Dietary fish oil associated with increased apoptosis and modulated expression of Bax and Bcl-2 during 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinogenesis in rats

The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on induction of apoptosis in mammary carcinogenesis model. Mammary carcinogenesis was initiated by a single, tail vein injection of 7,12-dimethylbenz(alpha)anthracene (DMBA) (0.5mg/0.2ml corn oil/100g body weight) at 7 weeks of animal age. Ninety female Sprague-Dawley rats were divided into two parts: part one was used for histology and immunohistochemical study and part two for morphological analysis. Each part consists of three experimental groups having 15 animals, i.e., Group A (DMBA control), Group B (DMBA+fish oil) and Group C (DMBA+corn oil). Rats were fed either fish oil or corn oil (0.5ml/day/rat) by oral gavage, 2 weeks prior to DMBA injection. Treatment was continued 25 weeks, studying histopathology, expression of Bax and Bcl-2 proteins by immunohistochemistry and apoptosis by TUNEL assay and morphological study at 36 weeks. Results showed that the fish oil-treated group exhibited a substantial increase in Bax (p<0.05) immunolabelling and a reduction of Bcl-2 immunopositivity (p<0.05), and increased TUNEL-positive apoptotic cells (p<0.05); however, corn oil treatment did not show these beneficial effects toward mammary preneoplasia. We conclude that fish oil has the potential to play a significant role in limiting mammary tumourigenesis in vivo.     

Effect of transforming growth factor-alpha on enterocyte apoptosis is correlated with EGF receptor expression along the villus-crypt axis during methotrexate-induced intestinal mucositis in a rat

The purpose of the present study was to evaluate the effect of transforming-growth factor-alpha (TGF-α) on enterocyte apoptosis following methotrexate (MTX) induced intestinal mucositis in a rat and in Caco-2 cells. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of TGF-α. Cell apoptosis was determined by FACS cytometry. Adult rats were divided into four groups: Control, Control-TGF-α, MTX, and MTX- TGF-α rats. Three days later rats were sacrificed. Enterocyte apoptosis were measured at sacrifice. RT-PCR and Western Blotting was used to determine the level of Bax and Bcl-2 mRNA and protein. Real time PCR was used to measure epidermal growth factor receptor (EGFr) expression along the villus-crypt axis. The in vitro experiment has shown that treatment with TGF-α of Caco-2 cells results in a significant inhibition of cell apoptosis in a dose-dependent manner. In vivo experiment, a decreased levels of apoptosis in MTX- TGF-α rats corresponded with the decrease in Bax and with the increase in Bcl-2 at both mRNA and protein levels. The inhibiting effect of TGF-α on enterocyte apoptosis was strongly correlated with EGFr expression along the villus-crypt axis. In conclusion, treatment with TGF-α inhibits enterocyte apoptosis following MTX- injury in the rat.     

Cornel Iridoid Glycoside Inhibits Inflammation and Apoptosis in Brains of Rats with Focal Cerebral Ischemia

The capacity of cornel iridoid glycoside (CIG) to suppress the manifestations of ischemic stroke was investigated. CIG was administered to rats by the intragastric route once daily for 7 days. Focal cerebral ischemia was induced by 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. In non-treated rats large infarct areas were observed within 24 h of reperfusion. Examination of the ischemic cerebral cortex revealed microglia and astrocyte activation, increased interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) concentrations, increased DNA fragmentation in the ischemia penumbra, elevated Bax expression, increased caspase-3 cleavage, and decreased Bcl-2 expression. Pretreatment with CIG decreased the infarct area, DNA fragmentation, IL-1β and TNF-α concentrations, microglia and astrocyte activation, Bax expression, and caspase-3 cleavage while increasing Bcl-2 expression. CIG exerts anti-neuroinflammatory and anti-apoptotic effects which should prove beneficial for prevention or treatment of stroke.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

Effect of soybean oil on atherogenic metabolic risks associated with estrogen deficiency in ovariectomized rats

