Mouse Model Of Lipopolysaccharide Research Articles

The tyrosine kinase inhibitor imatinib prevents lung injury and death after intravenous LPS in mice.

Severe sepsis and septic shock are frequent causes of the acute respiratory distress syndrome, and important sources of human mortality. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, plays a major role in the pathogenesis of severe sepsis and septic shock. LPS exposure induces the production of harmful reactive oxygen species, and the resultant oxidant injury has been implicated in the pathogenesis of both severe sepsis and ARDS. We previously showed that the tyrosine kinase inhibitor imatinib increases lung endothelial antioxidant enzymes and protects against pulmonary endothelial antioxidant injury. In the present study, we tested the hypothesis that imatinib would protect against lung injury and systemic inflammation caused by intravenous LPS in an intact mouse model of endotoxemia mimicking early sepsis. We found that intravenous LPS induced a significant increase in the activity of lung xanthine oxidoreductase (XOR), an enzyme which is a major source of reactive oxygen species and implicated in the pathogenesis of acute lung injury. Imatinib had no effect of LPS-induced XOR activity. However, pretreatment of mice with imatinib increased lung catalase activity and decreased intravenous LPS-induced lung oxidant injury as measured by γ-H2AX, a marker of oxidant-induced DNA damage, lung apoptosis, and pulmonary edema. Imatinib also attenuated systemic cytokine expression after intravenous LPS exposure. Finally, imatinib completely prevented mortality in an in vivo, intravenous LPS mouse model of endotoxemia and lung injury. These results support the testing of imatinib as a novel pharmacologic agent in the treatment of Gram-negative sepsis and sepsis-induced ARDS.

Selective inhibition of TNFR1 reduces osteoclast numbers and is differentiated from anti-TNF in a LPS-driven model of inflammatory bone loss

The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening.

TIIA attenuates LPS-induced mouse endometritis by suppressing the NF-κB signaling pathway.

Endometritis is one of the main diseases that harms the dairy cow industry. Tanshinone IIA (TIIA), a fat-soluble alkaloid isolated from Salviae miltiorrhizae, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effects of TIIA on a mouse model of lipopolysaccharide (LPS)-induced endometritis remain to be elucidated. The purpose of the present study was to investigate the effects of TIIA on LPS-induced mouse endometritis. TIIA was intraperitoneally injected 1 h before and 12 h after perfusion of LPS into the uterus. A histological examination was then performed, and the concentrations of myeloperoxidase (MPO) and nitric oxide (NO) in the uterine tissue were determined. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in a homogenate of the uterus were detected by enzyme-linked immunosorbent assay. The extent of phosphorylation of IκBα and p65 was detected by Western blotting. TIIA markedly reduced the infiltration of neutrophils, suppressed MPO activity and the concentration of NO, and attenuated the expression of TNF-α and IL-1β. Furthermore, TIIA inhibited the phosphorylation of the nuclear factor-kappa B (NF-κB) p65 subunit and the degradation of its inhibitor IκBα. All the results suggest that TIIA has strong anti-inflammatory effects on LPS-induced mouse endometritis.

Cordyceps militaris mushroom and cordycepin inhibit RANKL-induced osteoclast differentiation.

Cordyceps militaris is a medicinal mushroom and its bioactive compound, cordycepin, is reported to have many pharmacological activities. The goal of this study was to investigate the effects of C. militaris extract (CME) and cordycepin on osteoclast differentiation in vitro and on an inflammatory bone loss in vivo. In RAW 264.7 cells, CME and cordycepin showed dose-dependent inhibition of receptor activator of the nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation by TRAP (tartrate-resistant acid phosphatase) staining. Moreover, the mRNA expression of osteoclastogenesis-related genes (TRAP, cathepsin K, MMP-9, and NFATc1) was also inhibited by CME and cordycepin. Also, cordycepin significantly inhibited RANKL-induced phosphorylation of p38 and NF-κB, but not that of other members of mitogen-activated protein kinase families. To examine the effect of CME on bone loss in vivo, we used a mouse model of lipopolysaccharide (LPS)-mediated bone loss. Micro-CT analysis of the femurs showed that LPS treatment caused bone loss. However, bone loss was significantly attenuated in mice treated with CME. These results suggest that cordycepin or/and CME have inhibitory effects on osteoclast differentiation in vitro and that they suppress inflammatory bone loss in vivo.

