Search results for: Wistar albino rat

Authors: Ogechi Nzeagwu, Assumpta Osuagwu, Charlse Nkwoala

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Malnutrition in infants due to poverty, poor feeding practices, and high cost of commercial complementary foods among others is a concern in developing countries. The study evaluated the proximate, vitamin and mineral compositions, antinutrients and functional properties, biochemical, haematological and sensory evaluation of complementary food made from sorghum, groundnut, crayfish and paw-paw flour blends using standard procedures. The blends were formulated on protein requirement of infants (18 g/day) using Nutrisurvey linear programming software in ratio of sorghum(S), groundnut(G), crayfish(C) and pawpaw(P) flours as 50:25:10:15(SGCP1), 60:20:10:10 (SGCP2), 60:15:15:10 (SGCP3) and 60:10:20:10 (SGCP4). Plain-pap (fermented maize flour)(TCF) and cerelac (commercial complementary food) served as basal and control diets. Thirty weanling male albino rats aged 28-35 days weighing 33-60 g were purchased and used for the study. The rats after acclimatization were fed with gruel produced with the experimental diets and the control with water ad libitum daily for 35days. Effect of the blends on lipid profile, blood glucose, haematological (RBC, HB, PCV, MCV), liver and kidney function and weight gain of the rats were assessed. Acceptability of the gruel was conducted at the end of rat feeding on forty mothers of infants’ ≥ 6 months who gave their informed consent to participate using a 9 point hedonic scale. Data was analyzed for means and standard deviation, analysis of variance and means were separated using Duncan multiple range test and significance judged at 0.05, all using SPSS version 22.0. The results indicated that crude protein, fibre, ash and carbohydrate of the formulated diets were either comparable or higher than values in cerelac. The formulated diets (SGCP1- SGCP4) were significantly (P>0.05) higher in vitamin A and thiamin compared to cerelac. The iron content of the formulated diets SGCP1- SGCP4 (4.23-6.36 mg/100) were within the recommended iron intake of infants (0.55 mg/day). Phytate (1.56-2.55 mg/100g) and oxalate (0.23-0.35 mg/100g) contents of the formulated diets were within the permissible limits of 0-5%. In functional properties, bulk density, swelling index, % dispersibility and water absorption capacity significantly (P<0.05) increased and compared favourably with cerelac. The essential amino acids of the formulated blends were within the amino acid profile of the FAO/WHO/UNU reference protein for children 0.5 -2 years of age. Urea concentration of rats fed with SGCP1-SGCP4 (19.48 mmol/L),(23.76 mmol/L),(24.07 mmol/L),(23.65 mmol/L) respectively was significantly higher than that of rat fed cerelac (16.98 mmol/L); however, plain pap had the least value (9.15 mmol/L). Rats fed with SGCP1-SGCP4 (116 mg/dl), (119 mg/dl), (115 mg/dl), (117 mg/dl) respectively had significantly higher glucose levels those fed with cerelac (108 mg/dl). Liver function parameters (AST, ALP and ALT), lipid profile (triglyceride, HDL, LDL, VLDL) and hematological parameters of rats fed with formulated diets were within normal range. Rats fed SGCP1 gained more weight (90.45 g) than other rats fed with SGCP2-SGCP4 (71.65 g, 79.76 g, 75.68 g), TCF (20.13 g) and cerelac (59.06 g). In all the sensory attributes, the control was preferred with respect to the formulated diets. The formulated diets were generally adequate and may likely have potentials to meet nutrient requirements of infants as complementary food.

