Tamoxifen Attenuates Lipopolysaccharide/Galactosamine-induced Acute Liver Failure by Antagonizing Hepatic Inflammation and Apoptosis

Bacterial lipopolysaccharide (LPS)-induced acute liver failure (ALF) is a common severe clinical syndrome in intensive care unit. No other methods are available for its prevention apart from supportive treatment and liver transplantation. Tamoxifen (TAM) was reported to attenuate ALF induced by excessive acetaminophen, while its effect on LPS-induced ALF remained unknown. For this, in the present study, we comprehensively assessed whether TAM can attenuate ALF induced by LPS/galactosamine (GaIN). Mice were given TAM once a day for three times. Twelve hours after the last treatment, mice were given LPS/GaIN (intraperitoneally [i.p.]). Survival, plasma transaminases, and histopathology were examined. Serum TNF-α and IL-1β were analyzed by ELISA. Hepatic apoptosis was analyzed by TUNEL and caspase-3 Western blotting, respectively. Compared to the model group, ALF induced by LPS/GaIN was alleviated remarkably following TAM administration, as evidenced by the improvement of survival (87.5% vs. 37.5%), hepatic swell, moderate transaminases, slightly increased serum TNF-α, IL-1β (P < 0.05), and moderate histopathology. In respect of apoptosis, severe hepatocellular apoptosis was reduced notably by TAM treatment confirmed by less TUNEL-positive hepatocytes and decreased caspase-3 cleavage. The results demonstrated that TAM could attenuate LPS/GaIN-induced ALF effectively, probably due to hepatic inflammation and apoptosis antagonism. Furthermore, it was the first report about the effect of TAM on LPS/GaIN-induced ALF.     

The antagonistic effect of tamoxifen against d-galactosamine/lipopolysaccharide-induced acute liver failure is associated with reactivation of hepatic nuclear factor-κB

Context: Tamoxifen (TAM) ameliorates D-galactosamine/lipopolysaccharide (Gal/LPS)-induced acute liver failure (ALF) through its antioxidative effect; thus, this study was designed to determine whether the effectiveness of TAM is related to nuclear factor-κB (NF-κB) reactivation.

Materials and methods: Experimental mice were injected with TAM once daily for 3 consecutive days intraperitoneally (i.p). Twelve hours after pretreatment, Gal/LPS was given to mice (i.p) for ALF induction. In the positive control group, N-acetylcysteine (NAC) was administered immediately after ALF establishment. Except for survival observation, other animals were sacrificed 7?h after Gal/LPS treatment. Survival and hepatic failure were evaluated. For the oxidation assessment, the reduced/oxidized glutathione (GSH/GSSG) ratio and hepatic superoxide dismutase (SOD) activity were analyzed using both colorimetry and Western blotting. Lastly, hepatic NF-κB activation was measured through Western blot analysis of p65 and IκBα.

Results: The results indicated that pretreatment with TAM dramatically attenuated Gal/LPS-induced ALF, as demonstrated by improved survival (70%), decreased transaminase levels, and reversed histopathological manifestation. In addition, the hepatic GSH/GSSG ratio and SOD activity were decreased in the ALF model. However, to some degree, TAM and NAC effectively prevented this undesirable phenomenon in contrast to the ALF model. Western blotting revealed that compared with mice in the ALF model group, mice treated with TAM or NAC showed reactivation of hepatic NF-κB.

Conclusions: Taking the results together with those of other studies, we conclude that TAM may attenuate Gal/LPS-induced ALF by antagonizing oxidative stress through NF-κB reactivation.     

Oxidative stress promotes d-GalN/LPS-induced acute hepatotoxicity by increasing glycogen synthase kinase 3β activity

Objective

Our previous studies have demonstrated that glycogen synthase kinase 3β (GSK3β) activity is increased in the progression of acute liver failure (ALF), which aggravates liver injury, while its regulatory mechanism remains elusive. This study is designated to address whether oxidative stress activates GSK3β to promote ALF.

Methods

In a murine model induced by d-galactosamine (d-GalN) (700 mg/kg) and LPS (10 μg/kg), N-acetylcysteine (300 mg/kg) or SB216763 (25 mg/kg) was used to inhibit oxidative stress or GSK3β activity, respectively. Serum alanine aminotransferase and aspartate aminotransferase levels were assessed. The parameters of oxidative stress were evaluated in liver tissue. Whether GSK3β inhibition protects hepatocytes from oxidative stress-induced cell apoptosis was investigated in vitro. Moreover, the activity of GSK3β was measured in the liver of chronic hepatitis B (CHB) patients and ALF patients.

