatremia was reported to occur without changes in plasma vasopressin in a patient with central diabetes insipidus, we hypothesized that cyclophosphamide or its active metabolite, 4-hydroperoxycyclophosphamide (4-HC), may directly dysregulate the expression of water channels or sodium transporters in the kidney. To investigate whether intrarenal mechanisms for urinary concentration are activated in vivo and in vitro by treatment with cyclophosphamide and 4-HC, respectively, we used water-loaded male Sprague-Dawley rats, primary cultured inner medullary collecting duct (IMCD) cells, and IMCD suspensions prepared from male Sprague-Dawley rats. In cyclophosphamide-treated rats, significant increases in renal expression of aquaporin-2 (AQP2) and Na-K-2Cl cotransporter type 2 (NKCC2) were shown by immunoblot analysis and immunohistochemistry. Apical translocation of AQP2 was also demonstrated by quantitative immunocytochemistry. In both rat kidney and primary cultured IMCD cells, significant increases in AQP2 and vasopressin receptor type 2 (V2R) mRNA expression were demonstrated by real-time quantitative PCR analysis. Confocal laser-scanning microscopy revealed that apical translocation of AQP2 was remarkably increased when primary cultured IMCD cells were treated with 4-HC in the absence of vasopressin stimulation. Moreover, AQP2 upregulation and cAMP accumulation in response to 4-HC were significantly reduced by tolvaptan cotreatment in primary cultured IMCD cells and IMCD suspensions, respectively. We demonstrated that, in the rat kidney, cyclophosphamide may activate V2R and induce upregulation of AQP2 in the absence of vasopressin stimulation, suggesting the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis (NSIAD).