Baicalein inhibits nuclear factor-κB and apoptosis via c-FLIP and MAPK in d-GalN/LPS induced acute liver failure in murine models

The hepatoprotective effects and molecular mechanisms of baicalein on acute liver failure induced by d-galactosamine (d-GalN)/lipopolysaccharides (LPS) were investigated in vivo. Mice were administered with different doses of baicalein (50, 100 or 150 mg/kg, p.o.) 1 h before injection of d-GalN (700 mg/kg)/LPS (10 μg/kg) and then sacrificed 6 h after treatment with d-GalN/LPS. Pretreatment with baicalein prevented d-GalN/LPS-induced liver damage by preventing associated increases of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and by reducing serum tumor necrosis factor α (TNF-α), nitric oxide (NO) or inducible nitric oxide synthase (iNOS) expressions. The molecular mechanisms involved in baicalein-induced inhibition of d-GalN/LPS-caused apoptosis were associated with the protection of mitochondria, increasing the Bcl-2/Bax ratio, blocking the release of cytochrome c, and suppressing the phosphorylation of IκBα, ERK and JNK. Moreover, baicalein activated c-FLIP_L, XIAP and cIAP2 proteins, potentially blocking the recruitment of NF-κB signaling molecules. The results support the investigation of baicalein as a therapeutic candidate for acute liver apoptosis or injury and indicate that baicalein might inhibit liver apoptosis by mediating one or more of these pathways.     

Anti-apoptotic activity of gentiopicroside in d-galactosamine/lipopolysaccharide-induced murine fulminant hepatic failure

This study investigated the hepatoprotective effects of gentiopicroside on d-galactosamine (d-GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were administrated orally with gentiopicroside (40 or 80 mg/kg body weight) at 12 h and 1 h before d-GalN (700 mg/kg)/LPS (10 μg/kg) injection. Gentiopicroside markedly reduced the increases in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in d-GalN/LPS alone group, and this decrease was attenuated by gentiopicroside. Increases in serum tumor necrosis factor-α (TNF-α), which were observed in d-GalN/LPS alone group, were significantly reduced by gentiopicroside. Importantly, gentiopicroside attenuated d-GalN/LPS-induced apoptosis of hepatocytes, as estimated by the caspase-3 cleavage, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. d-GalN/LPS-induced caspase-8 and -9 activation was significantly suppressed by gentiopicroside. Moreover, increased cytosolic cytochrome c protein was reduced by gentiopicroside. Also, the increased ratio of Bax and Bcl-2 protein was significantly attenuated by gentiopicroside. After 6 h of d-GalN/LPS injection, phosphorylated c-jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) was significantly increased, whereas phosphorylation JNK and ERK were attenuated by gentiopicroside. Our results suggest that gentiopicroside offers remarkable hepatoprotection against damage induced by d-GalN/LPS related with its anti-apoptotic activities.     

Lipopolysaccharide (LPS) induction of nitric oxide synthase-2 and cyclooxygenase-2 is impaired in fructose overloaded rats

AimsFructose (F) overload in rats induces metabolic dysfunctions that resemble the human metabolic syndrome. In this paper, we aimed to investigate the response of F overload rats to lipopolysaccharide (LPS) challenge in terms of nitric oxide (NO) production and prostanoids (PR) release.Main methodsNO blood steady-state concentration was monitored through the detection of nitrosyl–hemoglobin complexes (NO–Hb) by electronic spin resonance. Production of 6-keto PGF₁α, PGE₂, PGF₂α and TXB₂ was measured in aorta and mesenteric beds by HPLC. Western blot analysis was used to examine the changes in the expression levels of NOS-2 and COX-2 in aorta.Key findingsOur results showed that increases in NO circulating steady-state concentration and PR production by aorta and mesenteric beds 6 h after LPS administration were significantly attenuated in F overload rats with respect to control animals. Oxidative stress parameters were equally affected in the presence or absence of the F treatment. Aorta protein levels of NOS-2 and COX-2, two enzymes inducible by LPS, were significantly lower in F overload rats with respect to control rats at the end of the treatment (?39% and ?61% for NOS-2 and COX-2 respectively).SignificanceThese results suggest that the metabolic alterations established by 15 weeks of F overload should affect the response to LPS challenge due to an attenuation in the induction of NOS-2 and COX-2. This effect would be one of the components contributing to abnormalities in the course of the inflammatory response in other conditions associated to insulin resistance, such as diabetes.     

