Thymoquinone ameliorates testicular tissue inflammation induced by chronic administration of oral sodium nitrite.

Although sodium nitrite has been widely used as food preservative, building bases of scientific evidence about nitrite continues to oppose the general safety in human health. Moreover, thymoquinone (TQ) has therapeutic potential as antioxidant, anti-inflammatory, antibacterial and anticancer. Therefore, we investigated the effects of both sodium nitrite and TQ on testicular tissues of rats. Forty adult male Sprague Dawley rats were used. They received either 80 mg kg(-1) sodium nitrite or 50 mg kg(-1) TQ daily for twelve weeks. Serum testosterone was measured. Testis were weighed and the testicular tissue homogenates were used for measurements of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4, IL-6, IL10, caspase-3, caspase-8 and caspase-9. Sodium nitrite resulted in significant reduction in serum testosterone concentration and elevation in testis weight and Gonado-Somatic Index. We found significant reduction in testicular tissues levels of IL-4 and IL-10 associated with elevated levels of TNF-α, IL-1ß, IL-6, caspase-3, caspase-8 and caspase-9. In conclusion, chronic oral sodium nitrite induced changes in the weight of rat testis accompanied by elevation in the testicular tissue level of oxidative stress markers and inflammatory cytokines. TQ attenuated sodium nitrite-induced testicular tissue damage through blocking oxidative stress, restoration of normal inflammatory cytokines balance and blocking of apoptosis.

Epicatechin blocks pro-nerve growth factor (proNGF)-mediated retinal neurodegeneration via inhibition of p75 neurotrophin receptor expression in a rat model of diabetes [corrected].

AIMS/HYPOTHESIS: Accumulation of pro-nerve growth factor (NGF), the pro form of NGF, has been detected in neurodegenerative diseases. However, the role of proNGF in the diabetic retina and the molecular mechanisms by which proNGF causes retinal neurodegeneration remain unknown. The aim of this study was to elucidate the role of proNGF in neuroglial activation and to examine the neuroprotective effects of epicatechin, a selective inhibitor of tyrosine nitration, in an experimental rat model of diabetes. METHODS: Expression of proNGF and its receptors was examined in retinas from streptozotocin-induced diabetic rats, and in retinal Müller and retinal ganglion cells (RGCs). RGC death was assessed by TUNEL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in diabetic retinas and cell culture. Nitrotyrosine was determined using Slot-blot. Activation of the tyrosine kinase A (TrkA) receptor and p38 mitogen-activated protein kinase (p38MAPK) was assessed by western blot. RESULTS: Diabetes-induced peroxynitrite impaired phosphorylation of TrkA-Y490 via tyrosine nitration, activated glial cells and increased expression of proNGF and its receptor, p75 neurotrophin receptor (p75(NTR)), in vivo and in Müller cells. These effects were associated with activation of p38MAPK, cleaved poly-(ADP-ribose) polymerase and RGC death. Treatment of diabetic animals with epicatechin (100 mg kg(-1) day(-1), orally) blocked these effects and restored neuronal survival. Co-cultures of RGCs with conditioned medium of activated Müller cells significantly reduced RGC viability (44%). Silencing expression of p75(NTR) by use of small interfering RNA protected against high glucose- and proNGF-induced apoptosis in RGC cultures. CONCLUSIONS/INTERPRETATION: Diabetes-induced peroxynitrite stimulates p75(NTR) and proNGF expression in Müller cells. It also impairs TrkA receptor phosphorylation and activates the p75(NTR) apoptotic pathway in RGCs, leading to neuronal cell death. These effects were blocked by epicatechin, a safe dietary supplement, suggesting its potential therapeutic use in diabetic patients.

Cannabinoid 1 receptor activation contributes to vascular inflammation and cell death in a mouse model of diabetic retinopathy and a human retinal cell line.