The aim of the present study was to investigate the cardiac biomarker changes in experimental bilateral ovariectomized (OVX) rats in addition to evaluating the role of soybean oil-supplemented diet to attenuate these alterations. Female rats were divided into four groups and treated for 2 months as follows: groups 1 and 2 fed with standard diet with or without 15% soybean oil. Groups 3 and 4 were bilateral OVX and received the standard diet with or without 15% soybean oil. The results revealed that rats subjected to ovariectomy exhibited an inhibition in estrogen and high-density lipoprotein cholesterol levels and marked increase of lipid profile, low-density lipoprotein cholesterol, and VLDL-C accompanied with a marked elevation in atherogenic index, cardiac enzyme activity, tumor necrosis factor-α, and C-reactive protein. Signs of cardiovascular injury which included an increase in cardiac thiobarbituric acid-reactive substances were concomitantly noticed with a reduction in the reduced glutathione, total antioxidant capacity, and superoxide dismutase. However, supplementation of soybean oil resulted in the restoration of the changed lipid profile and improved cardiac biomarkers near to normal values as well as improved inflammatory and antioxidant status. It was concluded that consumption of soybean oil may have a role in retarding atherosclerosis and risk of cardiovascular disorders associated with estrogen deficiency in ovariectomy status.     

The timing of re-institution of good blood glucose control affects apoptosis and expression of Bax and Bcl-2 in the retina of diabetic rats

Hyperglycemia initiates a sequence of events that leads to the development of diabetic retinopathy. We explored the effect of re-institution of good blood glucose control on apoptosis and apoptosis related genes (Bax and Bcl-2) in the retina of diabetic rats. Fifty male Wistar rats randomly divided into five groups : normal control group (CON), diabetic rats with high blood glucose levels for 8 months group (DM) ,diabetic rats with good blood glucose control for 8 months group (DM₁),diabetic rats with poor blood glucose control for 2 month followed by good blood glucose control for six additional months group (DM₂), rats with poor blood glucose control for 4 months followed by good blood glucose levels for four additional months group (DM₃). Expression of Bax and Bcl-2 in the retina was studied by immunohistochemistry and the apoptotic cells were stained using the TUNEL method. The apoptotic cell, expression of Bax and Bcl-2 and the ratio of Bax to Bcl-2 in the retina was increased in DM group compared with normal rats’ (P < 0.01). There was no significant difference in apoptotic cells and the ratio of Bax to Bcl-2 between DM₁ group and CON group. The number of TUNEL positive cells and Bax to Bcl-2 ratio was partially reversed in DM₂ group. But glucose control had no effect on the apoptotic cells and the expression of Bax and Bcl-2 in DM₃ group. There was a positive correlation between apoptotic cells and Bax/Bcl-2 ratio in the retina (r = 0.808, P < 0.01). Good blood glucose control at early stage can decrease the number of apoptotic cells in the retina; the decreased apoptosis is correlated with the down-regulation of Bax to Bcl-2 ratio.     

Effect of Chronic Lead Exposure on Pro-Apoptotic Bax and Anti-Apoptotic Bcl-2 Protein Expression in Rat Hippocampus In Vivo

Despite reduction in environmental lead, chronic lead exposure still possess a public health hazard, particularly in children, with devastating effects on developing CNS. To investigate the mechanism of this neurotoxicity, young and adult rats were used to study whether exposure to 500 ppm concentrations of lead could induce apoptosis in hippocampus. 2–4 and 12–14-week-old rats received lead acetate in concentration of 500 ppm for 40 days. Control animals received deionized distilled water. In lead-treated groups, the blood lead levels were increased by 3–4 folds. Light and electron microscopical study of hippocampus revealed increased apoptotic cells. Western blot analysis of Bax and Bcl-2 (pro- and anti-apoptotic gene products, respectively) indicated higher expression of Bax protein and no significant change in bcl-2 expression and accordingly increased the Bax/Bcl-2 ratio compared to control group, confirming the histological study. In conclusion, these data suggest that neurotoxicity of chronic lead exposure in hippocampus in vivo may partly be due to facilitation of apoptosis.     

Protective role of fish oil (Maxepa) on early events of rat mammary carcinogenesis by modulation of DNA-protein crosslinks,cell proliferation and p53 expression

Background  

Fish oil is known to protect from many types of cancers of the colon, liver, breast, prostate and lung [1–3]. The objective of the present study was to evaluate the role of fish oil [Maxepa, supplemented at a dose of 0.5 ml is equivalent to 90 mg eicosapentaenoic acid (EPA) and 60 mg docosahexaenoic acid (DHA)] on cell proliferation, expression of p53 tumor suppressor protein and DNA protein crosslinks (DPCs) in a defined model of chemical rat mammary carcinogenesis. Mammary carcinogenesis was initiated by a single, intravenous (i.v.) tail vein injection of 7,12 dimethylbenz(α)anthracene (DMBA) at a dose of 5 mg DMBA/2 ml corn oil/kg body weight in female Sprague-Dawley rats at 7 weeks of age. Fish oil supplementation was started daily, 2 weeks prior to DMBA injection and continued for 24 (31 weeks of animal age) weeks and 35 (42 weeks of animal age) weeks of post DMBA injection, for histopathological and immunohistochemical and for morphological studies, respectively.     