Flavonoids from the aerial parts of Houttuynia cordata attenuate lung inflammation in mice.

The aerial parts of Houttuynia cordata used for treating inflammation-related disorders contain flavonoids as major constituents. Since certain flavonoids possess anti-inflammatory activity, especially in the lung, the pharmacological activities of H. cordata and the flavonoid constituents were evaluated using in vitro and in vivo models of lung inflammation. The 70 % ethanol extract of the aerial parts of H. cordata inhibited the production of inflammatory biomarkers IL-6 and NO in lung epithelial cells (A549) and alveolar macrophages (MH-S), respectively. And the same plant material, administered orally (100 and 400 mg/kg), significantly inhibited lung inflammatory response in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury. From the extract, major flavonoids including afzelin, hyperoside and quercitrin were successfully isolated and they also attenuated LPS-induced lung inflammation in mice by oral administration. In particular, quercitrin showed most potent activity at 100 mg/kg. These results demonstrate for the first time that H. cordata and three flavonoid constituents have a therapeutic potential for treating lung inflammatory disorders.

Rubicon deficiency enhances cardiac autophagy and protects mice from lipopolysaccharide-induced lethality and reduction in stroke volume.

: Rubicon has been suggested to suppress autophagosome maturation by negatively regulating PI3KC3/Vps34 activity. However, the physiological function of Rubicon remains elusive. We hypothesized that Rubicon deficiency enhances autophagic flux in the heart and affects cardiac function. Rubicon knockout (KO) mice were generated by piggyBac transposition. Loss of Rubicon was demonstrated at both mRNA and protein levels. Rubicon KO mice were born in Mendelian ratios. Autophagic flux, assessed by bafilomycin A1-induced changes in LC3 II protein abundance, was enhanced in the heart of Rubicon KO mice compared with wild-type (WT) controls. Hematoxylin-eosin staining and picrosirius red staining showed that Rubicon KO mice exhibited normal baseline cardiac morphology. Echocardiography revealed that ejection fraction and fractional shortening, 2 indices of cardiac function, were comparable between Rubicon KO mice at 2, 8, and 12 months of age (n = 6-8 for each age group) and the corresponding WT controls (n = 6-8 for each age group). In a mouse model of lipopolysaccharide (LPS)-induced sepsis, the survival time of LPS-treated Rubicon KO mice (n = 10) was prolonged compared with LPS-treated WT controls (n = 11). Echocardiography revealed that Rubicon deficiency partially normalized LPS-induced reduction in stroke volume and cardiac output 12 hours after LPS administration compared with LPS-treated WT controls (n = 6 for each group). Autophagic flux was enhanced in Rubicon-deficient hearts 12 hours after LPS treatment compared with LPS-treated WT controls. Real-time quantitative polymerase chain reaction suggested that proinflammatory cytokine expression was not significantly different between LPS-treated Rubicon KO mice and WT controls (n = 3 for each group). Our data demonstrate for the first time that Rubicon deficiency enhances autophagic flux in the heart and protects mice from lethality and reduction in stroke volume induced by LPS.

Deferoxamine attenuates lipopolysaccharide-induced neuroinflammation and memory impairment in mice.