Acute and chronic nitrite toxicity in juvenile pike-perch (Sander lucioperca) and its compensation by chloride

Pike-perch Sander lucioperca is currently considered as one of the most promising candidates for production in freshwater recirculation aquaculture systems (RAS). Here, due to the lack of studies on nitrite (NO₂^?) toxicity in pike-perch, a flow-through exposure at 0, 0.44, 0.88, 1.75, 3.5, 7, 14 and 28 mg/L NO₂^?–N was carried out to determine the acute and chronic toxicity over a period of 32 days. In juvenile pike-perch, 120 h LC₅₀ was 6.1 mg/L NO₂^?–N and at ≥ 14 mg/L NO₂^?–N all fish had died within 24 h. Chronic exposure revealed a significant build up of NO₂^? in the plasma as well as in the muscles at ≥ 0.44 mg/L NO₂^?–N peaking in fish exposed to the highest concentration of 3.5 mg/L NO₂^?–N after 32 days. Still, due to high individual variation methemoglobin (MetHb) was only significantly increased (p < 0.01) at 3.5 mg/L NO₂^?–N. No adverse effects on red blood cells (RBC) and hematocrit were observed in any of the treatments. In a second experiment, compensation of NO₂^? toxicity at increasing chloride concentrations (40 (freshwater), 65, 90, 140, 240, 440 mg/L Cl^?) was observed at a constant exposure of 10 mg/L NO₂^?–N for 42 days. At ≥ 240 mg/L Cl^?, NO₂^? build-up in blood plasma and muscle was completely inhibited. At lower Cl^? concentrations (≤ 140 mg/L), NO₂^? was significantly increased in plasma, but only insignificantly elevated in muscle due to high individual variation. MetHb was increased significantly difference only at 40 mg/L Cl^? (freshwater control) compared to the control. Again, high individual variations were observed. As a conclusion, S. lucioperca is moderately sensitive towards NO₂^? and acceptable levels in RAS should hence not exceed 1.75 mg/L NO₂^?–N to avoid MetHb formation. However, based on the 120 h LC₅₀ and a factor of 0.01 according to Sprague (1971), a NO₂^? concentration of ≤ 0.061 mg/L NO₂^?–N is considered as “safe.” Thereby, no NO₂^? should accumulate in the plasma or muscle tissue during chronic exposure. For 10 mg/L NO₂^?–N, ≥ 240 mg/L chloride compensates for NO₂^? uptake in plasma and muscle.     

Nitrate removal processes in a constructed wetland treating drainage from dairy pasture

¹⁵N-labelled NO₃^? was used in a surface-flow constructed wetland in spring to examine the relative importance of competing NO₃^? removal processes. In situ mesocosms (0.25 m²) were dosed with 2 l of ¹⁵NO₃^? (NaNO₃, 300 mg N l^?1, 99 atom% ¹⁵N) and bromide (Br^?) solution (LiBr, 4.3 g l^?1, as a conservative tracer). Concentrations of NO₃^?, Br^?, dissolved oxygen and ¹⁵N₂ were monitored periodically and replicate mesocosms were destructively sampled prior to and 6 days after ¹⁵N addition. Denitrification, immobilisation, plant uptake and dissimilatory NO₃^? reduction to NH₄⁺ (DNRA) accounted for 77, 11, 9 and 2% of ¹⁵NO₃^? transformed during the experiment. Only 6% of denitrification gases were directly measured as atmospheric or dissolved ¹⁵N₂; the remainder (71%) was determined via ¹⁵N mass balance. This indicated that a large proportion of the denitrification gases were entrapped within the soil matrix and/or plant aerenchyma. The floating plant Lemna minor exhibited a significantly higher NO₃^? uptake rate (221 mg kg^?1 d^?1) than Typha orientalis (10 mg kg^?1 d^?1), but periodic harvest of plants would remove <3% of annual NO₃^? inputs. Our results suggest that this 6-year-old constructed wetland functions effectively as a sink for NO₃^? during the growing season with less than one-quarter of the NO₃^? processed sequestered into wetland plant, algal and microbial N pools and the balance permanently removed by denitrification.     