AIMS/HYPOTHESIS: Recent studies have demonstrated that cannabinoid-1 (CB(1)) receptor blockade ameliorated inflammation, endothelial and/or cardiac dysfunction, and cell death in models of nephropathy, atherosclerosis and cardiomyopathy. However the role of CB(1) receptor signalling in diabetic retinopathy remains unexplored. Using genetic deletion or pharmacological inhibition of the CB(1) receptor with SR141716 (rimonabant) in a rodent model of diabetic retinopathy or in human primary retinal endothelial cells (HREC) exposed to high glucose, we explored the role of CB(1) receptors in the pathogenesis of diabetic retinopathy. METHODS: Diabetes was induced using streptozotocin in C57BL/6J Cb(1) (also known as Cnr1)(+/+) and Cb(1)(-/-) mice aged 8 to 12 weeks. Samples from mice retina or HREC were used to determine: (1) apoptosis; (2) activity of nuclear factor kappa B, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), poly (ADP-ribose) polymerase and caspase-3; (3) content of 3-nitrotyrosine and reactive oxygen species; and (4) activation of p38/Jun N-terminal kinase/mitogen-activated protein kinase (MAPK). RESULTS: Deletion of CB(1) receptor or treatment of diabetic mice with CB(1) receptor antagonist SR141716 prevented retinal cell death. Treatment of diabetic mice or HREC cells exposed to high glucose with SR141716 attenuated the oxidative and nitrative stress, and reduced levels of nuclear factor κB, ICAM-1 and VCAM-1. In addition, SR141716 attenuated the diabetes- or high glucose-induced pro-apoptotic activation of MAPK and retinal vascular cell death. CONCLUSIONS/INTERPRETATION: Activation of CB(1) receptors may play an important role in the pathogenesis of diabetic retinopathy by facilitating MAPK activation, oxidative stress and inflammatory signalling. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of this devastating complication of diabetes.

Diabetes-induced peroxynitrite impairs the balance of pro-nerve growth factor and nerve growth factor, and causes neurovascular injury.

AIMS/HYPOTHESIS: Diabetic retinopathy, the leading cause of blindness in working-age Americans, is characterised by reduced neurotrophic support and increased proinflammatory cytokines, resulting in neurotoxicity and vascular permeability. We sought to elucidate how oxidative stress impairs homeostasis of nerve growth factor (NGF) and its precursor, proform of NGF (proNGF), to cause neurovascular dysfunction in the eye of diabetic patients. METHODS: Levels of NGF and proNGF were examined in samples from human patients, from retinal Müller glial cell line culture cells and from streptozotocin-induced diabetic animals treated with and without atorvastatin (10 mg/kg daily, per os) or 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride (FeTPPs) (15 mg/kg daily, i.p.) for 4 weeks. Neuronal death and vascular permeability were assessed by TUNEL and extravasation of BSA-fluorescein. RESULTS: Diabetes-induced peroxynitrite formation impaired production and activity of matrix metalloproteinase-7 (MMP-7), which cleaves proNGF extracellularly, leading to accumulation of proNGF and reducing NGF in samples from diabetic retinopathy patients and experimental models. Treatment of diabetic animals with atorvastatin exerted similar protective effects that blocked peroxynitrite using FeTPPs, restoring activity of MMP-7 and hence the balance between proNGF and NGF. These effects were associated with preservation of blood-retinal barrier integrity, preventing neuronal cell death and blocking activation of RhoA and p38 mitogen-activated protein kinase (p38MAPK) in experimental and human samples. CONCLUSIONS/INTERPRETATION: Oxidative stress plays an unrecognised role in causing accumulation of proNGF, which can activate a common pathway, RhoA/p38MAPK, to mediate neurovascular injury. Oral statin therapy shows promise for treatment of diabetic retinopathy.

Prominent chemopreventive and chemoenhancing effects for resveratrol: unraveling molecular targets and the role of C-reactive protein.

BACKGROUND: Resveratrol (RSVL) claims health benefits that pertain to the consumption of red wine/grapes. We currently evaluated the chemopreventive effects of RSVL, as well as its possible chemoenhancing effects when given with cisplatin (CP), in the Ehrlich ascites carcinoma (EAC) solid tumor model. Further, we monitored concomitant changes in serum levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), leukocytic count (LC) and lipid peroxidation (measured as malondialdehyde, MDA). RESULTS: EAC-bearing mice exhibited a markedly elevated LC (2 fold), CRP (11 fold) and MDA levels (2.7 fold). RSVL (20 or 40 mg/kg) elicited significant, dose-dependent reductions in tumor size (58 and 78%, respectively), as well as in LC (normalized), CRP (down to 2 fold), TNF-alpha (down to near control levels) and MDA levels (normalized). The chemopreventive effects for CP (55% reduction in cell growth) was significantly lower than that of RSVL (40 mg/kg, 79% inhibition). Interestingly, coadministration of RSVL (20 mg/kg) markedly enhanced the chemoprevention of CP. Correlation studies revealed a high degree of positive association between tumor growth and CRP (r = 0.89) and leukocytosis (r = 0.86), thus attesting to a diagnostic/prognostic role for CRP in this solid tumor. CONCLUSION: RSVL elicited remarkable cytotoxicity on its own and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels. RSVL acts prominently by reducing inflammatory cytokines, leukocytosis and oxidative stress.