Keywords: biochemical, chemical evaluation, complementary food, quadrimix

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Authors: Natalia V. Naryzhnaya, Alexandr V. Mukhomedzyanov, Ivan A. Derkachev, Boris K. Kurbatov, Leonid N. Maslov

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Background: Techniques of adaptation to hypoxia and remote postconditioning (RPost) have great prospects for use in the clinic. However, recent studies have shown low efficacy of remote postconditioning in patients with AMI. We hypothesize that the reasons for this inefficiency may be metabolic disorders, which are very common, especially in patients with cardiovascular disease, and age of patients. The purpose of the study was to reveal the effectiveness of adaptation to chronic hypoxia and RPost. To determine the possible relationship between the decrease in the effectiveness of projective impacts and disorders of carbohydrate and lipid metabolism. Design: The study was carried out on Wistar rats 60 day old. MetS was induced by high-carbohydrate, high-fat diet (HСHFD). Modeling MS led to the formation of obesity, hypertension, impaired lipid and carbohydrate metabolism, hyperleptinemia, and moderate stress. Groups with adaptation to chronic hypoxia were subjected to hypoxia for 21 days at 12% O2 and 0.3% CO2 after complete of HСHFD. All animals were subjected to 45 min coronary occlusion and 120 min reperfusion. Groups with RPost, immediately after the end of ischemia, tourniquets were applied to the hind limbs in the area of the hip joint (3 times in the mode of 5 min ischemia, 5 min reperfusion). Results: RPost led to a twofold reduction of infarct size in rats with intact metabolism (р < 0.0001), while in rats with MetS, a decrease in infarct size during RPost was 25 % (p = 0.00003). A direct correlation was found between of infarct size during RPost and the serum leptin level of rats with MetC (r = 0.85). The presented data suggested that a decrease in the efficiency of remote postconditioning in rats with diet-induced metabolic syndrome depends on serum leptin. Chronic hypoxia resulted in a 38% reduced in infarct size in metabolically intact rats. The decrease of cardioprotection was observed in rats with chronic hypoxia and MetS. Infarct size showed a direct correlation with impaired glucose tolerance (AUC, glucose tolerance test, r = 0.034) and serum triglyceride levels (r = 0.39). Our study showed the dependence of cardioprotection in rats with metabolic syndrome during chronic hypoxia and DPost on opioids in the blood serum and myocardium, protein kinase C and NO synthase activity. Conclusion: The results obtained showed that the infarct-limiting efficiency of adaptation to hypoxia and remote postconditioning is reduced or completely absent in animals with metabolic syndrome. The increase in the infarction, in this case, directly depends on the disturbances in carbohydrate. lipid metabolism and opioids signaling. Funding: Investigation of effectiveness of chronic hypoxia at the metabolic syndrome was carried out within the support of Russian Science Foundation Grant 22-15-00048. Studies of the mechanisms of arterial hypertension in induced metabolic syndrome were carried out within the framework of the state assignment (122020300042-4). The work was performed using the Center for Collective Use "Medical Genomics".

Keywords: chronic hypoxia, opioids, remote postconditioning, metabolic syndrome

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Authors: Tamar Bikashvili, Tamar Lordkipanidze, Ilia Lazrishvili

