On the 3rd, 7th, 14th, and 21st day after PQ administration, ten rats were selected from the PQ, bosentan therapy and control groups. These rats were anesthetized with an intraperitoneal injection of 4% chloral hydrate and immediately sacrificed. After a blunt dissection of each inferior vena cava, approximately 4 ml of blood was collected and placed in a disposable plastic tube. The blood samples were centrifuged at 5000 r/min for 5 minutes, and the serum was stored at 270uC to detect ET-1 and TGF-b1. To distinguish the rats’ left and right lungs, each left lung was placed in a frozen pipette and stored in a 270uC liquid nitrogen freezer to detect the ET-1,TGF-b1 and HYP. The lower right lung was immersed in 10% buffered formaldehyde and then embedded in paraffin. The lung lobes were subsequently separated and sectioned sagittally into 5- m m-thick sections, then stained with hematoxylin, eosin (HE) and Masson’s trichrome. The sections Table 1. Dynamic change of ET-1 in the serum from the three
The left lung harvested from each rat was infused with 5 mL PBS at room temperature, which was withdrawn, and reinfused two more times. There were no differences in the volume of saline recovered (4.2 ± 0.4 mL fluid) after the lung lavage process between the three experi- mental groups. Bronchoalveolar lavage fluid (BALF) was centrifuged at 1200 g for 10 min at 4 6 C. The supernatant was separated into aliquots and frozen at -30 6 C for batch analysis via enzyme-linked immunosorbent assay (ELISA). The levels of tumor necrosis factor (TNF)-a and interleukin (IL)-1b were later determined using rat TNF-a and IL-1b ELISA kits (both from R&D Systems Inc., USA) according to manufacturer instructions. Samples taken from each animal (8 per group) were analyzed in triplicate for TNF-a and IL-1b.
using a ready -to-use two-step (non-biotin) test kit. Rabbit anti-ratprimary antibodies, goat anti-rabbit secondary antibodies, and DAB chromogenic agent were purchased from Beijing ZSGB-Bio Origene Co., Ltd. A Leica DM1000 microscope (LeicaDFCT6.5.0 Switzerland Ltd.) was used to obtain images, which were analyzed for the integral optical density of HO-1 protein using the image-Pro Plus 6.0 optical analysis system. Each tissue section was sampled at five different fields and the average value was calculated.
Lider-7-tang, a medicine used for the treatment of respiratory diseases especially pneumonia and fever in Mongolian Traditional Medicine, was selected for this phytochemical and pharmacological study. The objectives of the study were to determine total biological active substances and analyze the effects of Lider-7-tang treatment inrats with acutelunginjury (ALI). Quantitative determination of the total active constituents (phenolic, ﬂavonoid, iridoid and alkaloid) of the methanol extract of Lider-7-tang was performed using Folin-Ciocalteu reagent, aluminum chloride reagent, Trim-Hill reagent, and Bromocresol green reagent, respectively. A total of ﬁfty 8–10-week-old male Wistar rats (200–240 g) were randomized into three groups: control group, lipopolysaccharide (LPS) group (7.5 mg/kg) and LPS+Lider-7 group (90 mg/kg Lider-7-tang before LPS administration). The total content of alkaloids was 0.2±0.043%, total phenols 7.8±0.67%, ﬂavonoids 3.12±0.206%, and iridoids 0.308±0.0095%. This study also evaluated the effects of Lider-7 on levels of inﬂammatory mediators by observing histopathological features associated with LPS-induced ALI. The rats pretreated with Lider-7 had signiﬁcantly lower levels of IL-6 (at 3 and 6 h), and TNF-a (at 3, 6, 9, and 12 h). The current study showed that Lider-7 exerted a preventive effect against LPS-induced ALI, which appeared to be mediated by inhibiting the release of pro-inﬂammatory cytokines.