Results

In vivo, N-acetylcysteine ameliorated the d-GalN/LPS-induced hepatotoxicity and reduced GSK3β activity; GSK3β inhibition increased hepatic superoxide dismutase activity and the glutathione content, decreased malondialdehyde production in the liver tissues; while GSK3β inhibition suppressed the JNK activation in the liver and decreased cytochrome c release from mitochondria. In vitro, GSK3β inhibition lessened hepatocytes apoptosis induced by H₂O₂ or Antimycin A, as demonstrated by decreased LDH activity, and reduced cleavage of caspase-3 expression. Furthermore, GSK3β activity in the CHB patients was increased in the phase of ALF.

Conclusions

Results indicate that GSK3β activation contributes to liver injury by participating in oxidative stress response in ALF and is, therefore, a potential therapeutic target for ALF.     

Association between activation of phase 2 enzymes and down-regulation of dendritic cell maturation by c9,t11-conjugated linoleic acid

Antioxidant and cytoprotective enzymes (phase 2) exert protective activity against reactive oxygen species (ROS)-induced injury. We have recently shown how the beneficial effects of conjugated linoleic acid (CLA) in a mouse model of an autoimmune disease are parallel with the activation of phase 2 enzymes. In the present study we found that c9,t11-CLA isomer activates cytoprotective enzymes and down-regulates LPS- or gliadin-induced maturation in dendritic cells (DCs) obtained from a murine model of celiac disease.

As expected, the enhancement of LPS-induced maturation (increased NFκB p65 nuclear translocation, CD86 expression and decreased CD11c+ cell number) was exacerbated by specific glutathione (GSH) inhibitor (buthionine sulphoximine; BSO). Conversely, the down-regulation of DC maturation by antioxidant N-acetylcysteine (NAC) was associated with the marked increase of intracellular thiol concentration. c9,t11-CLA activation of phase 2 enzymes in mouse DCs was observed first. Next, we found that the significant reduction of LPS- and gliadin-induced DC maturation in cultures pre-treated with c9,t11-CLA improved cellular redox status (decreased ROS and higher antioxidant defenses). Finally, the process of DC maturation triggered by gliadin, in contrast with that exhibited by LPS, was not associated with enhanced NFκB nuclear translocation and pro-inflammatory cytokines synthesis. These results demonstrate that c9,t11-CLA renders DCs more resistant to gliadin- or LPS-induced maturation, thus indicating that a cytoprotective mechanism elicited by c9,t11-CLA may modulate DC responsiveness.     

N-乙酰半胱氨酸对脂多糖诱导的小鼠肝MAPK磷酸化的影响

目的： 探讨N-乙酰半胱氨酸（NAC）对脂多糖（LPS）诱导的肝MAPK磷酸化的影响。方法: 雄性昆明种小鼠54只随机分为对照组(n=6)：0.9 % NaCl 0.2 mL ip；LPS组（n=24）：LPS 5 mg ip；NAC+LPS组（n=24）：NAC 150 mg·kg-1·d-1ip，连续3 d；第3 d NAC灌胃后1 h时，LPS 5 mg ip。将小鼠分别在注射LPS或生理盐水后0.5 h、1 h、2 h和6 h时，在戊巴比妥钠麻醉下开腹取肝，测定肝MDA和还原型谷胱甘肽（GSH）含量；Western blotting方法测定肝脏MEK1/2、ERK1/2、p38MAPK磷酸化水平，放免法测定肝TNF-α含量。结果: NAC预处理使肝MDA含量明显下降，使肝GSH含量升高。NAC预处理显著抑制了LPS所致的肝MEK1/2、ERK1/2、p38MAPK磷酸化，同时使肝TNF-α水平显著降低。结论: 在LPS诱导的急性肝损伤过程中，活性氧（ROS）在激活MAPK信号转导中起重要作用。NAC通过其抗氧化作用部分抑制了LPS诱导的MAPK磷酸化，使TNF-α生成减少，从而发挥抗损伤作用。     

Peroxisome proliferator-activated receptor alpha mediates C/EBP homologous protein to protect mice from acute liver failure

Objective

Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to ameliorate liver injury in cases of acute liver failure (ALF). However, its intrinsic protective molecular mechanisms remain largely undetermined. C/EBP homologous protein (CHOP) is an important mediator of lipopolysaccharide (LPS)-induced inflammation. The aim of the present study was to test the hypothesis that PPARα activation alleviates liver inflammation to protect mice from acute liver failure (ALF) mediated by CHOP.

