Fugen Aktan

Gypenosides derived from Gynostemma pentaphyllum suppress NO synthesis in murine macrophages by inhibiting iNOS enzymatic activity and attenuating NF-κB-mediated iNOS protein expression

Gypenosides isolated from Gynostemma pentaphyllum are widely used in traditional Chinese medicine, with beneficial effects reported in numerous diseases, including inflammation and atherosclerosis, although the mechanism underlying these therapeutic effects is unknown. Because increased nitric oxide (NO) plays a role in these pathological conditions, we investigated whether the pharmacological activity of gypenosides is due to suppression of NO synthesis. The markedly increased production of nitrite by stimulation of RAW 264.7 murine macrophages with 1 microg/mL lipopolysaccharide (LPS) for 20 h (unstimulated: 0.3+/-0.3 microM vs. LPS: 32.5+/-1.2 microM) was dose-dependently inhibited by gypenosides (0.1-100 microg/mL). When cells were pretreated with gypenosides (for 1h) prior to LPS stimulation, subsequent NO production was significantly attenuated (IC(50) of 3.1+/-0.4 microg/mL) (P<0.05). Gypenosides (25 microg/mL) produced the same maximum inhibition of LPS-induced NO production as aminoguanidine, a standard inhibitor of NOS enzymes. Suppression of NO production occurred both by direct inhibition of the activity and expression of iNOS. Inhibition of iNOS protein expression appears to be at the transcriptional level, since gypenosides decreased LPS-induced NF-kappaB activity in a dose-dependent manner (P<0.05), with significant inhibition achieved following pretreatment with 10 microg/mL gypenoside. Taken together, these results suggest that gypenosides derived from G. pentaphyllum suppress NO synthesis in murine macrophages by inhibiting iNOS enzymatic activity and attenuating NF-kappaB-mediated iNOS protein expression, thereby implicating a mechanism by which gypenosides may exert their therapeutic effects.

In vivo treatment with stobadine prevents lipid peroxidation, protein glycation and calcium overload but does not ameliorate Ca2+ -ATPase activity in heart and liver of streptozotocin-diabetic rats: comparison with vitamin E.

Hyperglycemia leads to excess production of reactive oxygen species (ROS), lipid peroxidation and protein glycation that may impair cellular calcium homeostasis and results in calcium sequestration and dysfunction in diabetic tissues. Stobadine (ST) is a pyridoindole antioxidant has been postulated as a new cardio- and neuroprotectant. This study was undertaken to test the hypothesis that the treatment with ST inhibits calcium accumulation, reduces lipid peroxidation and protein glycation and can change Ca2+,Mg2+-ATPase activity in diabetic animals. The effects of vitamin E treatment were also evaluated and compared with the effects of combined treatment with ST. Diabetes was induced by streptozotocin (STZ, 55 mg/kg i.p.). Some of diabetic rats and their age-matched controls were treated orally with a low dose of ST (24.7 mg/kg/day), vitamin E (400-500 IU/kg/day) or ST plus vitamin E for 10 weeks. ST and vitamin E separately produced, in a similar degree, reduction in diabetes-induced hyperglycemia. Each antioxidant alone significantly lowered the levels of plasma lipid peroxidation, cardiac and hepatic protein glycation in diabetic rats but vitamin E treatment was found to be more effective than ST treatment alone. Diabetes-induced increase in plasma triacylglycerol levels was not significantly altered by vitamin E treatment but markedly reduced by ST alone. The treatment with each antioxidant completely prevented calcium accumulation in diabetic heart and liver. Microsomal Ca2+,Mg2+-ATPase activity significantly decreased in both tissues of untreated diabetic rats. ST alone significantly increased microsomal Ca2+,Mg2+-ATPase activity in the heart of normal rats. However, neither treatment with ST nor vitamin E alone, nor their combination did change cardiac Ca2+,Mg2+-ATPase activity in diabetic heart. In normal rats, neither antioxidant had a significant effect on hepatic Ca2+,Mg2+-ATPase activity. Hepatic Ca2+,Mg2+-ATPase activity of diabetic rats was not changed by single treatment with ST, while vitamin E alone completely prevented diabetes-induced inhibition in microsomal Ca2+,Mg2+-ATPase activity in liver. Combined treatment with ST and vitamin E provided more benefits in the reduction of hyperglycemia and lipid peroxidation in diabetic animals. This study describes potential mechanisms on cellular effects of ST in the presence of diabetes-induced hyperglycemia that may delay or inhibit the development of diabetic complications. The use of ST together with vitamin E can better control hyperglycemia-induced oxidative stress.