BackgroundNeuroinflammation often results in enduring cognitive impairment and is a risk factor for postoperative cognitive dysfunction. There are currently no effective treatments for infection-induced cognitive impairment. Previous studies have shown that the iron chelator deferoxamine (DFO) can increase the resistance of neurons to injury and disease by stimulating adaptive cellular stress responses. However, the impact of DFO on the cognitive sequelae of neuroinflammation is unknown.MethodsA mouse model of lipopolysaccharide (LPS)-induced cognitive impairment was established to evaluate the neuroprotective effects of DFO against LPS-induced memory deficits and neuroinflammation. Adult C57BL/6 mice were treated with 0.5 μg of DFO 3 days prior to intracerebroventricular microinjection of 2 μg of LPS. Cognitive function was assessed using a Morris water maze from post-injection days 1 to 3. Animal behavioral tests, as well as pathological and biochemical assays were performed to evaluate the LPS-induced hippocampal damage and the neuroprotective effect of DFO.ResultsTreatment of mice with LPS resulted in deficits in cognitive performance in the Morris water maze without changing locomotor activity, which were ameliorated by pretreatment with DFO. DFO prevented LPS-induced microglial activation and elevations of IL-1β and TNF-α levels in the hippocampus. Moreover, DFO attenuated elevated expression of caspase-3, modulated GSK3β activity, and prevented LPS-induced increases of MDA and SOD levels in the hippocampus. DFO also significantly blocked LPS-induced iron accumulation and altered expression of proteins related to iron metabolism in the hippocampus.ConclusionsOur results suggest that DFO may possess a neuroprotective effect against LPS-induced neuroinflammation and cognitive deficits via mechanisms involving maintenance of less brain iron, prevention of neuroinflammation, and alleviation of oxidative stress and apoptosis.

Calcium-binding protein, spermatid-specific 1 is expressed in human salivary glands and contains an anti-inflammatory motif.

Salivary glands are involved in the production and exocrine and endocrine secretion of biologically active proteins, polypeptides, and hormones involved in growth and differentiation, homeostasis, and digestion. We have previously studied the prohormone submandibular rat 1 (SMR1), product of the Vcsa1 gene, which is highly expressed in the testes and salivary glands of rats, and can be cleaved to produce polypeptides with analgesic, erectile function, and anti-inflammatory activities. Humans lack the Vcsa1 gene, but homologous sequences and functions for analgesia and erectile function exist in the human genes Prol1, SMR3a, and SMR3b located on the human chromosomal region close to where Vcsa1 lies in the rat. Here we show the human protein calcium-binding protein spermatid-specific 1 (CABS1) contains a similar sequence to the anti-inflammatory sequence in rat SMR1, thus CABS1 may be another human gene with homologous function to Vcsa1. Using Western blot and PCR, we discovered that the human protein CABS1, previously thought to only be expressed in the testes, is also expressed in the salivary glands and lung, in a tissue-specific manner. Peptides derived from CABS1 were tested in an in vivo mouse model of lipopolysaccharide (LPS)-induced neutrophilia and an ex vivo rat model of antigen-induced intestinal anaphylaxis and significantly reduced both neutrophil accumulation in bronchoalveolar lavage fluid and antigen-induced ileal contractions, respectively. Thus human CABS1 has a peptide motif homologous to the anti-inflammatory peptide sequence of rat SMR1. Whether this similarity of CABS1 extends to the neuroendocrine regulation of the anti-inflammatory activity seen for SMR1 remains to be determined.

Methylene Blue Improves Brain Mitochondrial ABAD Functions and Decreases Aβ in a Neuroinflammatory Alzheimer's Disease Mouse Model.