Methods

In a murine model induced by d-galactosamine (d-GalN, 700 mg/kg) and LPS (10 μg/kg), Wy-14643 (6 mg/kg) was administered to activate PPARα. The mice of different groups were killed 6 h after d-GalN/LPS injection, and the liver and blood were collected for analysis. To find out whether PPARα activation protects the liver from injury due to inflammation by regulating CHOP, we used expression plasmid to increase CHOP expression and demonstrated how PPARα mediated CHOP to regulate inflammation in vivo and in vitro.

Results

The expression of PPARα was downregulated and the expression of CHOP was upregulated with the development of d-GalN/LPS-induced liver injury. The protective molecular mechanisms of PPARα activation were dependent on the expression of CHOP. Indeed, (1) PPARα activation decreased the expression of CHOP; on the other hand, PPARα knockdown increased the expression of CHOP in vivo; (2) the depressed liver inflammation by PPARα activation was due to the downregulation of CHOP expression, because overexpression of CHOP by transfect plasmid reversed liver protection and increased liver inflammation again; (3) in vitro, PPARα inhibition by siRNA treatment increased the expression of proinflammatory cytokines, and CHOP siRNA co-transfection reversed the expression of proinflammatory cytokines.

Conclusions

Here, we demonstrated that PPARα activation contributes to liver protection and decreases liver inflammation in ALF, particularly through regulating CHOP. Our findings may provide a rationale for targeting PPARα as a potential therapeutic strategy to ameliorate ALF.

     

Omega-3 Polyunsaturated Fatty Acids Delay the Progression of Endotoxic Shock-Induced Myocardial Dysfunction

Septic shock has a high mortality rate, partially related to myocardial dysfunction. Polyunsaturated fatty acids (omega-3 PUFAs) possess anti-inflammatory and antioxidant properties, but whether omega-3 PUFAs exert beneficial effects on myocardial function is unknown. We investigated, in a rat model of endotoxic shock, the effects of omega-3 PUFAs pretreatment on cardiac hemodynamics, function, and oxidative stress as well as intestinal barrier. Endotoxic shock was induced by lipopolysaccharide (LPS; 20 mg/kg IP) administered to rats pretreated or not with omega-3 PUFAs (Omegaven®; 0.5 g/kg IP, 90 min before injection of LPS). Two or 5 h after LPS, left ventricular function and arterial pressure were measured, followed by assessment left ventricular total glutathione as well as tumor necrosis factor alpha expression, occuldin expression, and proteasome activities. LPS reduced mean arterial blood pressure to the same extent 2 and 5 h after its administration, but cardiac output was more markedly decreased after 5 h. Omega-3 PUFAs pretreatment did not significantly modify the effect of LPS on mean arterial pressure and total peripheral resistance, but prevented the decrease in cardiac output 2 h after LPS. LPS increased oxidized glutathione after 2 h, and this increase was significantly attenuated by omega-3 PUFAs. Simultaneously, omega-3 PUFAs increased myocardial hemeoxygenase-1 (HO-1) mRNA expression. Finally, omega-3 PUFAs prevented the reduction of intestinal occludin expression. Omega-3 PUFAs pre-treatment improves myocardial dysfunction during endotoxemia and increases myocardial HO-1 expression. Moreover, the preservation of the intestinal occludin induced by omega-3 PUFAs precedes myocardial protection, suggesting the involvement of the intestinal barrier in the myocardial improvement observed with omega-3 PUFAs parenteral supplementation.     

Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro

Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

Effect of Cannabis sativa on oxidative stress and organ damage after systemic endotoxin administration in mice