Nitrogen dioxide oxidizes mitochondrial cytochrome c

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9–19). However, the subsequent re-evaluation of the redox potential of the NO/NO^- couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO₂^? a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3 × 10⁶ M^-1 s^-1 for the reaction of NO₂^? with ferrocytochrome c. The importance of NO₂^? was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO₂^? into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO^- or HNO) or of peroxynitrite.     

Nitrate removal from three different effluents using large-scale denitrification beds

Simple technologies that remove nitrate from effluents and other point discharges need to be developed to reduce pollution of receiving waters. Denitrification beds are lined containers filled with organic carbon (typically wood chip or coarse sawdust) and are a technology that is proving promising. Water containing NO₃^? (treated effluent or agricultural drainage) is passed through the bed and the wood chips act as an energy source for denitrifying bacteria that convert NO₃^? to N gases. There are few data on the efficiency of NO₃ removal in large-scale beds. We report here NO₃^? removal results from three large denitrification beds with volumes of 83, 294, and 1320 m³ treating dairy shed effluent, treated domestic effluent and glasshouse effluent, respectively. Nitrate was nearly completely removed from the dairy shed effluent (annual load of 31 kg N) and domestic effluent (annual load 365 kg N). In these beds, NO₃^? removal, presumably by denitrification, was limited by NO₃^? concentration. However, the bed treating glasshouse effluent was overwhelmed by very high NO₃^? concentration (about 250 g N m^?3) and high flow rates (about 150 m³ d^?1) but still reduced NO₃^? concentration to about 150 g N m^?3. For this bed, long-term NO₃^? removal was between 5 and 10 g N m^?3 of bed material when NO₃^? was non-limiting and was similar to rates reported for other smaller denitrification beds. As expected, organic N, ammonium and phosphorus were not removed from any of the effluents following passage through the beds. Our results suggest that denitrification beds are a relatively inexpensive system to construct and operate, and are suitable for final treatment of a range of NO₃^?-laden effluents.     

Heme oxygenase-1 induction by cobalt protoporphyrin enhances fever and inhibits pyrogenic tolerance to lipopolysaccharide

Heme oxygenase-1 (HO-1) is an enzyme that catalyzes degradation of the heme and regulates its availability for newly synthetized hemeproteins such as cyclooxygenases, NO synthases and cytochrome P450. Moreover, HO-1 activity modulates synthesis of cytokines and prostaglandins. All of these factors are well-defined components of fever and pyrogenic tolerance mechanisms. We examine the effect of HO-1 induction and activation using cobalt protoporphyrin (CoPP) on changes in body temperature (T_b), plasma levels of interleukin-6 (IL-6), prostaglandin E₂ (PGE₂) and HO-1 protein in the course of these processes. Intraperitoneally (i.p.) pre-treatment of rats with CoPP (5 mg kg⁻¹) significantly accelerated and enhanced the early stage of lipopolysaccharide (LPS)-induced fever and shortened a post-fever recovery to normal temperature. Pre-treatment with CoPP significantly potentiated the increase in plasma IL-6, PGE₂ and HO-1 levels measured 4 h after the LPS administration. Furthermore, induction of HO-1 attenuated the development of pyrogenic tolerance to repeated injections of LPS. Based on these data we conclude that heme oxygenase-1 may act as a physiological regulator of the febrile response intensity to bacterial infections.     