Is Vitamin E Toxic to Neuron Cells?

Besides acting as potent free radical scavengers, tocopherols and tocotrienols have been known to have non-antioxidant properties such as the involvement of α-tocopherol (αT) in PKC pathway and the anti-cancer properties of γ-tocotrienol (γT3). This study aims to elucidate whether protective effects shown by αT and γT3 in H₂O₂-induced neuron cultures have anti-apoptotic or pro-apoptotic tendency toward the initiation of neuronal apoptosis. H₂O₂ is used to induce apoptosis in primary cerebellar neuron cultures which is attenuated by pretreatment of αT or γT3 at concentrations ≤10 μM. Similar to our previous work, γT3 was found to be neurotoxic at concentrations ≥100 μM, whereas αT showed no neurotoxicity. Cellular uptake of γT3 was higher than that of αT. Treating cells simultaneously with either γT3 or αT and with then H₂O₂ led to higher expression of Bax and Bcl-2 than in neurons exposed to H₂O₂ alone. Analysis of Bcl-2/Bax ratio as ‘survival index’ showed that both pretreatment of γT3 and αT followed by H₂O₂ increase the ‘survival index’ of Bcl-2/Bax ratio compared to H₂O₂-treated cells, while treatment of γT3 alone decrease the ratio compared to unchanged Bcl2/Bax ratio of similar treatment with αT alone. Similar treatment of γT3 decreased p53 expression and activates p38 MAPK phosphorylation, whereas αT did not alter its expression compared to H₂O₂-treated cells. Treating neurons with only γT3 or αT increased the expression of Bax, Bcl-2, p53, and p38 MAPK compared to control with γT3 exerting stronger expression for proteins involved than αT. In conclusion, low doses of γT3 and αT confer neuroprotection to H₂O₂-treated neurons via their antioxidant mechanism but γT3 has stronger pro-apoptosis tendency than αT by activating molecules involved in the neuronal apoptotic pathway in the absence of H₂O₂.     

The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax,modulate functional interactions with the anti-apoptotic protein Bcl-x<Subscript>L</Subscript>

Background  

Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L.     

Mechanism of Monoclonal Antibody-Coupled Staphylococcus Superantigen-A Induced Apoptosis in Human Bladder Cancer Cells

To investigate the mechanism of apoptosis induced by BDI-1 monoclonal antibody (MAb) coupled Staphylococcal superantigen-A (SEA) in human bladder cancer cell line BIU-87. Human PBMC (effector cells) mediated cytotoxic killing of BIU-87 cells (target cells) was studied by culturing the BIU-87 cells in the presence of effector cells plus medium after their activation by treatment with SEA-targeted by MAb, SEA alone or vehicle (control). Proliferation and apoptosis of BIU-87 cells was measured after the treatments. Expression of Bax and Bcl-2 and cytokine concentration in co-culture supernatants were detected by Western blot and ELISA, respectively. Proliferation of MAb-targeted SEA BIU-87 cells decreased significantly (P < 0.05) as compared to control and SEA groups. Flow cytometry revealed apoptosis in SEA alone and more prominently in targeted-SEA treated in BIU-87 cells, which is significantly more than in controI cells (P < 0.05). In addition, Western blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased by targeted SEA treatment, even at low concentration, as compared to cells treated with SEA alone or control cells (P < 0.05). However, there were no significant differences in IL-2, TNF-α and IFN-γ levels in the culture medium between SEA and targeted SEA groups, even though they are several folds higher than in control cells. SEA targeting by MAb significantly increases apoptosis in BIU-87 cells, possibly through the up-regulation of proapoptotic protein Bax and down regulation of antiapoptotic protein, Bcl-2.     