Epigallocatechin-3-Gallate Upregulates miR-221 to Inhibit Osteopontin-Dependent Hepatic Fibrosis.

Osteopontin (OPN) promotes hepatic fibrosis, and developing therapies targeting OPN expression in settings of hepatic injury holds promise. The polyphenol epigallocatechin-3-gallate (EGCG), found in high concentrations in green tea, downregulates OPN expression through OPN mRNA degradation, but the mechanism is unknown. Previous work has shown that microRNAs can decrease OPN mRNA levels, and other studies have shown that EGCG modulates the expression of multiple microRNAs. In our study, we first demonstrated that OPN induces hepatic stellate cells to transform into an activated state. We then identified three microRNAs which target OPN mRNA: miR-181a, miR-10b, and miR-221. In vitro results show that EGCG upregulates all three microRNAs, and all three microRNAs are capable of down regulating OPN mRNA when administered alone. Interestingly, only miR-221 is necessary for EGCG-mediated OPN mRNA degradation and miR-221 inhibition reduces the effects of EGCG on cell function. In vivo experiments show that thioacetamide (TAA)-induced cell cytotoxicity upregulates OPN expression; treatment with EGCG blocks the effects of TAA. Furthermore, chronic treatment of EGCG in vivo upregulates all three microRNAs equally, suggesting that in more chronic treatment all three microRNAs are involved in modulating OPN expression. We conclude that in in vitro and in vivo models of TAA-induced hepatic fibrosis, EGCG inhibits OPN-dependent injury and fibrosis. EGCG works primarily by upregulating miR-221 to accelerate OPN degradation. EGCG may therefore have utility as a protective agent in settings of liver injury.

Cod liver oil in sodium nitrite induced hepatic injury: does it have a potential protective effect?

OBJECTIVES: Exposure to sodium nitrites, a food additive, at high levels has been reported to produce reactive nitrogen and oxygen species that cause dysregulation of inflammatory responses and tissue injury. In this work, we examined the impact of dietary cod liver oil on sodium nitrite-induced inflammation in rats. METHODS: Thirty-two adult male Sprague-Dawely rats were treated with 80 mg/kg sodium nitrite in presence/absence of 5 ml/kg cod liver oil. Liver sections were stained with hematoxylin/eosin. We measured hepatic tumor necrosis factor (TNF)-α, interleukin-1 beta (IL)-1ß, C-reactive protein (CRP), transforming growth factor (TGF)-ß1, and caspase-3. RESULTS: Cod liver oil reduced sodium nitrite-induced hepatocyte damage. In addition, cod liver oil results in reduction of hepatic TNF-α, IL-1ß, CRP, TGF-ß1, and caspase-3 when compared with the sodium nitrite group. DISCUSSION: Cod liver oil ameliorates sodium nitrite-induced hepatic injury via multiple mechanisms including blocking sodium nitrite-induced elevation of inflammatory cytokines, fibrosis mediators, and apoptosis markers.

Contribution of TNF-α to the development of retinal neurodegenerative disorders.

During the late 1970s, tumor necrosis factor alpha (TNF-α) was initially recognized as an endotoxin-induced substance that was mainly produced by macrophages, and able to cause the lysis of certain tumor cells. Subsequent research demonstrated that TNF-α mediates a broad range of cellular activities, including proliferation, survival, differentiation and apoptosis. It is also considered to be essential for the induction and maintenance of the inflammatory immune responses. Meanwhile, visual impairment imposes a substantial disease burden on society. It is associated with both significant economic impact and reduction in quality of life. Visual impairment raises serious social challenges for both patients and their families, interfering with day-to-day life, and can limit employment possibilities. Many of the most common, irreversible blinding pathologies involve neuronal loss from the retina, which is the light-sensing tissue of the eye. The retina, being part of the central nervous system, is unable to regenerate neurons lost to disease. Therefore, in the current review we will discuss the association between increased expression of TNF-α with neurodegenerative disorders, downstream cellular signaling mechanisms following interaction of TNF-α with its receptors, and the role of TNF-α as a possible target in the treatment of retinal neurodegenerative disorders.