Short-term gemfibrozil treatment reverses lipid profile and peroxidation but does not alter blood glucose and tissue antioxidant enzymes in chronically diabetic rats

In this study, we investigated the efficiency of short-term treatment with gemfibrozil in the reversal of diabetes-induced changes on carbohydrate and lipid metabolism, and antioxidant status of aorta. Diabetes was induced by a single injection of streptozotocin (45 mg/kg, i.p.). After 12 weeks of induction of diabetes, the control and diabetic rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of gemfibrozil for 2 weeks. At 14 weeks, there was a significant increase in blood glucose, plasma cholesterol and triglyceride levels of untreated-diabetic animals. Diabetes was associated with a significant increase in thiobarbituric acid reactive substances (TBARS) in both plasma and aortic homogenates, indicating increased lipid peroxidation. Diabetes caused an increase in vascular antioxidant enzyme activity, catalase, indicating existence of excess hydrogen peroxide (H2O2). However, superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities in aortas did not significantly change in untreated-diabetic rats. In diabetic plus gemfibrozil group both plasma lipids and lipid peroxides showed a significant recovery. Gemfibrozil treatment had no effect on blood glucose, plasma insulin and vessel antioxidant enzyme activity of diabetic animals. Our findings suggest that the beneficial effect of short-term gemfibrozil treatment in reducing lipid peroxidation in diabetic animals does not depend on a change of glucose metabolism and antioxidant status of aorta, but this may be attributed to its decreasing effect on circulating lipids. The ability of short-term gemfibrozil treatment to recovery of metabolism and peroxidation of lipids may be an effective strategy to minimize increased oxidative stress in diabetic plasma and vasculature.

Effects of L-carnitine treatment on oxidant/antioxidant state and vascular reactivity of streptozotocin-diabetic rat aorta

In this study, the effects of L‐carnitine treatment on lipids, lipid peroxidation of plasma, reactivity and antioxidant enzyme activity of aorta were evaluated in streptozotocin (STZ)‐diabetic rats. Treatment with L‐carnitine (0.6 g kg−1 daily, i.p.) was started 8 weeks after the induction of diabetes and continued for 2 weeks. Diabetes was induced by a single injection of streptozotocin (45 mg kg−1, i.p.). Plasma cholesterol, triglyceride and thiobarbituric acid reactive substance (TBARS) levels and blood glucose levels were significantly increased, although free carnitine levels were markedly decreased in diabetic rats. L‐Carnitine treatment completely normalized plasma cholesterol, triglyceride, free carnitine and TBARS levels but partially restored blood glucose levels of diabetic rats. STZ‐diabetes caused a significant reduction in the endothelium‐dependent relaxation response to acetylcholine (ACh). In diabetic aorta, TBARS levels and catalase (CAT) activity were significantly increased but glutathione peroxidase (GSH‐Px) activity was unchanged. Treatment of diabetic rats with L‐carnitine resulted in partial restoration of the endothelium‐dependent relaxation response to ACh and completely normalized the oxidant/antioxidant state. These results suggested that the beneficial effects of L‐carnitine treatment partially improve vascular reactivity and antioxidant property beyond its reduction of plasma lipids and it may have an important therapeutic approach in the treatment of diabetic vascular complications.

Effect of L-carnitine on the synthesis of nitric oxide in RAW 264·7 murine macrophage cell line

L‐Carnitine (β‐hydroxy‐γ‐trimethyl aminobutyric acid) plays a critical role in inflammatory diseases by modulating inflammatory cell functions. Inducible nitric oxide synthase (iNOS), a proinflammatory enzyme responsible for the generation of nitric oxide (NO), has been implicated in the pathogenesis of inflammatory diseases. Mechanism of action of L‐carnitine on inflammation via iNOS and nuclear factor κB (NF‐κB) is unclear. In this study, we aimed to investigate the effect of L‐carnitine on nitric oxide synthesis in lipopolysaccharide (LPS)‐stimulated RAW 264·7 macrophage cells. For this purpose, cells were pretreated with various concentrations of L‐carnitine and subsequently incubated with LPS (1 µg·ml−1). NO levels, iNOS protein expression, and NF‐κB activity were determined using colorimetric detection, Western blotting and transfection assays. Our results showed that treatment with L‐carnitine suppressed nitric oxide production, iNOS protein expression and NF‐κB activity. We demonstrated that inhibitory effect of L‐carnitine on iNOS protein expression is at transcriptional level. This study may contribute to understanding the anti‐inflammatory effect of L‐carnitine. Copyright

Methylsulfonylmethane modulates apoptosis of LPS/IFN-γ-activated RAW 264.7 macrophage-like cells by targeting p53, Bax, Bcl-2, cytochrome c and PARP proteins