Methylene blue (MB) phase II clinical trials reported improvements in cognitive functions of Alzheimer's disease (AD) patients. Regarding MB mechanism of action, its antioxidant and mitochondrial protective effects have been previously described. In relation to AD, it has been recently reported that MB reduced amyloid beta (Aβ) levels in AD models. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) has been shown to bind Aβ inducing mitochondrial dysfunction, providing a direct relation between Aβ toxicity and mitochondrial dysfunction occurring in AD. Since it has been reported that inhibiting ABAD protects mitochondrial functions and prevents Aβ-induced toxicity, the aim of the current study was to investigate if the protective effects of MB could be associated with an effect on ABAD levels and functions. The current study shows that MB is able to enhance cell viability, reduce both reactive oxygen species levels and importantly Aβ oligomers in a lipopolysaccharide (LPS) mouse model. Interestingly, ABAD levels were increased in the brains of the LPS mouse model and MB treatment was able to reduce its levels. Given that regulation of the estradiol level is a well-established function of ABAD, brain estradiol level was compared in LPS mouse model and in MB-treated mice. The results of the current study show that MB treatment is able to improve significantly the LPS-induced decrease of estradiol levels in mice brains, indicating its ability to modulate both levels and function of ABAD. These results give a new insight to possible mechanisms of MB in AD.

The Role of Autophagy in Kidney Inflammatory Injury via the NF-κB Route Induced by LPS.

Acute kidney injury (AKI) is a systemic inflammatory response syndrome associated with poor clinical outcomes. No treatments effective for AKI are currently available. Thus, there is an urgent need of development of treatments effective for AKI. Autophagy, an intracellular proteolytic system, is induced in renal cells during AKI. However, whether autophagy is protective or injurious for AKI needs to be clearly clarified. We addressed this question by pharmacological inhibition of autophagy using a mouse model of lipopolysaccharide (LPS) induced-AKI. We found that autophagy was induced in renal cortex of mice during LPS-induced AKI as reflected by a dose-and time-dependent increased accumulation of light chain 3-II (LC3-II), the common marker of autophagy, compared to that of control group; 2) the occurrence of intensive, punctate and increased immunohistochemical staining image of LC3-II in renal cortex; 3) the significant increase in the expression levels of Beclin-1, another key marker of autophagy; 4) the significantly increased levels of plasma urea and serum creatinine and 5) the significant increase in autophagagosome area ratio. We observed that 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy, blocked autophagy flux, alleviated AKI and protected against LPS-induced AKI. LPS triggered kidney inflammation by activation of the canonical NF-κB pathway. This route can be modulated by autophagy. Activation of the canonical NF-κB pathway was reduced in 3-MA+LPS as compared to that in LPS-treated group of mice. Mice pretreated with 3-MA before exposure to LPS showed a reduction in p65 phosphorylation, resulting in the accumulation of ubiquitinated IκB. In conclusion, impairment of autophagy ameliorates LPS-induced inflammation and decreases kidney injury. The accumulation of ubiquitinated IκB may be responsible for this effect.

Suppression of mitochondrial biogenesis through toll-like receptor 4-dependent mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling in endotoxin-induced acute kidney injury.

Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis.

C-type natriuretic peptide attenuates lipopolysaccharide-induced acute lung injury in mice

BackgroundC-type natriuretic peptide (CNP), secreted by vascular endothelial cells, belongs to a family of peptides that includes atrial and brain natriuretic peptides. CNP exhibits many vasoprotective effects against pulmonary hypertension and pulmonary fibrosis. The objective of this study was to investigate the prophylactic effects of CNP in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Materials and methodsC57BL/6 mice were divided into three groups as follows: normal control mice (n = 13), LPS mice treated with vehicle (n = 12), and LPS mice treated with CNP (n = 12). Twenty-four hours after tail vein injection of LPS, histopathologic, gene expression, and bronchoalveolar lavage fluid (BALF) assessments were performed on the lungs. To examine the neutrophils in the lungs, cells positive for myeloperoxidase staining were detected by immunohistochemistry. BALF cytokine levels were analyzed by enzyme-linked immunosorbent assays. Gene expression in lung tissue was analyzed by real-time polymerase chain reaction. ResultsCNP significantly attenuated the elevation of leukocyte cell counts and levels of tumor necrosis factor-alpha, macrophage inflammatory protein-2, monocyte chemoattractant protein-1, interleukin-6, and keratinocyte-derived chemokine in the BALF after LPS injection. Furthermore, there were significantly fewer myeloperoxidase-positive cells in lungs treated with CNP after LPS injection. In lungs of CNP-treated mice, expression of the monocyte chemoattractant protein-1, S100A8, and E-selectin genes was significantly lower than that in vehicle-treated mice. ConclusionsCNP had a protective effect on ALI induced by LPS by reducing inflammatory cell infiltration. CNP may hold promise in therapeutic strategies for ALI after pulmonary resection surgery.