Evaluation of renal protective effects of the green-tea (EGCG) and red grape resveratrol: role of oxidative stress and inflammatory cytokines.

Epigallocatechin-gallate (EGCG) and resveratrol (RSVL) are two of the most promising natural medicines. We verified their capacity to ameliorate cisplatin (CP)-induced disruption of renal glomerular filtration rate (GFR) in rats, and sought the mediatory involvement of lipid peroxidation (malondialdehyde [MDA]-level) and inflammatory cytokine (TNF-α) therein. CP (10 mg kg⁻¹), a single i.p. dose, disrupted GFR (11-fold-rise in proteinuria, 2-5-fold rise in serum creatinine/urea levels) after 7 days, and killed all animals after 10 days. Kidney-homogenates from CP-treated rats displayed higher MDA and TNF-α, but lower reduced-glutathione (GSH) levels. Rats treated with EGCG (50 mg kg⁻¹, but not 25 mg kg⁻¹) had no fatalities and showed significantly-recovered GFR; while their kidney-homogenates had markedly reduced MDA, TNF-α and enhanced GSH levels at 7 days. Conversely, RSVL or quercetin (25, 50 mg kg⁻¹) neither improved GFR nor reduced (MDA)/TNF-α levels after 7 days. Resuming treatment with 50 mg kg⁻¹ for 10 days rescued only 25% of animals (p > 0.05). Correlation studies showed a significant association between creatinine level, and each of MDA (r = 0.91), GSH (r = -0.87), and TNF-α (0.91). The study showed for the first time that EGCG, unlike RSVL, can protect against CP-induced nephrotoxicity. At the molecular level, CP triggers a high level of oxidative stress and systemic inflammation, events that were all abrogated with EGCG; better than RSVL or quercetin.

Novel chemotherapeutic and renal protective effects for the green tea (EGCG): role of oxidative stress and inflammatory-cytokine signaling.

BACKGROUND: The green tea catechin, epigallocatechin-gallate (EGCG) is a superb nature's medicine candidate. We evaluated the chemotherapeutic/chemoenhancing effects of EGCG in mice bearing the solid Ehrlich ascites carcinoma (EAC) tumor, and jointly monitored levels of serum C-reactive protein (CRP), lipid peroxidation (as malondialdehyde: MDA) and leukocytosis (LC). Besides, we verified whether; and how then, EGCG would protect against a devastating CP-induced nephrotoxicity in rats. In particular, renal proinflammatory (TNF-α) and oxidant stress signals have been investigated. RESULTS: (EAC)-bearing mice displayed elevated serum-LC (2-fold), -CRP (11-fold) and -MDA levels (2.7-fold). EGCG (20, 40 mg/kg) significantly shrank tumors (by 48% and 92%, respectively), and reduced LC, CRP and MDA levels. Such responses for CP were less prominent than those of EGCG (40 mg/kg). Further, EGCG (20 mg/kg) markedly augmented such functional and biochemical responses to CP. Correlation studies showed positive association between tumor size and each of CRP (r=0.97) and LC (r=0.83). Additionally; in rats, CP (10 mg/kg) caused a prominent nephrotoxicity that was manifested as deteriorated glomerular filtration rate (GFR, 2-5-fold rise in serum creatinine/urea levels) after 4 days, and unanimous animal fatalities after 7 days. Kidney homogenates from CP-treated rats showed significantly higher MDA- and TNF-α-, and -depleted GSH levels. Rats treated with EGCG (50 mg/kg, but not 25 mg/kg) devoid the nephrotoxic effects of CP and their consequences; while their homogenates had appreciably lower MDA and TNF-α, and higher GSH levels. Notable correlation was detected between serum creatinine level and each of MDA (r=0.85), TNF-α (r=0.85) and GSH (r=-0.81). CONCLUSION: This study shows remarkable cytotoxic/chemoenhancing effects for EGCG and introduces CRP as a predictor of both tumor's progression and responsiveness to chemotherapy. Further, this study is the first to reveal that EGCG can obliterate the lethal CP-induced nephrotoxicity. Mechanistically, EGCG acts by suppressing leukocytosis, systemic inflammation, oxidative stress, and their sequelae.