The effect of Cannabis sativa extract on oxidative stress and organ tissue damage during systemic inflammation was studied. For this purpose, Swiss mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 μg/kg) to mimic aspects of mild systemic infection. Cannabis resin extract (5, 10, or 20 mg/kg) (expressed as Δ⁹-tetrahydrocannabinol) was given via subcutaneous route for 2 days prior to and at the time of endotoxin administration. Mice were euthanized 4 h after LPS injection. Malondialdehyde (MDA), reduced glutathione (GSH), and nitric oxide (nitrite/nitrate) in the brain, liver, kidney, lung, and heart as well as brain glucose were measured. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured in liver homogenates. Histopathological examination of different organs was performed, and immunohistochemical techniques were used to evaluate expression levels of inducible nitric oxide synthase (iNOS) and caspase-3 in the brain and liver. The administration of only cannabis (20 mg/kg) decreased MDA, increased GSH, and decreased glucose level in the brain. No significant effects were observed for cannabis alone on MDA, GSH, or nitric oxide in other organs or on liver enzymes. The administration of LPS increased MDA and nitric oxide, while GSH decreased in different organs. Brain glucose increased by endotoxin. AST, ALT, and ALP were markedly increased in the liver tissue. In LPS-treated mice, cannabis (20 mg/kg) decreased MDA. GSH increased in the brain, kidney, and lung, nitric oxide decreased in the brain and lung while brain glucose decreased after the highest dose of cannabis. Cannabis failed to alter the level of liver enzymes. Histological damage in the brain, kidney, heart, lung, and liver due to endotoxin is increased by cannabis. Increased immunoreactivity of caspase-3 in the cytoplasm of the hepatocytes was observed after LPS and cannabis cotreatment compared with the LPS only group. Caspase-3 immunoreactivity markedly increased in degenerating neurons of the cortex following cannabis and LPS cotreatment. iNOS inmmunoreactivity increased after LPS and more intense iNOS expression was detected in hepatocytes after cannabis and LPS cotreatment. iNOS expression increased after cannabis and LPS treatment especially in the cerebral cortex. Thus, the administration of cannabis decreased tissue oxidative stress but increased organ damage after endotoxin injection in mice.     

Inhibition of Lipopolysaccharide-Induced Inflammatory and Oxidative Responses by Trans-cinnamaldehyde in C2C12 Myoblasts

Background: Trans-cinnamaldehyde (tCA), a bioactive component found in Cinnamomum cassia, has been reported to exhibit anti-inflammatory and antioxidant effects, but its efficacy in muscle cells has yet to be found. In this study, we investigated the inhibitory effect of tCA on inflammatory and oxidative stress induced by lipopolysaccharide (LPS) in C2C12 mouse skeletal myoblasts.Methods: To investigate the anti-inflammatory and antioxidant effects of tCA in LPS-treated C2C12 cells, we measured the levels of pro-inflammatory mediator, cytokines, and reactive oxygen species (ROS). To elucidate the mechanism underlying the effect of tCA, the expression of genes involved in the expression of inflammatory and oxidative regulators was also investigated. We further evaluated the anti-inflammatory and antioxidant efficacy of tCA against LPS in the zebrafish model.Results: tCA significantly inhibited the LPS-induced release of pro-inflammatory mediators and cytokines, which was associated with decreased expression of their regulatory genes. tCA also suppressed the expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor, and attenuated the nuclear translocation of nuclear factor-kappa B (NF-κB) and the binding of LPS to TLR4 on the cell surface in LPS-treated C2C12 cells. Furthermore, tCA abolished LPS-induced generation of ROS and expression levels of ROS producing enzymes, NADPH oxidase 1 (NOX1) and NOX2. However, tCA enhanced the activation of nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in LPS-stimulated C2C12 myoblasts. In addition, tCA showed strong protective effects against NO and ROS production in LPS-injected zebrafish larvae.Conclusions: Our findings suggest that tCA exerts its inhibitory ability against LPS-induced inflammatory and antioxidant stress in C2C12 myoblasts by targeting the TLR4/NF-κB, which might be mediated by the NOXs and Nrf2/HO-1 pathways.     

The Nitrone Spin Trap 5,5-Dimethyl-1-pyrroline N-oxide Affects Stress Response and Fate of Lipopolysaccharide-Primed RAW 264.7 Macrophage Cells

The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is commonly used to study free radicals. Due to its free radical trapping properties, DMPO is thought to reduce free radial-mediated oxidative damage and other related cellular responses. The purpose of this study was to assess the effect of DMPO on lipopolysaccharide (LPS)-induced inflammation, endoplasmic reticulum (ER) stress, and apoptosis in RAW 264.7 cells. The results showed that DMPO at 50 mM inhibited inducible nitric oxide synthase expression when added shortly after LPS treatment (≤3 h). Interestingly, DMPO increased anti-inflammatory heme oxygenase-1 (HO-1) expression and reversed LPS-induced decrease in HO-1 expression. LPS could increase cellular ER stress as indicated by C/EBP homologous protein (CHOP) induction; DMPO reduced LPS effect on CHOP expression. Unexpectedly, DMPO had a synergistic effect with LPS on increased caspase-3 activity. Overall, DMPO harbors multiple modulating effects but may induce apoptosis in LPS-stressed cells when given at 50 mM, an effective dose for its anti-inflammatory activity in vitro. Our data provide clues for further understanding of the nitrone spin trap with therapeutic potential.     