Detection of Myeloperoxidase Activity inLung Tissue As an index for neutrophil sequestration inlung tissue, the activity of myeloperoxidase (MPO) was measured with an MPO assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) ac- cording to the manufacturer’s instructions. Briefly, frozen lung tissues were thawed and homogenized (1:10, w/v) in normal saline. The homogenate was diluted (1:1) with solution B, and then the samples were assayed spectrophotometrically for MPO activity following the recommendations of the manufacturer. MPO catalyzes the redox reaction of peroxide and 3,3 9,5,59-tetramethylbenzidine and produces yellow compounds . The absorbance at 460 nm was determined using a 721 spectrophotometer (Shanghai, China). One unit of MPO is defined as the amount of MPO capable of degrading one micromole of peroxide per gram of wet lung tissue at 37 uC. The results are expressed as MPO units per gram of wet lung tissue.
Lipopolysaccharide (LPS)-induced endotoxemia triggers the secretion of proinﬂammatory cytokines and can cause acutelunginjury (ALI). The high mobility group box 1 (HMGB1) protein plays an important role as a late mediator of sepsis and ALI. Galantamine (GAL) is a central acetylcholinesterase inhibitor that inhibits the expression of HMGB1. This study evaluated the effects of GAL by measuring levels of inﬂammatory mediators and observing histopathological features associated with LPS- induced ALI. Sixty 8–10 week old male Sprague-Dawley rats (200–240 g) were randomized into three groups as follows: control group, LPS group (7.5 mg/kg LPS), and LPS+GAL group (5 mg/kg GAL before LPS administration). Histopathological examination of lung specimens obtained 12 h after LPS administration was performed to analyze changes in wet-to-dry (W/D) weight ratio, myeloperoxidase (MPO) activity, and HMGB1 expression level. Additionally, plasma concentrations of tumor necrosis factor-a, interleukin-6, and HMGB1 were measured using an enzyme-linked immunosorbent assay at 0 (baseline), 3, 6, 9, and 12 h after LPS administration. Mortality in the three groups was recorded at 72 h. LPS-induced ALI was characterized by distortion of pulmonary architecture and elevation of MPO activity, W/D weight ratio, and levels of pro-inﬂammatory cytokines, including tumor necrosis factor-a, interleukin-6, and HMGB1. Pretreatment with GAL signiﬁcantly reduced the LPS-inducedlung pathological changes, W/D weight ratio, levels of pro-inﬂammatory cytokines and MPO activity (ANOVA). Moreover, GAL treatment signiﬁcantly decreased the mortality rate (ANOVA). In conclusion, we demonstrated that GAL exerted a protective effect on LPS-induced ALI inrats.
The aim of this study was to investigate the effect of propofol pretreatment on lipopolysaccharide (LPS)-inducedacutelunginjury (ALI) and the role of the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway in this procedure. Survival was determined 48 h after LPS injection. At 1 h after LPS challenge, the lung wet- to dry-weight ratio was examined, and concentrations of protein, tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) were determined using the bicinchoninic acid method or ELISA. Lunginjury was assayed via lung histological examination. PI3K and p-Akt expression levels in the lung tissue were determined by Western blotting. Propofol pretreatment prolonged survival, decreased the concentrations of protein, TNF-a, and IL-6 in BALF, attenuated ALI, and increased PI3K and p-Akt expression in the lung tissue of LPS-challenged rats, whereas treatment with wortmannin, a PI3K/Akt pathway specific inhibitor, blunted this effect. Our study indicates that propofol pretreatment attenuated LPS-induced ALI, partly by activation of the PI3K/Akt pathway.