Abstract Methylsulfonylmethane (MSM) is a non-toxic, natural organosulfur compound, which is known to possess antioxidant and anti-inflammatory activities. In recent years, MSM has been widely used as a dietary supplement for its beneficial effects against various diseases, especially arthritis. Despite being a popular supplement product, the mechanism of action of MSM is not well known. This study was designed to investigate the effects of MSM on cytotoxic signals induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in RAW 264.7 macrophage-like cells. The results showed that MSM reversed apoptosis of RAW 264.7 macrophage-like cells at non-cytotoxic concentrations probably through the modulation of apoptotic proteins. After pre-treatment of cells with non-toxic doses of MSM; caspase-3 activation, p53 accumulation, cytochrome c release and Bax/Bcl-2 ratio were significantly decreased and full length poly ADP-ribose polymerase (PARP) was significantly increased. In addition, the loss of mitochondrial membrane potential was decreased with MSM pretreatment in activated macrophages. Since excess nitric oxide production causes apoptosis of macrophages, anti-apoptotic effects of MSM are thought to be mediated by its inhibitor effects on inducible nitric oxide synthase (iNOS) protein and nitric oxide levels. More interestingly, higher doses of MSM exhibited biphasic effects, inhibited cell viability, induced apoptosis of macrophages, increased caspase-3 activity and PARP cleavage. Thus, our results reveal the molecular mechanism of of MSM indicating that MSM supplementation may be beneficial for complications related to nitric oxide-dependent apoptosis in inflammatory conditions. However, the optimum concentration of MSM must be chosen carefully to elicit the desired effect.

Methylsulfonylmethane Induces p53 Independent Apoptosis in HCT-116 Colon Cancer Cells

Methylsulfonylmethane (MSM) is an organic sulfur-containing compound which has been used as a dietary supplement for osteoarthritis. MSM has been shown to reduce oxidative stress and inflammation, as well as exhibit apoptotic or anti-apoptotic effects depending on the cell type or activating stimuli. However, there are still a lot of unknowns about the mechanisms of actions of MSM. In this study, MSM was tested on colon cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis revealed that MSM inhibited cell viability and increased apoptotic markers in both HCT-116 p53 +/+ and HCT-116 p53 −/− colon cancer cells. Increased poly (ADP-ribose) polymerase (PARP) fragmentation and caspase-3 activity by MSM also supported these findings. MSM also modulated the expression of various apoptosis-related genes and proteins. Moreover, MSM was found to increase c-Jun N-terminal kinases (JNK) phosphorylation in both cell lines, dose-dependently. In conclusion, our results show for the first time that MSM induces apoptosis in HCT-116 colon cancer cells regardless of their p53 status. Since p53 is defective in >50% of tumors, the ability of MSM to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy. Moreover, the remarkable effect of MSM on Bim, an apoptotic protein, also suggests its potential use as a novel chemotherapeutic agent for Bim-targeted anti-cancer therapies.

Effects of varying degrees of partial bladder outlet obstruction on urinary bladder function of rats: A novel link to inflammation, oxidative stress and hypoxia

The aim of the present study was to investigate the effects of different degrees of obstruction, and the roles of inflammation, oxidative stress, and hypoxia parameters on bladder function.

Apoptotic Effects of Some Tetrahydronaphthalene Derivatives on K562 Human Chronic Myelogenous Leukemia Cell Line

BACKGROUND Retinoids which are vitamin A (Retinol) derivatives have been suggested to mediate the inhibition of cancer cell growth and apoptosis. It has been reported that all trans retinoic acid (ATRA) exhibited suppressive effects on different types of leukemia including chronic myelogenous leukemia. OBJECTIVE In the present study, we aim to find out the effects of 6 synthetic N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalene-2-yl)-carboxamide derivatives (compound 6-12) on cell viability and apoptotic pathways in K562 human chronic myelogenous leukemia cell line. METHODS Cell viability and apoptosis were examined by spectrophotometric thiazolyl blue tetrazolium bromide (MTT) and caspase-3 assay, western blot, RT-PCR and flow cytometry. RESULTS Our results indicated that compound 6 (5-(1,2-Dithiolan-3-yl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)pentanamide), 8 (4-(3,4-Dimethoxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)butanamide) and 11 (E-3-(4-Hydroxy-3-methoxyphenyl)-N-(3,5,5,8,8-pentamethyl- 5,6,7,8-tetrahydronaphthalen-2-yl)acrylamide) exhibited apoptotic effects in K562 human chronic myelogenous leukemia cell line and induced caspase 3, PARP cleavage, Bax/Bcl-2 ratio, Bad and Bim gene expressions. CONCLUSION Some retinoid derivatives tested in this study induced apoptosis of K562 cells which suggest that these compounds may serve as potential agents in the treatment of chronic myelogenous leukemia.

Expression analysis of Akirin-2, NFκB-p65 and β-catenin proteins in imatinib resistance of chronic myeloid leukemia

ABSTRACT Objective: Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and β-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and β-catenin in imatinib resistance of CML and to test if any direct physical protein–protein interaction exists between NFkB and both β-catenin and Akirin-2. Methods: RT–PCR and western blot were performed to determine Akirin-2, NFκB-p65 and β-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and β-catenin proteins to MDR1 promoter region, respectively. Results: β-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein–protein interaction could not be found between NFκB-p65 and Akirin-2 or β-catenin proteins. Nuclear β-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells. Discussion: We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and β-catenin genes may provide an opportunity to overcome imatinib resistance in CML.