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Heavy metals remain one of serious environmental problems due to their toxic effects. The effect of arsenic and manganese compounds on rat behavior and neuromorphology was studied. Wistar rats were assigned to four groups: rats in control group were given regular water, while rats in other groups drank water with final manganese concentration of 10 mg/L (group A), 20 mg/L (group B) and final arsenic concentration 68 mg/L (group C), respectively, for a month. To study exploratory and anxiety behavior and also to evaluate aggressive performance in “home cage” rats were tested in “Open Field” and to estimate learning and memory status multi-branched maze was used. Statistically significant increase of motor and oriental-searching activity in experimental groups was revealed by an open field test, which was expressed in increase of number of lines crossed, rearing and hole reflexes. Obtained results indicated the suppression of fear in rats exposed to manganese. Specifically, this was estimated by the frequency of getting to the central part of the open field. Experiments revealed that 30-day exposure to 10 mg/ml manganese did not stimulate aggressive behavior in rats, while exposure to the higher dose (20 mg/ml), 37% of initially non-aggressive animals manifested aggressive behavior. Furthermore, 25% of rats were extremely aggressive. Obtained data support the hypothesis that excess manganese in the body is one of the immediate causes of enhancement of interspecific predatory aggressive and violent behavior in rats. It was also discovered that manganese intoxication produces non-reversible severe learning disability and insignificant, reversible memory disturbances. Studies of rodents exposed to arsenic also revealed changes in the learning process. As it is known, the distribution of metal ions differs in various brain regions. The principle manganese accumulation was observed in the hippocampus and in the neocortex, while arsenic was predominantly accumulated in nucleus accumbens, striatum, and cortex. These brain regions play an important role in the regulation of emotional state and motor activity. Histopathological analyzes of brain sections illustrated two morphologically distinct altered phenotypes of neurons: (1) shrunk cells with indications of apoptosis - nucleus and cytoplasm were very difficult to be distinguished, the integrity of neuronal cytoplasm was not disturbed; and (2) swollen cells - with indications of necrosis. Pyknotic nucleus, plasma membrane disruption and cytoplasmic vacuoles were observed in swollen neurons and they were surrounded by activated gliocytes. It’s worth to mention that in the cortex the majority of damaged neurons were apoptotic while in subcortical nuclei –neurons were mainly necrotic. Ultrastructural analyses demonstrated that all cell types in the cortex and the nucleus caudatus represent destructed mitochondria, widened neurons’ vacuolar system profiles, increased number of lysosomes and degeneration of axonal endings.

Keywords: arsenic, manganese, behavior, learning, neuron

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Authors: Tatsiana Ihnatovich, Darya Nizheharodava, Mikalai Halabarodzka, Tatsiana Savitskaya, Marina Zafranskaya

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Liver fibrosis is a complex of histological changes resulting from chronic liver disease accompanied by an excessive production and deposition of extracellular matrix components in the hepatic parenchyma. Liver fibrosis is a serious medical and social problem. Hepatic stellate cells (HSCs) make a significant contribution to the extracellular matrix deposition due to liver injury. Mesenchymal stem cells (MSCs) have a pronounced anti-inflammatory, regenerative and immunomodulatory effect; they are able to differentiate into hepatocytes and induce apoptosis of activated HSCs that opens the prospect of their use for preventing the excessive fibro-formation and the development of liver cirrhosis. The aim of the study is to evaluate the effect of MSCs therapy on the expression of fibrogenesis markers genes in liver tissue and HSCs cultures of rats with experimental liver cirrhosis (ELC). Materials and methods: ELC was induced by the common bile duct ligation (CBDL) in female Wistar rats (n = 19) with an average body weight of 250 (220 ÷ 270) g. Animals from the control group (n = 10) were sham-operated. On the 56th day after the CBDL, the rats of the experimental (n = 12) and the control (n = 5) groups received intraportal MSCs in concentration of 1×106 cells/animal (previously obtained from rat’s bone marrow) or saline, respectively. The animals were taken out of the experiment on the 21st day. HSCs were isolated by sequential liver perfusion in situ with following disaggregation, enzymatic treatment and centrifugation of cell suspension on a two-stage density gradient. The expression of collagen type I (Col1a1) and type III (Col3a1), matrix metalloproteinase type 2 (MMP2) and type 9 (MMP9), tissue inhibitor of matrix metalloproteinases type 1 (TIMP1), transforming growth factor β type 1 (TGFβ1) and type 3 (TGFβ3) was determined by real-time polymerase chain reaction. Statistical analysis was performed using Statistica 10.0. Results: In ELC rats compared to sham-operated animals, a significant increase of all studied markers expression was observed. The administration of MSCs led to a significant decrease of all detectable markers in the experimental group compared to rats without cell therapy. In ELC rats, an increased MMP9/TIMP1 ratio after cell therapy was also detected. The infusion of MSCs in the sham-operated animals did not lead to any changes. In the HSCs from ELC animals, the expression of Col1a1 and Col3a1 exceeded the similar parameters of the control group (p <0.05) and statistically decreased after the MSCs administration. The correlation between Col3a1 (Rs = 0.51, p <0.05), TGFβ1 (Rs = 0.6, p <0.01), and TGFβ3 (Rs = 0.75, p <0.001) expression in HSCs cultures and liver tissue has been found. Conclusion: Intraportal administration of MSCs to rats with ELC leads to a decreased Col1a1 and Col3a1, MMP2 and MMP9, TIMP1, TGFβ1 and TGFβ3 expression. The correlation between the expression of Col3a1, TGFβ1 and TGFβ3 in liver tissue and in HSCs cultures indicates the involvement of activated HSCs in the fibrogenesis that allows considering HSCs to be the main cell therapy target in ELC.