The results of the present study indicate that acuteparaquat intoxication inrats causes pulmonary lesions mainly due to surfactant dysfunction, sparing lung tissue, as shown in the PV curves obtained for air- and saline- filled lungs (Figures 2 and 3). Other models of acutelunginjury have been described and have confirmed the effectiveness of the PV curve to study pulmonary mechanics (13,14). Both tension work and hysteresis loop were greater in the paraquat group, and the extent of alteration increased as larger doses were administered (Figure 4). In the inspiratory limb, pressures were much higher in the paraquatlung since the degree of collapse due to surfactant dysfunction was intense. Hysteresis was almost eliminated in the sa- line-filled lungin both groups, since it was mainly due to an alveolar surface film. Alter- ations of work due to surface active proper- ties were observed both during inspiration and expiration. However, inspiratory work due to tension was different from control starting at the lowest dose of paraquat. Expi- ratory work due to tension changed signifi- cantly only at the highest dose of paraquat (Figure 5). These results suggest that in- spiratory tension work is a more sensitive estimator of alveolar instability inparaquatpoisoning.
the term recommendation was used when the level of evidence was high, i.e., derived from randomized studies conducted with more than 100 participants, meta-analyses, all-or-nothing effect, or patient safety. The term suggestion was used when the available evidence was weak, i.e., based on observational or case-control studies, case series, or on the experience of specialists to provide guidance for efficient and safe ventilatory support in Brazil. We therefore hoped that these evidence-based recommendations would help to avoid potential deleterious effects associated with inadequate ventilatory support in our patients. The 58 participating specialists were requested to answer the proposed questions during an eight-hour session conducted at the Brazilian Intensive Care Medicine Association (Associação de Medicina Intensiva Brasileira - AMIB) on August 3, 2013. The answers were formulated based on the evidence available in the literature and on the experience of the specialists and were then presented at a plenary session that included all 58 participating specialists, which was held on August 4, 2013 at AMIB headquarters. During that session, the answers were discussed, modified when needed, voted on, and approved in accordance with the suggestions and observations of the specialists who attended the meeting.
Left lung tissues were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), embedded in paraffin, and sectioned (5-μm thickness) for routine hematoxylin and eosin (H&E) and Sirius red staining, the latter highlighting histopathologic changes such as fibrosis. Degree of lung fibrosis was scored on a scale from 0 to 5, which was modified from a previous report . Grade 0 represents normal lung. Grade 1 to 5 means minimal fibrosis with thickening of alveolar wall to totally obliterative fibrosis. Histologic derangements were also scored (1, minimal; 2, moderate; 3, extensive), as previously described . Macrophage influx and parenchymal cell changes including collapse and consolidation, alveolar epithelial cell changes, and bronchial epithelial cell alterations were graded. Regarding macrophage influx, 1 denotes mild influx; 2 denotes moderate influx; 3 denotes extensive influx. Regarding parenchymal changes, 1 denotes less than 10% of lung parenchymal changes; 2 denotes 10– 50% of lung parenchymal changes; 3 denotes more than 50% of lung parenchymal changes. All scores were assigned by one expert pathologist and one clinician blinded to the study design and outcomes. To quantify the Sirius red stained collagen in an image of lung sections (n = 2–6 mice per group), we used the Image J (NIH image program, version 1.44) software. The per- centage of fibrosis was assessed by determining the total tissue areas occupied by pneumocytes and collagen and excluding empty spaces, as wells as excluding annotations of perivascular fibrosis and staining artefacts.