Inhibition of airway inflammation by the roots of Angelica decursiva and its constituent, columbianadin

Ethnopharmacological relevanceThe roots of Angelica decursiva Fr. Et Sav (Umbelliferae) have been frequently used in traditional medicine as anti-inflammatory, antitussive, analgesic agents and expectorant, especially for treating cough, asthma, bronchitis and upper respiratory tract infections. To establish the scientific rationale for the clinical use of Angelica decursiva and to identify new agents for treating inflammatory lung disorders, pharmacological evaluation of the roots of Angelica decursiva and the isolated constituents was performed. MethodsIn vitro study was carried out using two lung cells, lung epithelial cells (A549) and alveolar macrophages (MH-S). The inflammatory markers such as IL-6 and nitric oxide (NO) for each cell line were examined. For in vivo study, a mouse model of lipopolysaccharide (LPS)-induced acute lung injury was used and the effects on lung inflammation were established by measuring the cell numbers in bronchoalveolar lavage fluid (BALF) and by histological observation. ResultsWater and 70% ethanol extracts of the roots of Angelica decursiva showed considerable inhibitory activity against LPS-induced lung inflammation in mice following oral administration at a dose of 400mg/kg. Five coumarin derivatives including columbianadin, umbelliferone, umbelliferone 6-carboxylic acid, nodakenin and nodakenetin were isolated. Among the isolated compounds, columbianadin was found to possess strong inhibitory activity against the inflammatory response of IL-1β-treated A549 cells and LPS-treated MH-S cells. Columbianadin was found to inhibit NO production by down-regulation of inducible NO synthase. Moreover, columbianadin was also proved to possess significant inhibitory activity against LPS-induced lung inflammation following oral administration at a dose of 20–60mg/kg. ConclusionsThe roots of Angelica decursiva were proved to be effective in the treatment of lung inflammation. Columbianadin can be a potential new agent for treating inflammatory lung disorders.

Aspirin-triggered resolvin D1 down-regulates inflammatory responses and protects against endotoxin-induced acute kidney injury

The presence of endotoxin in blood can lead to acute kidney injury (AKI) and septic shock. Resolvins, the endogenous lipid mediators derived from docosahexaenoic acid, have been reported to exhibit potent anti-inflammatory action. Using a mouse model of lipopolysaccharide (LPS)-induced AKI, we investigated the effects of aspirin-triggered resolvin D1 (AT-RvD1) on inflammatory kidney injury. Administration of AT-RvD1 1h after LPS challenge protected the mice from kidney injury as indicated by the measurements of blood urea nitrogen, serum creatinine, and morphological alterations associated with tubular damage. The protective effects were evidenced by decreased neutrophil infiltration in the kidney indicating reduction in inflammation. AT-RvD1 treatment restored kidney cell junction protein claudin-4 expression, which was otherwise reduced after LPS challenge. AT-RvD1 treatment inhibited endotoxin-induced NF-κB activation and suppressed LPS-induced ICAM-1 and VCAM-1 expression in the kidney. Moreover, AT-RvD1 treatment markedly decreased LPS-induced IL-6 level in the kidney and blocked IL-6-mediated signaling including STAT3 and ERK phosphorylation. Our findings demonstrate that AT-RvD1 is a potent anti-inflammatory mediator in LPS-induced kidney injury, and AT-RvD1 has therapeutic potential against AKI during endotoxemia.