Effects of N-acetylcysteine on isolated mouse skeletal muscle: contractile properties,temperature dependence,and metabolism

The effects of the general antioxidant N-acetylcysteine (NAC) on muscle function and metabolism were examined. Isolated paired mouse extensor digitorum longus muscles were studied in the absence or presence of 20 mM NAC. Muscles were electrically stimulated to perform 100 isometric tetanic contractions (300 ms duration) at frequencies resulting in ～85 % of maximal force (70–150 Hz at 25–40 °C). NAC did not significantly affect peak force in the unfatigued state at any temperature but significantly slowed tetanic force development in a temperature-dependent fashion (e.g., time to 50 % of peak tension averaged 35?±?2 ms [control] and 37?±?1 ms [NAC] at 25 °C vs. 21?±?1 ms [control] and 52?±?6 ms [NAC, P?<?0.01] at 40 °C). During repeated contractions, NAC maximally enhanced peak force by the fifth tetanus at all temperatures (by ～30 %). Thereafter, the effect of NAC disappeared rapidly at high temperatures (35–40 °C) and more slowly at the lower temperatures (25–30 °C). At all temperatures, the enhancing effect of NAC on peak force was associated with a slowing of relaxation. NAC did not significantly affect myosin light chain phosphorylation at rest or after five contractions (～50 % increase vs. rest). After five tetani, lactate and inorganic phosphate increased about 20-fold and 2-fold, respectively, both in control and NAC-treated muscles. Interestingly, after five tetani, the increase in glucose 6-P was ～2-fold greater, whereas the increase in malate was inhibited by ～75 % with NAC vs. control, illustrating the metabolic effects of NAC. NAC slightly decreased the maximum shortening velocity in early fatigue (five to seven repeated tetani). These data demonstrate that the antioxidant NAC transiently enhances muscle force generation by a mechanism that is independent of changes in myosin light chain phosphorylation and inorganic phosphate. The slowing of relaxation suggests that NAC enhances isometric force by facilitating fusion (i.e., delaying force decline between pulses). The initial slowing of tension development and subsequent slowing of relaxation suggest that NAC would result in impaired performance during a high-intensity dynamic exercise.     

Involvement of P450s and nuclear receptors in the hepatoprotective effect of quercetin on liver injury by bacterial lipopolysaccharide

Abstract

Objective: Quercetin (Que), a flavonoid, possesses anti-inflammatory and antioxidant properties. It has been shown to protect against liver injury induced by various factors. This study was designed to investigate the underlying mechanism of its protective effect against lipopolysaccharide (LPS)- induced liver damage.

Methods: Mice were pretreated with Que for 7 consecutive days and then exposed to LPS. To study the hepatoprotective effect of Que, oxidative stress parameters, inflammatory cytokine levels in liver and serum liver function indexes were examined. Protein and mRNA expression of nuclear orphan receptors and cytochrome P450 enzymes were measured by Western Blotting and qPCR, respectively.

Results: Que significantly reduced circulating ALT, AST, ALP, and ameliorated LPS-induced histological alterations. In addition, Que obviously decreased markers of oxidative stress and pro-inflammatory cytokines. Furthermore, Que carried out the hepatoprotective effect via regulation of the expression of nuclear orphan receptors (CAR, PXR) and cytochrome P450 enzymes (CYP1A2, CYP2E1, CYP2D22, CYP3A11).

Conclusions: Our findings suggested that Que pretreatment could ameliorate LPS-induced liver injury.     

Propofol Protects Rats and Human Alveolar Epithelial Cells Against Lipopolysaccharide-Induced Acute Lung Injury via Inhibiting HMGB1 Expression

High-mobility group box 1 (HMGB1) plays a key role in the development of acute lung injury (ALI). Propofol, a general anesthetic with anti-inflammatory properties, has been suggested to be able to modulate lipopolysaccharide (LPS)-induced ALI. In this study, we investigated the effects of propofol on the expression of HMGB1 in a rat model of LPS-induced ALI. Rats underwent intraperitoneal injection of LPS to mimic sepsis-induced ALI. Propofol bolus (1, 5, or 10 mg/kg) was infused continuously 30 min after LPS administration, followed by infusion at 5 mg/(kg?·?h) through the left femoral vein cannula. LPS increased wet to dry weight ratio and myeloperoxidase activity in lung tissues and caused the elevation of total protein and cells, neutrophils, macrophages, and neutrophils in bronchoalveolar lavage fluid (BALF). Moreover, HMGB1 and other cytokine levels were increased in BALF and lung tissues and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. Propofol inhibited all the above effects. It also inhibited LPS-induced toll-like receptor (TLR)2/4 protein upexpression and NF-κB activation in lung tissues and human alveolar epithelial cells. Propofol protects rats and human alveolar epithelial cells against HMGB1 expression in a rat model of LPS-induced ALI. These effects may partially result from reductions in TLR2/4 and NF-κB activation.     