derstanding of pathophysiological mechanisms, parti- cularly in sepsis/septic shock and ALI/ARDS. An im- portant issue in ALI and ARDS is why some patients die from uncontrolled inlammation or sepsis, while others recover without major issues. his could be ex- plained, at least partially, by that cellular events invol- ved in inlammation mediation, tissue injury and re- pair, are controlled at a molecular level, and may not be completely explained without considering the genes and their products participating in this response. (9) In
Cissampelos sympodialis Eichl. (Menispermaceae) is an endemic species in Brazil, popularly known as "Milona", "Jarrinha" or "Orelha-de-onça". This species was selected for this study considering the chemotaxonomic (alkaloids and flavonoids) and ethnopharmacological criteria, due to this species be popularly used totreat inflammatory disorders. This study aimed to evaluate the acute preclinical toxicity and anti-ulcer activity of the ethanolic extract (EtOHE-Cs) and alkaloids total fraction (TAF-Cs) obtained from aerial parts of C. sympodialis. Inacute toxicity, doses of 300 and 2000 mg/kg EtOHE-Cs were administered orally (p.o.). Our experiment conditions shows that, this type of doses did not induce signs of toxicity in female mice and 50% of lethal dose (LD50) is equal to or greater 5000 mg/kg according to the OECD 423 guide. For TAF-Cs was founded that the animals treated with the dose of 2000 mg/kg showed straub tail and analgesia. In addition, the LD50 of this substance being approximately 1000 mg/kg. To evaluate the gastroprotective activity usinginducedacute models of gastric ulcers were used: ethanol and containment of gastric juice inrats, anti-inflammatory non- steroidal drug (NSAID - piroxicam) and stress (immobilization and cold) in mice. In ulcer model induced by ethanol the results showed that carbenoxolone (100 mg/kg), EtOHE-Cs or TAF-Cs (62.5; 125; 250 and 500 mg/kg – v.o.) reduced the ulcerative lesion area (ULA) of 97, 69, 75, 94, 98; 97, 88, 90, 94, 95% (p<0,001) respectively in comparison with the negative control. In the same model, treatment with carbenoxolone (100 mg/kg), EtOHE-Cs (500 mg/kg) or TAF-Cs (250 mg/kg) improved histological parameters analyzed. Considering ulcers induced by NSAIDs, cimetidine (100 mg/kg), the EtOHE-Cs or TAF-Cs (62.5; 125; 250 and 500 mg/kg
Inducible nitric oxide synthase (iNOS) and nitric oxide (NO), produced by iNOS, are elevated in many inflammatory diseases of the respiratory tract. Although NO is also involved in antimicrobial, anti-inflammatory and antioxidant effects, overproduction of NO may be harmful in conditions such as sepsis and infection . Excessive NO production may also enhance the generation of reactive nitrogen species (RNS), including peroxynitrite (ONOO-) and nitrogen dioxide (NO 2 ), and the formation of such RNS is thought to be the mechanism underlying the role of NO in the etiology of inflammatory lung disease . One of the potential mechanisms by which leptin pretreatment abolished LPS-inducedlung inflammation might be a decrease in iNOS expression, given that the inducible isoform of NOS is responsible for the excess production of NO in LPS-induced sepsis in animals and NO also increases lung chemokine expression, facilitating cell influx into the airways [37, 38].
38. Jauncey-Cooke JI, Bogossian F, East CE. Lung recruitment -- a guide for clinicians. Aust Crit Care. 2009;22(4):155-62. 39. Meade MO, Cook DJ, Guyatt GH, Slutsky AS, Arabi YM, Cooper DJ, Davies AR, Hand LE, Zhou Q, habane L, Austin P, Lapinsky S, Baxter A, Russell J, Skrobik Y, Ronco JJ, Stewart TE; Lung Open Ventilation Study Investigators. Ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acutelunginjury and acute respiratory distress syndrome: a randomized controlled trial. JAMA. 2008;299(6):637-45. 40. Brower RG, Morris A, MacIntyre N, Matthay MA, Hay-
Experimental studies have shown that, in the case of RILI, the immediate release of proinflammatory cytokines such as TNF-a, IL-1b, and IL-6 after IR is closely related tolung toxicity (24). In our study, OOP reduced the release of radiation-induced TNF-a and IL-1b. On the other hand, studies have shown that inhalation of ozone induces airway hyperactivity and interacts with airway epithelium and alveolar macrophages to produce inflam- mation via increasing inflammatory cytokines, including TNF-a and IL-1b (25,26). However, Bette et al. (27) evaluated OOP with ip administration of ozone, followed by a tazobactam/piperacillin regimen inrats submitted to peritonitis, and found an increase in survival rates and a decrease in proinflammatory cytokines, TNF-a and IL-1b. Similarly, Zamora et al. (28) observed a significant inhibitory effect of serum TNF-a release on mice pretreated with an ozone/oxygen mixture by the ip route before induction of endotoxic shock by lipopolysaccharides. These results suggest that ip injections of a toxic dose of an ozone/oxygen mixture might modify the production and/or release of TNF-a and IL-1b. The inhibitory effects of OOP on TNF-a and IL-1b levels in the serum of irradiated rats might be a consequence of stimulation of antioxidant defenses induced by ozone therapy.