CT Imaging Biomarker for Evaluation of Emodin as a Potential Drug on LPS-mediated Osteoporosis Mice

To identify micro-computed tomography (CT) imaging biomarkers for evaluating the effects of emodin, a potential drug to treat osteoporosis, in the mouse model of lipopolysaccharide (LPS)-mediated osteoporosis. Forty male imprinting control region (ICR) mice with LPS-induced bone resorption were equally divided into four experimental groups: phosphate-buffered saline-treated (control), emodin-treated, LPS-treated, and LPS + emodin-treated groups. Emodin (50 mg/kg) was administered orally on alternate days for 8 days, and LPS (5 mg/kg) was injected intraperitoneally on days 1 and 4. After 8 days, the mice were sacrificed, and micro-CT images of the left proximal femurs were obtained. Three-dimensional images were analyzed by using commercial software to measure the bone volume to total volume fraction (BV/TV), trabecular number (Tb-N), trabecular thickness (Tb-Th), and trabecular separation (Tb-Sp) as CT imaging biomarkers. Histologic analyses of the femurs were performed using hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining. The LPS + emodin-treated group demonstrated marked suppression of LPS-induced bone resorption compared to the LPS-treated group (BV/TV, 28.84% vs. 40.76%; Tb-N, 2.65 vs. 3.45 mm(-1); Tb-Sp, 300.81 vs. 212.31 μm; Tb-Th, 116.94 vs. 131.25 μm). TRAP immunohistochemical analysis showed fewer osteoclasts per field of tissue in the LPS + emodin-treated group than in the LPS-treated group (27.8 vs. 41.8). The BV/TV, Tb-N, and Tb-Sp data correlated well with the histomorphometric findings. The findings reveal a novel effect of emodin on bone remodeling in the LPS-mediated osteoporotic mouse model. The ex vivo micro-CT imaging is a promising tool for assessing the therapeutic effects of potential drugs on osteoporosis.

Effects of somatostatin against oxidative stress in mouse model of acute lung injury

Objective To investigate the effects and potential mechanisms of somatostatin (SST)against oxidative stress in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods A total of 96 ICR male mice of specific pathogen free (SPF) were divided randomly (random number) into four groups:normal control group (n =24),SST control group (n =24),ALI group (n =24) and SST treated group (n =24).The mice of ALI group were given the intra-peritoneal injection of LPS in dose of 12 mg in solution of 40 mL/kg.Mice of normal control group were given the intra-peritoneal injection of equivalent volume of normal saline (NS 40 mL/kg) instead,and mice of SST control group were given the intra-peritoneal injection of equivalent volume of normal saline (40 mL/kg) instead,then hypodermic injection of SST (20 μg:20 mL/kg) at 0.5,2,6 or 12 h after normal saline administration,and mice of SST treated group were given the intra-peritoneal injection of LPS (12 mg:40 mL/kg),then hypodermic injection of SST (20 μg:20 mL/kg) at 0.5,2,6 or 12 h after LPS administration.All mice were sacrificed at 3,8 h or 16 h after the first injection of LPS or NS.The lung wet/dry ratios were calculated.Pathological changes of lung tissues were observed under light microscope after HE staining.The levels of malondialdehyde (MDA),superoxide dismutase (SOD) and catalase (CAT) in the lung tissues were determined by chemical colorimetry; quinone oxidoreductase (NQO-1) levels in the lung tissues were measured by ELISA ; the expressions of nuclear factor-E2-related factor2 (Nrf2) and its protein levels in the lung tissues were measured by RT-PCR and Western blotting,respectively.One-way analysis of variance (ANOVA) was employed for statistical analysis by using SPSS version 19.0 to compare values among all groups.Results There were no significant differences in all biomarker variables between normal control group and SST control group (P ＜ 0.05).Lung wet/dry ratios in ALI group at 8 h and 16 h were significantly higher than those in normal control group (P =0.000) and obviously lower in SST treated group (5.21 ±0.13) vs.(5.78±0.20),(5.39±0.29) vs.(6.17 ±0.17) (P=0.000).Compared with normal control group,the damage of lung tissues in ALI group was more severe.In contrast,histopathological lesions observed in SST treated group were milder significantly.The MDA and NQO-1 in the lung tissues were much higher in ALI group than those in normal control group,while the activities of SOD and CAT were much lower in ALI group (P =0.000).Compared with ALI group,the MDA level (ng/mg) in SST treated group decreased remarkably (2.66 ±0.18) vs.(3.58 ±0.26),(3.52 ±0.31)vs.(4.37 ±0.19),(3.81 ±0.38) vs.(4.92 ±0.25) (P=0.000),whereas CAT activity (U/mg)increased significantly (9.21± 0.47) vs.(7.90±0.32),(10.06±0.51) vs.(8.39±0.41),(10.98 ± 0.33) vs.(9.52 ± 0.55) (P =0.000) ; SOD activity and NQO-1 level at 3 h had no obvious increase (P =0.359、0.111),but SOD activities and NQO-1 levels were markedly increased at 8 h and 16 h [SOD (U/mg):(75.34±5.5) vs.(67.89±3.8),P=0.021; (64.82±4.2) vs.(50.31 ±5.0),P=0.000],[NQO-1 (ng/mg):(1.052 ±0.041) vs.(0.715±0.038),(1.338±0.027)vs.(0.532 ± 0.028),P =0.000].Compared with normal control group,the Nrf2 mRNA and protein levels in ALI group were higher markedly (P =0.000),and Nrf2 mRNA in SST treated group were much higher (P =0.023-0.000),while Nrf2 protein level at 3 h showed no significant difference (P =0.101)but increased obviously at 8 h,16 h (P =0.000).Conclusions Our study has shown that SST can upregulate Nrf2 signal pathway,which may have the effects on regulating the immune response and protections against oxidative damages in ALI. Key words: Lipopolysaccharide ; Acute lung injury; Somatostatin ; Oxidative Stress ; Nuclear factor-E2-related factor 2 ; Mice