Protective effect of cannabidiol against cadmium hepatotoxicity in rats

The protective effect of cannabidiol, the non-psychoactive component of Cannabis sativa, against liver toxicity induced by a single dose of cadmium chloride (6.5 mg kg^?1 i.p.) was investigated in rats. Cannabidiol treatment (5 mg kg^?1/day, i.p.) was applied for five days starting three days before cadmium administration. Cannabidiol significantly reduced serum alanine aminotransferase, and suppressed hepatic lipid peroxidation, prevented the depletion of reduced glutathione and nitric oxide, and catalase activity, and attenuated the elevation of cadmium level in the liver tissue resulted from cadmium administration. Histopathological examination showed that cadmium-induced liver tissue injury was ameliorated by cannabidiol treatment. Immunohistochemical analysis revealed that cannabidiol significantly decreased the cadmium-induced expression of tumor necrosis factor-α, cyclooxygenase-2, nuclear factor-κB, caspase-3, and caspase-9, and increased the expression of endothelial nitric oxide synthase in liver tissue. It was concluded that cannabidiol may represent a potential option to protect the liver tissue from the detrimental effects of cadmium toxicity.     

Nitrate removal and greenhouse gas production in a stream-bed denitrifying bioreactor

Denitrifying bioreactors are currently being tested as an option for treating nitrate (NO₃^?) contamination in groundwater and surface waters. However, a possible side effect of this technology is the production of greenhouse gases (GHG) including nitrous oxide (N₂O) and methane (CH₄). This study examines NO₃^? removal and GHG production in a stream-bed denitrifying bioreactor currently operating in Southern Ontario, Canada. The reactor contains organic carbon material (pine woodchips) intended to promote denitrification. Over a 1 year period, monthly averaged removal of influent (stream water) NO₃^? ranged from 18 to 100% (0.3–2.5 mg N L^?1). Concomitantly, reactor dissolved N₂O and CH₄ production, averaged 6.4 μg N L^?1 (2.4 mg N m^?2 d^?1), and 974 μg C L^?1 (297 mg C m^?2 d^?1) respectively, where production is calculated as the difference between inflow and effluent concentrations. Gas bubbles entrapped in sediments overlying the reactor had a composition ranging from 19 to 64% CH₄, 1 to 6% CO₂, and 0.5 to 2 ppmv N₂O; however, gas bubble emission rates were not quantified in this study. Dissolved N₂O production rates from the bioreactor were similar to emission rates reported for some agricultural croplands (e.g. 0.1–15 mg N m^?2 d^?1) and remained less than the highest rates observed in some N-polluted streams and rivers (e.g. 110 mg N m^?2 d^?1, Grand R., ON). Dissolved N₂O production represented only a small fraction (0.6%) of the observed NO₃^? removal over the monitoring period. Dissolved CH₄ production during summer months (up to 1236 mg C m^?2 d^?1), was higher than reported for some rivers and reservoirs (e.g. 6–66 mg C m^?2 d^?1) but remained lower than rates reported for some wastewater treatment facilities (e.g. sewage treatment plants and constructed wetlands, 19,500–38,000 mg C m^?2 d^?1).     

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5 s), therefore direct NO quantification is challenging. An indirect method – based on quantifying conversion of an l-Arg- to l-Cit-derivative by ¹H NMR spectroscopy – is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a–j and 11a–c were better or equivalent substrates for the eNOS enzyme (NO₂^? production = 19–46 μM) than native l-Arg (NO₂^? production = 25 μM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by ¹H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.     