Keywords: cell therapy, experimental liver cirrhosis, hepatic stellate cells, mesenchymal stem cells

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Authors: A. Abdi, A. Abbasi Daloee, A. Barari

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Aim: The adaptations that occur in human body after doing exercises training are a factor to help healthy people stay away from certain diseases. One of the main adaptations is a change in blood circulation, especially in vessels. The increase of capillary density is dependent on the balance between angiogenic and angiostatic factors. Most studies show that the changes made to angiogenic developmental factors resulted from physical exercises indicate the low level of stimulators compared with inhibitors. It is believed that the plasma level of VEGF-A, the important angiogenic factor, is reduced after physical exercise. Findings indicate that the extract of crataegus plant reduces the platelet-derived growth factor receptor (PDGFR) autophosphorylation in human's fibroblast. More importantly, crataegus (1 to 100 mg in liter) clearly leads to the inhibition of PDGFR autophosphorylation in vascular smooth muscle cells (VSMCs). Angiogenesis is a process that can be classified into physiological and pathophysiological forms. collagen XVIII is a part of extracellular protein and heparan sulfate proteoglycans in vascular epithelial and endothelial basement membrane cause the release of endostatin from noncollagenous collagen XVIII. Endostatin inhibits the growth of endothelial cells, inhibits angiogenesis, weakens different types of cancer, and the growth of tumors. The purpose of the current study was to investigate the effect of intensive aerobic running with or without aqueous extraction of black Crataegus elbursensis on Collagen XVIII in male rats. Design: Thirty-two Wistar male rats (4-6 weeks old, 125-135 gr weight) were acquired from the Pasteur's Institute (Amol, Mazandaran), and randomly assigned into control (n = 16) and training (n = 16) groups. Rats were further divided into saline-control (SC) (n=8), saline-training (ST) (n=8), crataegus pentaegyna extraction -control (CPEC) (n=8), and crataegus pentaegyna extraction - training (CPET) (n=8). The control (SC and CPEC) groups remained sedentary; whereas the training groups underwent a high running exercise program. plasma were excised and immediately frozen in liquid nitrogen. Statistical analysis was performed using a one way analysis of variance and Tukey test. Significance was accepted at P = 0.05. Results: The results show that aerobic exercise group had the highest concentration collagen XVIII compared to other groups and then respectively black crataegus, training-crataegus and control groups. Conclusion: In general, researchers in this study concluded that the increase of collagen XVIII (albeit insignificant) as a result of physical activity and consumption of black crataegus extract could possibly serve as a regional inhibitor of angiogenesis and another evidence for the anti-cancer effects of physical activities. Since the research has not managed in this study to measure the amount of plasma endostatin, it is suggested that both indices are measured with important angiogenic factors so that we can have a more accurate interpretation of changes to angiogenic and angiostatic factors resulted from physical exercises.