Recent changes were introduced inacute hypoxemic respiratory failure children ventilation methods. There are evidences that less aggressive ven- tilation strategies can improve severe pulmonary injury patients’ survival. Experimental trials evidenced a rela- tionship between inappropriate ven- tilatory measures and delayed acute pulmonary injury improvement, or even worsening. From this, a protec- tive ventilatory measure arises in com- bination with alveolar recruitment maneuver. This association is believed in clinical practice to determine im- portantly reduced morbidity and mortality as well as reduced mechanic ventilation-induced injuries. It is in- dicated for acutelunginjury patients, generally from pneumonia or sep- sis, with severe hypoxemia. Its main contraindications are homodynamic instability, pneumothorax and intra- cranial hypertension. Experimental trials showed beneficial maneuver effects on both oxygenation and al-
Triterpenes are natural compounds with anti-inflammatory and cytoprotective effects relatively non-toxic. Acute pancreatitis, an inflammation of the pancreas, may lead to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), conditions that can lead the patient to death. In this study, a mixture of triterpenes isolated from Protium hepthaphyllum was investigated for their effects in models of acute pancreatitis. In the model of acute pancreatitis induced by L-arginine, male Wistar rats were treated with a mixture of α,β-amyrin (10, 30 and 100 mg/kg, p.o.) or with vehicle (2% Tween 80 in distilled water, 10 ml/kg) 48, 24 and 1.5 h before the administration of L-arginine (2 x 2.5 g/kg, 1 h apart) or with metilprednisolone (30 mg/kg, i.m.) 30 min before the administration of L-arginine. In cerulein-induced pancreatitis, male Swiss mice were treated with a mixture of α,β- amyrin (10, 30 and 100 mg/kg, p.o.) or with vehicle (2% Tween 80 in distilled water, 10 ml/kg) 48, 24 and 1.5 h before administration of cerulein (5 x 50 mg/kg, 1 h apart) or with thalidomide (200 mg/kg, p.o.) 1h before administration of cerulein. Animals treated with saline (0.9% NaCl) were included in both models. We analyzed the pancreatic edema, serum amylase, lipase and cytokines (TNF-α, IL-6), myeloperoxidase, reactive substances to thiobarbituric acid (TBARS), histology and immunohistochemistry (TNF-α, iNOS and nitrotyrosine) pancreatic. L-arginine and cerulein significantly increased pancreatic edema and serum levels of amylase, lipase, TNF-α and IL-6. Histopathologic evaluation of pancreas revealed the presence of edema, neutrophil infiltration, hemorrhage, acinar vacuolization and necrosis. We observed a marked increase in the expression of TNF-α, iNOS and nitrotyrosine in the evaluation by immunohistochemistry. The pre-treatment with alpha and beta-amyrin (10, 30 and 100 mg/kg, p.o.), metilprednisolone (30 mg/kg, i.m.) or thalidomide (200 mg/kg, p.o.) significantly attenuated the severity of acute pancreatitis induced by either L-arginine, and by cerulein, as evidenced by the reduction of pancreatic edema, amylase, lipase and serum cytokines, myeloperoxidase and pancreatic TBARS. Furthermore, treatment with α,β-amyrin and the reference drugs suppressed the histopathological changes and expression of cytokines and nitrotyrosine pancreatic. Together, these results indicate that the mixture of α,β-amyrin reduces the severity of acute pancreatitis induced by L-arginine or cerulein acting as anti-inflammatory and antioxidant agent.