Denitrifying bioreactors—An approach for reducing nitrate loads to receiving waters

Low-cost and simple technologies are needed to reduce watershed export of excess nitrogen to sensitive aquatic ecosystems. Denitrifying bioreactors are an approach where solid carbon substrates are added into the flow path of contaminated water. These carbon (C) substrates (often fragmented wood-products) act as a C and energy source to support denitrification; the conversion of nitrate (NO₃^?) to nitrogen gases. Here, we summarize the different designs of denitrifying bioreactors that use a solid C substrate, their hydrological connections, effectiveness, and factors that limit their performance. The main denitrifying bioreactors are: denitrification walls (intercepting shallow groundwater), denitrifying beds (intercepting concentrated discharges) and denitrifying layers (intercepting soil leachate). Both denitrifcation walls and beds have proven successful in appropriate field settings with NO₃^? removal rates generally ranging from 0.01 to 3.6 g N m^?3 day^?1 for walls and 2–22 g N m^?3 day^?1 for beds, with the lower rates often associated with nitrate-limitations. Nitrate removal is also limited by the rate of C supply from degrading substrate and removal is operationally zero-order with respect to NO₃^? concentration primarily because the inputs of NO₃^? into studied bioreactors have been generally high. In bioreactors where NO₃^? is not fully depleted, removal rates generally increase with increasing temperature. Nitrate removal has been supported for up to 15 years without further maintenance or C supplementation because wood chips degrade sufficiently slowly under anoxic conditions. There have been few field-based comparisons of alternative C substrates to increase NO₃^? removal rates but laboratory trials suggest that some alternatives could support greater rates of NO₃^? removal (e.g., corn cobs and wheat straw). Denitrifying bioreactors may have a number of adverse effects, such as production of nitrous oxide and leaching of dissolved organic matter (usually only for the first few months after construction and start-up). The relatively small amount of field data suggests that these problems can be adequately managed or minimized. An initial cost/benefit analysis demonstrates that denitrifying bioreactors are cost effective and complementary to other agricultural management practices aimed at decreasing nitrogen loads to surface waters. We conclude with recommendations for further research to enhance performance of denitrifying bioreactors.     

Effects of wetland construction on water quality in a semi-arid catchment degraded by intensive agricultural use

Many factors can influence the improvement of water quality in surface-flow constructed wetlands (SFW). To test if water quality was improved, especially in nutrient and salt content, after passage through SFW, 11 wetland plots of various sizes (50, 200, 800 and 5000 m²) were established within constructed wetlands on agricultural soils in the Ebro River basin (NE Spain) that had been affected by salinization. A set of 15 water quality parameters (e.g., nutrients, salts, sediments, and alkalinity) was obtained from samples collected at the inflow and outflow of the wetlands during the first 4 years after the wetlands were constructed. NO₃-N retention rates were as high as 99% in the largest (5000 m²) wetlands. After 4 years, total phosphorus was still being released from the wetlands but not salts. Over the same period, in small wetlands (50, 200, and 800 m²), retention rate relative to the input of NO₃-N increased from 40% to almost 60%. Retention of NO₃-N amounted to up to 500 g N m^?2 per year, for an average load concentration at inflow of ～20 mg l^?1. Release of Na⁺ declined from 16% to 0–2% by volume, for an average load concentration at inflow of ～70 mg l^?1. At the current retention rate of NO₃-N (76–227 g m^?2 per year), 1.5–4% of the catchment should be converted into wetlands to optimize the elimination of NO₃-N.     

Nonspecific inhibition of nitric oxide synthesis evokes endothelin-dependent increases in myocardial contractility

The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure–diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n = 7) was the saline-treated control, while in groups 2 (n = 7) and 3 (n = 7) N-nitro-l-arginine (NNA, 4 mg kg^?1), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100 nmol kg^?1 iv). Plasma ET-1, nitrite/nitrate (NO_x) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120 min decreased the plasma NO_x, significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30 mm Hg mm^?1]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism.     