The anhydrous milk fat, ghee, lowers serum prostaglandins and secretion of leukotrienes by rat peritoneal macrophages

Ghee, the anhydrous milk fat, is one of the most important sources of dietary fat in India. Male Wistar rats were fed diets containing 2.5, 5.0 and 10 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. The results showed that serum thromboxane levels decreased by 27-35%, and 6-keto-prostaglandin F1alpha by 23-37% when ghee was incorporated at level of 10% in the diet. Prostaglandin E2 levels in serum and secretion of leukotrienes B4, C4 and D4 by peritoneal macrophages activated with calcium ionophore decreased when increased amounts of ghee from 2.5 to 10% were included in the diet. Arachidonic acid levels in macrophage phospholipids decreased when incremental amounts of ghee were fed to rats. These studies indicate that ghee in the diet not only lowers the prostaglandin levels in serum but also decreases the secretion of leukotrienes by macrophages.     

Aminoguanidine Changes Hippocampal Expression of Apoptosis-Related Genes,Improves Passive Avoidance Learning and Memory in Streptozotocin-Induced Diabetic Rats

Cognitive dysfunction occurs in patients with diabetes mellitus. The objective of this study was to examine whether bilateral intrahippocampal CA1 (intra-CA1) injection of aminoguanidine (AG) can either affect the Bcl-2 family gene expression or reduce the diabetic imposing abnormalities of passive avoidance learning (PAL) and memory. Rats were divided into five groups: control (C), control treated with normal saline (CS), control treated with AG (S-AG), diabetics (D), and diabetics treated with AG (D-AG). Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg). AG (30 μg/rat) or vehicle was administered intra-CA1 bilaterally at the onset of hyperglycemia. PAL was assessed 7 weeks later. Animals were killed, and hippocampus was dissected following the behavioral test. The expressions of Bax, Bcl-2, and Bcl-xl mRNAs were measured using semiquantitative RT-PCR technique. The result of passive avoidance task showed that AG significantly improved the cognitive performance in diabetic rats. Moreover, AG treatment decreased the levels of Bcl-2 and Bcl-x_L expressions in diabetic group. The ratio of Bax/Bcl-2 and Bax/Bcl-x_L decreased significantly in AG-treated diabetic animals. In conclusion, initial treatment with AG by intra-CA1 micro-injection improves the impaired passive avoidance task in STZ-induced diabetic rats which may be related to the decreased Bax/Bcl-2 and Bax/Bcl-x_L ratios.     

Hypocholesterolemic effect of anhydrous milk fat ghee is mediated by increasing the secretion of biliary lipids

The anhydrous milk fat ghee is one of the important sources of fat in the Indian diet. Our earlier studies showed that rats fed diets containing greater than 2.5 wt% of ghee had lower levels of serum cholesterol compared with rats fed diets containing groundnut oil. To evaluate the mechanism of the hypocholesterolemic effect of ghee, male Wistar rats were fed a diet containing 2.5 or 5.0 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. Both native and ghee heated at 120 degrees C containing oxidized lipids were included in the diet. The ghee in the diet did not affect the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity in the liver microsomes, but it significantly increased biliary excretion of cholesterol, bile acids, uronic acid, and phospholipids. The rats fed ghee had lower levels of cholesterol esters in the serum as well as in the intestinal mucosa. Both native and oxidized ghee influenced cholesterol metabolism. These results indicate that supplementation of diets with ghee lipids would increase the excretion of bile constituents and lower serum cholesterol levels.     

Effect of dietary ghee – the anhydrous milk fat on lymphocytes in rats

Lymphocytes are important components of the immune system. Dietary lipids affect the functioning of the immune system. Changes in the lipid composition of the lymphocyte membrane is a case in point. Membrane structural changes are reflected in the altered function of the cell. Lymphocyte proliferation and lymphocyte rosetting are membrane associated phenomena. Ghee, is a clarified butter product, commonly used in the Indian diet. It is rich in saturated fatty acids and also contain oxysterols which are generated on prolonged heating of ghee. Male weanling rats were fed 2.5% (of the total fat levels) of fresh or thermally oxidized ghee for a period of 8 weeks. The control rats were fed groundnut oil. Lipid composition of lymphocytes in ghee fed rats showed changes. In vitro lipid peroxidation of lymphocyte membranes increased by 26% in oxidized ghee fed rats. Na⁺K⁺ ATPase activity was decreased in oxidized ghee fed rats (18%). Lymphocyte proliferation was reduced in ghee fed rats (32%), compared to the controls, irrespective of the mitogens used (ConA or PHA), or the tissue (splenocytes or peripheral blood lymphocytes). Oxysterols present in oxidized ghee are the likely agents inhibiting lymphoproliferation. Rosetting of lymphocytes decreased in the fresh ghee fed rats by 16% and in oxidized ghee fed rats by 25%. Membrane fluidity declined in the oxidized ghee fed rats. It is concluded that feeding ghee results in decreased proliferation of lymphocytes. Also, feeding oxidised ghee results in decreased proliferation of lymphocytes through alterations in the structure of the lymphocyte membranes in the rat.     