Keywords: aerobic running, Crataegus elbursensis, Collagen XVIII

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Authors: S. N. T. I. Sampath, J. M. S. Jayasinghe, A. P. Attanayake, V. Karunaratne

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Diabetes mellitus is one of the major public health posers throughout the world today that incidence and associated with increasing mortality. Insufficient regulation of the blood glucose level might be serious effects for health and its necessity to identify new therapeutics that have ability to reduce hyperglycaemic condition in the human body. Even though synthetic antidiabetic drugs are more effective to control diabetes mellitus, there are considerable side effects have been reported. Thus, there is an increasing demand for searching new natural products having high antidiabetic activity with lesser side effects. The purposes of the present study were to evaluate different physical parameters and in-vitro and in-vivo antidiabetic potential of the selected combined medicinal plant extracts mixture composed of leaves of Murraya koenigii, cloves of Allium sativum, fruits of Garcinia queasita and seeds of Piper nigrum. The selected plants parts were mixed and ground together and extracted sequentially into the hexane, ethyl acetate and methanol. Solvents were evaporated and they were further dried by freeze-drying to obtain a fine powder of each extract. Various physical parameters such as moisture, total ash, acid insoluble ash and water soluble ash were evaluated using standard test procedures. In-vitro antidiabetic activity of combined plant extracts mixture was screened using enzyme assays such as α-amylase inhibition assay and α-glucosidase inhibition assay. The acute anti-hyperglycaemic activity was performed using oral glucose tolerance test for the streptozotocin induced diabetic Wistar rats to find out in-vivo antidiabetic activity of combined plant extracts mixture and it was assessed through total oral glucose tolerance curve (TAUC) values. The percentage of moisture content, total ash content, acid insoluble ash content and water soluble ash content were ranged of 7.6-17.8, 8.1-11.78, 0.019-0.134 and 6.2-9.2 respectively for the plant extracts and those values were less than standard values except the methanol extract. The hexane and ethyl acetate extracts exhibited highest α-amylase (IC50 = 25.7 ±0.6; 27.1 ±1.2 ppm) and α-glucosidase (IC50 = 22.4 ±0.1; 33.7 ±0.2 ppm) inhibitory activities than methanol extract (IC50 = 360.2 ±0.6; 179.6 ±0.9 ppm) when compared with the acarbose positive control (IC50 = 5.7 ±0.4; 17.1 ±0.6 ppm). The TAUC values for hexane, ethyl acetate, and methanol extracts and glibenclamide (positive control) treated rats were 8.01 ±0.66; 8.05 ±1.07; 8.40±0.50; 5.87 ±0.93 mmol/L.h respectively, whereas in diabetic control rats the TAUC value was 13.22 ±1.07 mmol/L.h. Administration of plant extracts treated rats significantly suppressed (p<0.05) the rise in plasma blood glucose levels compared to control rats but less significant than glibenclamide. The obtained results from in-vivo and in-vitro antidiabetic study showed that the hexane and ethyl acetate extracts of selected combined plant mixture might be considered as a potential source to isolate natural antidiabetic agents and physical parameters of hexane and ethyl acetate extracts will helpful to develop antidiabetic drug with further standardize properties.

Keywords: diabetes mellitus, in-vitro antidiabetic assays, medicinal plants, standardization

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Authors: Khelladi Hadj Mostefa, Krouf Djamil, Taleb-Dida Nawel