Cepharanthine Attenuates Lipopolysaccharide-Induced Mice Mastitis by Suppressing the NF-κB Signaling Pathway

Cepharanthine (CEP), a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effects of CEP on a mouse model of lipopolysaccharide (LPS)-induced mastitis and its underlying molecular mechanisms remain to be elucidated. The purpose of the present study was to investigate the effects of CEP on LPS-induced mouse mastitis. The mouse model of mastitis was induced by inoculation of LPS through the canals of the mammary gland. CEP was administered intraperitoneally at 1 h before and 12 h after induction of LPS. The results show that CEP significantly attenuates the infiltration of neutrophils, suppresses myeloperoxidase activity, and reduces the levels of TNF-α, IL-1β, and IL-6 in LPS-induced mouse mastitis. Furthermore, CEP inhibited the phosphorylation of NF-κB p65 subunit and the degradation of its inhibitor IκBα. All the results suggest that CEP exerts potent anti-inflammatory effects on LPS-induced mouse mastitis. Accordingly, CEP might be a potential therapeutic agent for mastitis.

Activation of PPARα by Wy-14643 ameliorates systemic lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a major cause of mortality and morbidity worldwide. The activation of peroxisome proliferator-activated receptor-α (PPARα) by its ligands, which include Wy-14643, has been implicated as a potential anti-inflammatory therapy. To address the beneficial efficacy of Wy-14643 for ALI along with systemic inflammation, the in vivo role of PPARα activation was investigated in a mouse model of lipopolysaccharide (LPS)-induced ALI. Using age-matched Ppara-null and wild-type mice, we demonstrate that the activation of PPARα by Wy-14643 attenuated LPS-mediated ALI. This was evidenced histologically by the significant alleviation of inflammatory manifestations and apoptosis observed in the lung tissues of wild-type mice, but not in the corresponding Ppara-null mice. This protective effect probably resulted from the inhibition of LPS-induced increases in pro-inflammatory cytokines and nitroxidative stress levels. These results suggest that the pharmacological activation of PPARα might have a therapeutic effect on LPS-induced ALI.