Nitrate removal rates in woodchip media of varying age

A variety of low-cost carbonaceous solids have been successfully tested in bioreactors designed for nitrate treatment. In many agricultural and wastewater settings, however, such reactors may be practical only if they are maintenance free for a number of years after installation. Although field installations have demonstrated consistent treatment over multi-year timeframes, the ability to accurately quantify slowly declining reaction rates in field settings is problematic because of variations in reactor flow rates, ambient temperatures and influent chemistry. In this study, laboratory column tests were undertaken on four samples of coarse wood particle media (woodchips), two that were fresh and two that had been in continuous operation in subsurface denitrifying bioreactors for periods of 2 and 7 years respectively. Four experimental runs were undertaken at increasing influent NO₃-N concentrations of from 3.1 to 48.8 mg N L^?1. Nitrate mass removal rates remained relatively constant and did not systematically increase in successive runs at higher NO₃ concentrations indicating that NO₃ was not the rate-limiting substrate at these concentrations. Thus, zero-order reaction kinetics were used to model the attenuation reaction (presumably denitrification). The 7-year-old media had a mean NO₃-N removal rate of 9.1 mg N L^?1 d^?1 (6.4 g N m^?3 media d^?1), which remained within 75% of the rate for the 2-year-old media (12.1 mg N L^?1 d^?1 or 8.5 g N m^?3 media d^?11) and within 40–59% of the rate for the fresh chips (15.4–23.0 mg N L^?1 d^?1 or 10.8–16.1 g N m^?3 media d^?1). Results support field experience indicating that woodchips loose about 50% of their reactivity during their first year of operation as soluble organic compounds are leached out, but then relatively stable rates persist for a considerable number of years thereafter.     

Neohesperidin dihydrochalcone down-regulates MyD88-dependent and -independent signaling by inhibiting endotoxin-induced trafficking of TLR4 to lipid rafts

Fulminant hepatic failure (FHF) is a lethal clinical syndrome characterized by the activation of macrophages and the increased production of inflammatory mediators. The purpose of this study was to investigate the effects of neohesperidin dihydrochalcone (NHDC), a widely-used low caloric artificial sweetener against FHF. An FHF experimental model was established in mice by intraperitoneal injection of D-galactosamine (d-GalN) (400 mg/kg)/lipopolysaccharides (LPS) (10 μg/kg). Mice were orally administered NHDC for 6 continuous days and at 1 h before d-GalN/LPS administration. RAW264.7 macrophages were used as an in vitro model. Cells were pre-treated with NHDC for 1 h before stimulation with LPS (10 μg/ml) for 6 h. d-GalN/LPS markedly increased the serum transaminase activities and levels of oxidative and inflammatory markers, which were significantly attenuated by NHDC. Mechanistic analysis indicated that NHDC inhibited LPS-induced myeloid differentiation factor 88 (MyD88) and TIR-containing adapter molecule (TRIF)-dependent signaling. Transient transfection of TLR4 or MyD88 siRNA inhibited the downstream inflammatory signaling. This effect could also be achieved by the pretreatment with NHDC. The fluorescence microscopy and flow cytometry results suggested that NHDC potently inhibited the binding of LPS to TLR4 in RAW264.7 macrophages. In addition, the inhibitory effect of NHDC on LPS-induced translocation of TLR4 into lipid raft domains played an important role in the amelioration of production of downstream pro-inflammatory molecules. Furthermore, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) by NHDC inhibited TLR4 signaling. In conclusion, our results suggest that NHDC attenuates d-GalN/LPS-induced FHF by inhibiting the TLR4-mediated inflammatory pathway, demonstrating a new application of NHDC as a hepatoprotective agent.     

The effects of land use change and irrigation water resource on nitrate contamination in shallow groundwater at county scale