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Background: Obesity results from a prolonged imbalance between energy intake and energy expenditure, as depending on basal metabolic rate. Oils and proteins from sea have important therapeutic (such as obesity and hypercholesterolemia) and antioxidant effects. Sardine are a widely consumed fish in the Mediterranean region. Its consumption provides humans with various nutrients such as oils (rich in omega 3 plyunsaturated fatty acids)) and proteins. Methods: Sardine oil (SO) and sardine proteins (SP) were extracted and purified. Mixture of vegetable oils (olive-walnut-sunflower) were prepared from oils produced in Algeria. Eighteen wistar rats are fed a high fat diet enriched with 1% cholesterol for 30 days to induce obesity and hypercholesterolemia. The rats are divided into 3 groups. The first group consumes 20% sardine protein combined with 5% sardine oil (38% SFA (saturated fatty acids), 31% MIFA (monounsaturated fatty acids) and 31% PIFA (polyunsaturated fatty acids)) (SPso). The second group consumes 20% sardine protein combined with 5% of a mixture of vegetable oils (VO) containing 13% SFA, 58% MIFA and 29% PIFA (PSvo), and the third group consuming 20% casein combined with 5% of the mixture of vegetable oils and serves as a semi-synthetic reference (CASvo). Body weights and glycaemia are measured weekly After 28 days of experimentation, the rats are sacrificed, the blood and the liver removed. Serum assays of total cholesterol (TC) and triglycerides (TG) were performed by enzymatic colorimetric methods. Evaluation of lipid peroxidation was performed by assaying thiobarbituric acid reactive species (TBARS) and hydroperoxides values. The protein oxidation was performed by assaying carbonyl derivatives values. Finally, evaluation of antioxidant defense is made by measuring the activity of antioxidant enzymes, the superoxide dismutase (SOD) and the catalase (CAT).Results: After 28 days, the body weight (BW) of the rats increased significantly in SPso and SPvo groups compared to CAS group, by +11% and 7%, respectively. Cholesterolemia (TC) increased significantly in the SPso and SPvo groups compared to the CAS group (P<0.01), while triglyceridemia (TG) decreased significantly in the SPso group compared to SPvo and CAS groups (P<0.01). Albumin (marker of inflammation) increased in the PSs group compared to SPvo and CAS groups by +35% and +13%, respectively. The serum TBARS levels are -40% lower in SPso group compared to SPvo group, and they are -80% and -76% lower in SPso compared to SPvo and CAS groups, respectively. The level of carbonyls derivatives in the serum and liver are significantly reduced in the SPso group compared to the SPvo and CAS groups. Superoxide dismutase (SOD) activity decreased in liver of SPso group compared to SPvo group (P<0.01). While that of CAT is increased in liver tissue of SPso group compared to SPvo group (P<0.01). Conclusion: Sardine oil combined with sardine protein has a hypotriglyceridemic effect, reduces body weight, attenuates inflammation and seems to protect against lipid peroxidation and protein oxidation and increases antioxidant defense in hypercholesterolemic and obese rats. This could be in favor of a protective effect against obesity and cardiovascular diseases.

Keywords: rat, obesity, hypercholesterolemia, sardine protein, sardine oil, vegetable oils mixture, lipid peroxidation, protein oxidation, antioxidant defense

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Authors: A. Newman-Tancredi, R. Depoortère, P. Gruca, E. Litwa, M. Lason, M. Papp