Myeloperoxidase-Derived Oxidants Induce Blood-Brain Barrier Dysfunction In Vitro and In Vivo

Peripheral leukocytes can exacerbate brain damage by release of cytotoxic mediators that disrupt blood-brain barrier (BBB) function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) formed via the myeloperoxidase (MPO)-H2O2-Cl− system. In the present study we examined the role of leukocyte activation, leukocyte-derived MPO and MPO-generated oxidants on BBB function in vitro and in vivo. In a mouse model of lipopolysaccharide (LPS)-induced systemic inflammation, neutrophils that had become adherent released MPO into the cerebrovasculature. In vivo, LPS-induced BBB dysfunction was significantly lower in MPO-deficient mice as compared to wild-type littermates. Both, fMLP-activated leukocytes and the MPO-H2O2-Cl− system inflicted barrier dysfunction of primary brain microvascular endothelial cells (BMVEC) that was partially rescued with the MPO inhibitor 4-aminobenzoic acid hydrazide. BMVEC treatment with the MPO-H2O2-Cl− system or activated neutrophils resulted in the formation of plasmalogen-derived chlorinated fatty aldehydes. 2-chlorohexadecanal (2-ClHDA) severely compromised BMVEC barrier function and induced morphological alterations in tight and adherens junctions. In situ perfusion of rat brain with 2-ClHDA increased BBB permeability in vivo. 2-ClHDA potently activated the MAPK cascade at physiological concentrations. An ERK1/2 and JNK antagonist (PD098059 and SP600125, respectively) protected against 2-ClHDA-induced barrier dysfunction in vitro. The current data provide evidence that interference with the MPO pathway could protect against BBB dysfunction under (neuro)inflammatory conditions.

Abstract 199: Anticoagulant Heparan Sulfate Conveys Anti-Inflammatory Activity of Antithrombin

Antithrombin (AT) therapy reduces morbidity/mortality in systemic inflammatory response syndromes, such as septic shock. These effects stem from the ability of AT to directly initiate cell signaling, which requires AT engagement with heparan sulfate proteoglycans. It is presently unclear if this process involves anticoagulant heparan sulfate (HS AT+ ), a specific motif with high affinity for AT that is produced by endothelial cells. If HS AT+ regulates anti-inflammatory signaling, then Hs3st1 -/- mice, which lack endothelial HS AT+ , should exhibit a proinflammatory state and should not benefit from AT treatment. We tested these hypotheses using a mouse model of lipopolysaccharide (LPS)-induced septic shock. Hemodynamics were monitored with a left ventricle pressure-volume catheter. Both Hs3st1 +/+ and Hs3st1 -/- mice die of cardiogenic shock. However, Hs3st1 -/- , compared to Hs3st1 +/+ , mice exhibited a much more rapid onset of death. Left ventricular gene expression profiles of mice with systemic LPS challenge showed enhanced expression of leukocyte recruitment genes (P-selectin, Cxcl10) in Hs3st1 -/- mice. Consistent with these data, LPS-treated Hs3st1 -/- mice, compared to Hs3st1 +/+ , exhibited enhanced leukocyte rolling flux within cremaster muscle venules. Thus, mice lacking HS AT+ exhibit a proinflammatory phenotype. AT treatment of Hs3st1 +/+ mice reduced both LPS-induced lethality and leukocyte-endothelial interactions. Conversely AT treatment of Hs3st1 -/- mice enhanced both LPS-induced lethality and leukocyte-endothelial interactions. Thus, AT failed to elicit anti-inflammatory effects in mice lacking HS AT+ . Our results indicate that endothelial HS AT+ is one component that mediates AT’s anti-inflammatory activity. These findings provide an initial step towards elucidating the role of HS AT+ and AT in a novel natural anti-inflammatory pathway of the blood vessel wall. This pathway is an ideal target for developing therapeutics to combat the devastating outcomes of septic shock.