NF-κB在大鼠急性肺损伤模型肺组织中的表达及N-乙酰半胱氨酸的影响

目的 :检测NF κB在LPS诱导的急性肺损伤 (ALI)肺组织中的表达 ,以及N 乙酰半胱氨酸 (NAC)对ALI的抑制作用。方法 :采用免疫组化染色 (ABC法 )和Westernblot,检测NF κB在急性肺损伤大鼠气道和肺组织中的表达 ,以及NAC干预后活性NF κB表达的变化。结果 :正常对照组大鼠气道黏膜上皮和肺间质中 ,仅见少量散在的NF κB核阳性细胞 ;而LPS诱导ALI后 ,气道黏膜、肺间质、肺泡腔及血管内皮细胞中NF κB核阳性的细胞明显增多 (P <0 .0 1)。NF κB核阳性反应细胞主要为气道黏膜上皮细胞、浸润的炎症细胞、肺泡上皮细胞和血管内皮细胞。NAC治疗组NF κB核阳性细胞较LPS诱导的ALI组及对照组均明显减少 (P <0 .0 1)。Westernblot的结果显示 ,LPS诱导的ALI后不同时间点 ,NF κB的表达不同 ,于急性肺损伤 3h达高峰。各时间点NF κB的表达均较正常对照组高。结论 :LPS诱发的大鼠急性肺损伤的气道和肺组织内NF κB的表达增加 ,肺组织内的多数细胞参与了NF κB的激活。NAC可通过抑制NF κB的激活减轻急性肺损伤的炎症程度     

The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA)

Resin monomers like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation, or induce cell death via apoptosis. These phenomena are associated with oxidative stress and a reduction in the concentration of the antioxidant glutathione (GSH), resulting in imbalanced redox homeostasis. Thus far, the precise mechanism of how resin monomers interfere with cellular redox regulation is unknown. The present study provides insight into the induction of apoptosis and the differential expression of antioxidant enzymes depending on the availability of GSH. Buthionine sulfoximine (BSO) was used to inhibit GSH synthesis, while 2-oxothiazolidine-4-carboxylate (OTC), and N-acetylcysteine (NAC) as prodrugs supported GSH synthesis in RAW264.7 mouse macrophages exposed to HEMA (0-8 mm) for 24 h. The level of GSH was significantly decreased after cells were preincubated with BSO, and the formation of reactive oxygen species (ROS) increased in cultures subsequently exposed to HEMA. Apoptosis was drastically increased by BSO in HEMA-exposed cell cultures as well, but OTC and NAC retracted HEMA-induced cell death. These results show that dental monomer-induced apoptosis is causally related to the availability of GSH. The hydrogen peroxide decomposing enzymes glutathione peroxidase (GPx1/2) and catalase were differentially regulated in HEMA-exposed cultures. Expression of GPx1/2 was inhibited by HEMA and further reduced in the presence of BSO. SOD1 (superoxide dismutase) expression was inhibited in the presence of HEMA, and was decreased to an even greater extent by BSO, possibly due to H(2)O(2)-feedback inhibition. The expression of catalase was considerably up-regulated in HEMA-exposed cultures, implying that H(2)O(2) is the type of ROS that is significantly increased in monomer-exposed cells. OTC and NAC counteracted the effect of HEMA on GPx1/2, SOD1, and catalase expression. HO-1 (heme oxygenase) expression was strongly enhanced by HEMA, suggesting the need for further antioxidants like bilirubin to support enzyme activities that directly regulate H(2)O(2) equilibrium. Expression of the oxidoreductase thioredoxin (TRX1), the second major thiol-dependent antioxidant system in eukaryotic cells, was slightly reduced, while the oxygen-sensing protein HIF-1α was downregulated in HEMA-exposed cell cultures. These results indicate that cells and tissues actively respond to monomer-induced oxidative stress by the differential expression of enzymatic antioxidants.