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The N-methyl-D-aspartic acid (NMDA) receptor antagonist, ketamine, can elicit rapid-acting antidepressant (RAAD) effects in treatment-resistant patients, but it requires parenteral co-administration with a classical antidepressant under medical supervision. In addition, ketamine can also produce serious side effects that limit its long-term use, and there is much interest in identifying RAADs based on ketamine’s mechanism of action but with safer profiles. Ketamine elicits GABAergic interneuron inhibition, glutamatergic neuron stimulation, and, notably, activation of serotonin 5-HT1A receptors in the prefrontal cortex (PFC). Direct activation of the latter receptor subpopulation with selective ‘biased agonists’ may therefore be a promising strategy to identify novel RAADs and, consistent with this hypothesis, the prototypical cortical biased agonist, NLX-101, exhibited robust RAAD-like activity in the chronic mild stress model of depression (CMS). The present study compared the effects of a novel, selective 5-HT1A receptor-biased agonist, NLX-204, with those of ketamine and NLX-101. Materials and methods: CMS procedure was conducted on Wistar rats; drugs were administered either intraperitoneally (i.p.) or by bilateral intracortical microinjection. Ketamine: 10 mg/kg i.p. or 10 µg/side in PFC; NLX-204 and NLX-101: 0.08 and 0.16 mg/kg i.p. or 16 µg/side in PFC. In addition, interaction studies were carried out with systemic NLX-204 or NLX-101 (each at 0.16 mg/kg i.p.) in combination with intracortical WAY-100635 (selective 5-HT1A receptor antagonist; 2 µg/side) or muscimol (GABA-A receptor agonist, 12.5 ng/side). Anhedonia was assessed by CMS-induced decrease in sucrose solution consumption; anxiety-like behavior was assessed using the Elevated Plus Maze (EPM), and cognitive impairment was assessed by the Novel Object Recognition (NOR) test. Results: A single administration of NLX-204 was sufficient to reverse the CMS-induced deficit in sucrose consumption, similarly to ketamine and NLX-101. NLX-204 also reduced CMS-induced anxiety in the EPM and abolished CMS-induced NOR deficits. These effects were maintained (EPM and NOR) or enhanced (sucrose consumption) over a subsequent 2-week period of treatment. The anti-anhedonic response of the drugs was also maintained for several weeks Following treatment discontinuation, suggesting that they had sustained effects on neuronal networks. A single PFC administration of NLX-204 reversed deficient sucrose consumption, similarly to ketamine and NLX-101. Moreover, the anti-anhedonic activities of systemic NLX-204 and NLX 101 were abolished by coadministration with intracortical WAY-100635 or muscimol. Conclusions: (i) The antidepressant-like activity of NLX-204 in the rat CMS model was as rapid as that of ketamine or NLX-101, supporting targeting cortical 5-HT1A receptors with selective, biased agonists to achieve RAAD effects. (ii)The anti-anhedonic activity of systemic NLX-204 was mimicked by local administration of the compound in the PFC, confirming the involvement of cortical circuits in its RAAD-like effects. (iii) Notably, the effects of systemic NLX-204 and NLX-101 were abolished by PFC administration of muscimol, indicating that they act by (indirectly) eliciting a reduction in cortical GABAergic neurotransmission. This is consistent with ketamine’s mechanism of action and suggests that there are converging NMDA and 5-HT1A receptor signaling cascades in PFC underlying the RAAD-like activities of ketamine and NLX-204. Acknowledgements: The study was financially supported by NCN grant no. 2019/35/B/NZ7/00787.

Keywords: depression, ketamine, serotonin, 5-HT1A receptor, chronic mild stress

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Authors: Kuladip Jana

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Kuladip Jana, Bhaswati Banerjee, Parimal C. Sen

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems along with carcinogenesis in skin, prostate, ovary, lung and mammary glands. Our study was focused on elucidating the molecular mechanism of B(a)P induced male reproductive toxicity and its prevention with phytochemical like resveratrol. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of Wistar rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased Reactive Oxygen Species (ROS), altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38 mitogen activated protein kinase (p38MAPK), cytosolic translocation of cytochrome-c, upregulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of poly ADP ribose polymerase (PARP) and down regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like steroidogenic acute regulatory protein (StAR), cytochrome P450 IIA1 (CYPIIA1), 3β hydroxy steroid dehydrogenase (3β HSD), 17β hydroxy steroid dehydrogenase (17β HSD) showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis. Next we investigated the role of resveratrol on B(a)P induced male reproductive toxicity. Our study highlighted that resveratrol co-treatment with B(a)P maintained testicular redox potential, increased serum testosterone level and prevented steroidogenic dysfunction with enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3β HSD,17β HSD) relative to treatment with B(a)P only. Resveratrol suppressed B(a)P-induced testicular activation of p38 MAPK, ATF2, iNOS and ROS production; cytosolic translocation of Cytochome c and Caspase 3 activation thereby prevented oxidative stress of testis and inhibited apoptosis. Resveratrol co-treatment also decreased B(a)P-induced AhR protein level, its nuclear translocation and subsequent CYP1A1 promoter activation, thereby decreased protein and mRNA levels of testicular cytochrome P4501A1 (CYP1A1) and prevented BPDE-DNA adduct formation. Our findings cumulatively suggest that resveratrol prevents activation of B(a)P by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Keywords: benzo(a)pyrene, resveratrol, testis, apoptosis, cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), p38 MAPK/ATF2/iNOS

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