Retrospective analysis of protein kinase C-beta (PKC-β) expression in lymphoid malignancies and its association with survival in diffuse large B-cell lymphomas

Background  

Both mechanistic features and recent correlative findings suggest a potential role for protein kinase C-beta (PKC-β) in tumor pathogenesis, particularly in B-cell malignancies. To evaluate the role of this gene in lymphoid malignancies, we analyzed global gene expression data to quantify PKC-β expression across diagnostic groups and, when possible, determined correlations between PKC-β expression and survival.     

参麦注射液对肠缺血/再灌注肺损伤大鼠肺组织p38MAPK及凋亡相关基因表达的影响

目的:观察参麦注射液(SM)对肠缺血/再灌注(I/R)肺损伤大鼠肺组织p38MAPK和凋亡相关基因Bax、Bcl-2蛋白表达的影响,探讨其保护机制。方法:采用夹闭肠系膜上动脉(SMA)方法建立大鼠肠I/R损伤模型。24只SD大鼠随机分为对照组(Control组)、肠缺血/再灌注组(I/R组)、参麦注射液组(SM+I/R组),每组8只。比较各组大鼠肺湿/干比(W/D)、肺表面活性物质主要成分卵磷脂(PC)及总磷脂(TPL)含量的变化;同时免疫组织化学法检测各组大鼠肺组织中p38MAPK、Bax及Bcl-2蛋白的表达水平。结果:与对照组比较,I/R组肺组织W/D明显升高,而PC和TPL的含量显著降低,肺组织p38MAPK、Bcl-2和Bax蛋白表达明显增强(P均＜0.01),其中Bax的增强比Bcl-2的增强更为明显,Bcl-2/Bax比值降低(P＜0.01);与I/R组比较,SM+I/R组大鼠肺组织W/D明显降低,PC和TPL的含量增加,肺组织p38MAPK和Bax蛋白表达下降(P均＜0.01),Bcl-2的表达增强,Bcl-2/Bax比值明显升高(P＜0.01)。相关分析显示,肠I/R时肺组织p38MAPK蛋白表达水平与肺表面活性物质主要功能成分PC含量及凋亡基因Bcl-2/Bax比值呈负相关(r分别为-0.787,-0.731,P均＜0.01)。结论:SM可能通过抑制p38MAPK信号通路的激活,提高Bcl-2/Bax比值来阻抑细胞凋亡,从而减轻肠I/R时的肺损伤。     

Effects of 17β-estradiol replacement on the apoptotic effects caused by ovariectomy in the rat hippocampus

AimsThe aim of the present study was to investigate the effects of different periods of ovariectomy and 17β-estradiol replacement on apoptotic cell death and expression of members of the Bcl-2 family in the rat hippocampus.Main methodsHippocampi were obtained from rats in proestrus, ovariectomized (15 days, 21 days and 36 days), ovariectomized for 15 days and then treated with 17β-estradiol for 7 or 21 days, and rats ovariectomized and immediately treated with 17β-estradiol for 21 days.The expression of Bcl-2 and Bax and the number of apoptotic cells were determined.Key findingsOvariectomy decreased Bcl-2 expression and increased Bax expression and the number of apoptotic cells. Replacement with 17β-estradiol (21 days) throughout the post-ovariectomy period reduced the number of apoptotic cells to the control levels, and prevented the effects of ovariectomy on Bax expression, but only partially restored the Bcl-2 expression. After 15 days of ovariectomy, the replacement with 17β-estradiol for 21 days, but not for 7 days, restored the Bcl-2 and Bax expression and the percentage of apoptotic cells to the levels found in the proestrus control.SignificanceThe present results show that a physiological concentration of 17β-estradiol may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Even after a period of hormonal deprivation, treatment with 17β-estradiol is able to restore the expression of Bax and Bcl-2 to control levels, but the duration of the treatment is a key factor to obtain the desired effect. These data provide new understanding into the mechanisms contributing to the neuroprotective action of estrogen.