But why is VEGF responsible for the onset of malaria- associated ALI? VEGF has long been known for its activity as a regulator of vessel permeability . In fact VEGF was primarily termed vascular permeability factor, for its ability to induce vascular leakage, rather than for its growth factor activity . VEGF increases vascular permeability 50,000 times more efficiently than does histamine . Interestingly, VEGF also plays a central role in the formation and maintenance of lung vasculature . However, when VEGF levels are altered, lung disease frequently follows. Plasma VEGF levels in subjects with non-malaria ALI/ARDS are strongly elevated compared to controls and values higher than two-fold have been associated with mortality . The association between VEGF levels and mortality due to respiratory failure does not mean that VEGF effects are restricted to the lung, but simply highlights the importance of vascular integrity for lung function. Another example in which VEGF and lunginjury are involved in response to a pathogenic microorganism has recently been reported . Pseudomonas aeruginosa is a pathogenic bacterium that colonizes the lungs and may lead tolung disease in immunocompromized patients. Interestingly, while aerosol delivery of this bacterium causes fatal disease in DBA/2 mice, other mouse strains are able to resolve infection. DBA/2 mice display progressive deteriora- tion of lung pathology with extensive alveolar exsudate and edema formation together with significantly increase levels of VEGF that seem to result from an uncontrolled host inflamma- tory response . Indeed, a cross-talk between angiogenesis and inflammation has long been proposed . Similarly, P. berghei ANKA-infected DBA/2 mice treated with a potent anti- inflammatory molecule prior to the onset of ALI show significantly reduced levels of VEGF in sera and are fully protected from this syndrome of severe malaria.
RT-PCR quantitation of TNFα, PCI, PCIII, and NF-κB gene expression Total RNA was extracted from liver tissue with TRIzol reagent (Invitrogen Co. USA) and the SV Total RNA Isolation System (Promega, Madison, WI). First-strand cDNA was synthesized from 1 μg of total RNA with the ImProm-IITM Reverse Transcriptase System for RT-PCR (Promega) according to the manufacturer’s protocol. Real-time quantitative PCR analyses of TNF-α, PCI, PCIII, and GAPDH mRNAs were performed with SYBR Green Mix in the Applied Biosystems1 7500 FAST system (Applied Biosystems, Foster City, CA), according to a standard protocol with the following primers: GAPDH (forward: 5’ AAC TTT GGC ATC GTG GAA GG 3’; reverse: 5’ GTG GAT GCA GGG ATG ATG TTC 3’, annealing primer tem- perature: 60°C), TNFα (forward: 5’ AGT CCG GGC AGG TCT ACT TT3’; reverse: 5’ TTC AGC GTC TCG TGT GTT TC 3’, annealing primer temperature: 56.5°C), PCI (forward:5’ CAG GGA GTA AGG GAC ACG AA 3’; reverse: 5’TCC CAC AGC AGT TAG GAA CC 3’, annealing primer temperature: 56.8°C), PCIII (forward: 5’ ATG GTG GCT TTC AGT TCA GC 3’; reverse: 5’ TGG GGT TTC AGA GAG TTT GG 3’, annealing primer temperature: 55.2°C), and NF-κB (forward: 5’ TCT GCT TCC AGG TGA CAG TG 3; reverse: 5’ ATC TTG AGC TCG GCA GTG TT 3’, annealing primer temperature: 55.2°C. The standard PCR condi- tions were as follow: 50°C for 2 min and 95°C for 10 min, followed by forty 30-s cycles at 94°C, a variable annealing primer temperature for 30 s, and 72°C for 1 min. The experiments were performed in triplicate and repeated at least three times. Mean Ct values were used to calculate the relative expression levels of the target genes for the experimental groups, relative to those in the negative control group; expression data were normalized relative to the housekeeping gene GAPDH using the 2–ΔΔCt formula.
Groups 2 and 3 revealed no alterations of all measured parameters. Groups 4, 5 and 6 showed a significant increase in serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and total bilirubin, together with a significant decrease in the serum concentrations of total protein and albumin, when compared with the control group. In addition, a significant decrease in the albumin/globulin ratio was reported in the group to which both ethanol and thermally treated oil had been administered (Table I). Groups 7, 8 and 9 which received curcumin in addition to liver oxidants, showed significantly less alterations in the various analytes when compared with the corresponding groups that had not been treated with curcumin.