The objective of this study was to assess the impacts of land use changes and irrigation water resource on the nitrate contamination in shallow groundwater. 394 water samples were sampled from the same irrigation wells during a period of five years (from 2002 to 2007) in Huantai County in the North China Plain. NO₃-N concentration in irrigation wells was measured. Geostatistical method combined with GIS technique was used to analyze the spatio-temporal distribution of groundwater NO₃-N concentrations in Huantai County. Land use type and irrigation water resource were combined with the variation of NO₃-N concentrations by statistical approach to investigate the relationship between them. The distribution map showed that the percentages of area increased by 13.06%, 14.37%, 12.23% and 3.85% for that had nitrate concentrations of 10–15, 15–20, 20–30 mg L^?1 and greater than 30 mg L^?1 for shallow groundwater, respectively, while decreased by 28.87% and 14.63% for 0–5 and 5–10 mg L^?1. In the well-irrigated field, the NO₃-N concentrations in shallow groundwater had increased for vegetables, wheat–vegetables and wheat–maize rotations. In contrast, fast-growing tree system could act as a buffer to retain shallow groundwater nitrate content which resulted in reduced NO₃-N concentrations. Under the same land use condition, irrigation with sewage, or well and sewage by turns would both enormously add nitrate to groundwater.     

Nitrogen transformations in vertical flow systems with and without rice (Oryza sativa) studied with a high-resolution soil–water profiler

Nitrogen transformations were studied in flooded and non-flooded vertical flow columns with and without a rice plant. Influent (average concentration: NH₄⁺-N: 40 mg L^?1; NO₃^?-N: 0.15 mg L^?1; and NO₂^?-N: 4.0 mg L^?1) was supplied at 1.25 cm d^?1 during stage 1 (20 May–5 August) and at 2.50 cm d^?1 at stage 2 (6 August–26 October), which resulted in an average nitrogen loading of 156 g m^?2 during the entire experimental period. Total nitrogen (T-N) removal efficiencies exceeded 90% in vertical flow systems with rice plants. Nitrogen assimilated by the rice plants in the flooded column accounted for 60% of the total input nitrogen, while that in the non-flooded column accounted for 36% of the total input. The remaining nitrogen appeared to be removed through biogeochemical pathways. Although some nitrogen flowed out, most input nitrogen was also removed even in the flooded and non-flooded unplanted columns.A high-resolution vertical distribution investigation showed the changes of nitrogen forms in soil water. In the flooded condition, there were high ammonium and high nitrite concentrations in the upper layers. The concentrations of ammonium and nitrite simultaneously decreased with depth increasing, suggesting that anaerobic ammonia oxidation (anammox) may occur in these anaerobic conditions. In contrast, the distributions of nitrogen in the non-flooded columns with elevated water level suggested that nitrification–denitrification route was the major removal mechanism, whether or not rice plants were present.     

Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.     

Influence of resveratrol on liver fibrosis induced by dimethylnitrosamine in male rats

Liver fibrosis is a significant health problem which represents the liver’s scarring process and response to injury through deposition of collagen and extracellular matrix, and ultimately leads to cirrhosis. Resveratrol is a naturally occurring phytoalexin found predominantly in grapes. This study aimed to investigate the antifibrotic role of resveratrol on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Rats were divided into four groups and treated for three weeks; control, resveratrol administered orally (20 mg/kg daily), DMN intraperitoneally injected (10 mg/kg 3 days/week), and the last group was pre-treated daily with resveratrol then injected with DMN, 3 days/week. DMN administration induced severe liver pathological alterations. However, oral administration of resveratrol before DMN significantly prevented the induced loss in body weight, as well as the increase in liver weight which arise from DMN administration. Resveratrol has also inhibited the elevation of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin levels. Furthermore, resveratrol significantly increased hepatic reduced glutathione (GSH) levels and reduced the levels of malondialdehyde (MDA) due to its antioxidants effect as well as increased serum protein levels. In addition, DMN induced elevation in hydroxyproline content. On the other hand, hydroxyproline level was significantly reduced in the resveratrol pretreated rats. Resveratrol has also remarkably maintained the normal liver lobular architecture. Moreover, resveratrol had displayed potent potentials to prevent collagen deposition, lymphocytic infiltration, necrosis, steatosis, vascular damage, blood hypertention, cholangiocyte proliferation. It can be concluded that resveratrol has a marked protective role on DMN-induced liver fibrosis in rats, and can be considered as antiproliferative, antihypertensive, as well as antifibrotic agent and may be used to block the development of liver fibrosis.