Inhibition of matrix metalloproteins 9 attenuated Candida albicans induced inflammation in mouse cornea.

Since the severe corneal ulceration of mouse cornea is known to occur with inflammation. As one of imperative matrix metalloproteinase, the potential roles of matrix metalloproteins 9 (MMP9) in corneal ulceration and keratitis are still unveiled caused by fungal invasion. In this study, Candida albicans (CA) inoculated wild-type KM mice cornea was used as a model pathogen in corneal inflammation.  CA invasion significantly stimulated the expression of collagen IV and MMP9 detected by RT-PCR, Real-time PCR and Immunofluorescent staining in mouse cornea as soon as 6 hours post infection, and relatively decreased at 1 day post infection. For examining the role of MMP9 in fungal keratitis, the mice corneas were subconjunctivally injected MMP9 antibody or recombinant MMP9 protein 6 hours prior to CA inoculation, using rabbit IgG as control. Subconjunctival injection of recombinant MMP9 protein prior to CA inoculation enhanced, whereas MMP9 antibody attenuated corneal ulceration and inflammation, examining basement membrane, fungal load, myeloperoxidase (MPO) and proinflammatory cytokines including Macrophage inflammatory protein 2 (MIP2), Interleukin-1ß (IL-1ß) and Tumor necrosis factor-α (TNF-α). Inhibition of MMP9 could potentially attenuate Candida albicans induced inflammation in mouse cornea.

Bubble CPAP Support after Discontinuation of Mechanical Ventilation Protects Rat Lungs with Ventilator-Induced Lung Injury.

BACKGROUND: Bubble continuous positive airway pressure (BCPAP) has been used in neonates with respiratory distress for decades, but its lung-protective effect and underlying mechanism has not been investigated. OBJECTIVES: To test the hypothesis that BCPAP use after extubation decreases lung injury and that alterations to lung nitric oxide synthase (NOS) 3 expression may be one of the underlying mechanisms. METHODS: We compared gas exchange, lung injury severity, and lung NOS expression among rats with ventilator-induced lung injury (VILI) treated with either BCPAP or spontaneous breathing. After high tidal volume ventilation for 30 min, the rats were randomly divided to three groups: a control group underwent spontaneous breathing (n = 7), and two BCPAP groups were treated with the bubble technique with either a 2.5-mm-diameter (n = 7) or a 5.5-mm-diameter (n = 7) expiratory limb for 2 h. RESULTS: The bubble technique (2.5 and 5.5 mm diameter combined) resulted in a significantly higher PaO2, decreased alveolar protein levels (1.01 vs. 1.43 mg/kg, p < 0.05), a lower lung injury score (3.87 vs. 4.86, p < 0.05), and decreased NOS3 expression (1.99 vs. 3.32, p < 0.05) compared to spontaneous breathing in the control group. BCPAP with a 2.5-mm-diameter and with a 5.5-mm-diameter expiratory limb was not different with regard to gas exchange, alveolar protein levels, and lung injury scores, but there was a trend for lower NOS3 expression in the 5.5-mm group (1.41 vs. 2.56, p = 0.052). CONCLUSIONS: BCPAP decreases lung injury in rats with VILI after stopping mechanical ventilation. Attenuation of lung NOS3 expression may be one of the underlying mechanisms.

Pravastatin protects hyperbaric hyperoxia-induced lung injury via inhibiting inflammation in mice.

This study aimed to investigate the protective effects of pravastatin on hyperbaric hyperoxia-induced lung injury (HILI). C57BL/6 mice were randomly assigned into three groups: control group, HILI group and pravastatin (Pra) group. Mice in the HILI and Pra groups were subjected to exposure to pure oxygen at 2.5 atm abs for six hours. Mice in the Pra group were intraperitoneally treated with pravastatin at 15 mg/kg. immediately after exposure. At 24 hours, the lungs were collected for HE staining, TUNEL staining and detection of lung edema, myeloperoxidase (MPO) activity and cytokines, and bronchoalveolar lavage fluid (BALF) was harvested for cell-counting. Pravastatin treatment significantly improved the pathology of the lung after HILI (reduction in thickness of alveolar septum, attenuation of lung edema, fracturing of alveolar septa and decrease in infiltrated leukocytes); reduced the number of apoptotic cells; inhibited lung MPO activity; and regulated the balance between pro-inflammatory and anti-inflammatory cytokines. Our findings suggest that pravastatin may exert a protective effect on lung injury after hyperbaric hyperoxia exposure by inhibiting inflammation.

Radicicol, an Hsp90 inhibitor, inhibits intestinal inflammation and leakage in abdominal sepsis.

BACKGROUND: Intestinal injury is a key feature in sepsis. Inhibitors of heat shock protein 90 (Hsp90) have been shown to exert protective effects in models of inflammation. Herein, we hypothesized that Hsp90 might regulate intestinal inflammation and leakage in abdominal sepsis. MATERIALS AND METHODS: Male C57BL/6 mice were pretreated with radicicol (60 mg/kg), which is a specific inhibitor of Hsp90, prior to cecal ligation and puncture (CLP). Intravital fluorescence microscopy was used to quantify leukocyte-endothelium interactions in the colonic microcirculation 6 h after CLP. Colonic tissue was harvested to determine levels of myeloperoxidase, tumor necrosis factor-α and CXC chemokines. Intestinal injury was examined by histology. Intestinal barrier function was quantified by leakage of fluorescein isothiocyanate-dextran from the vascular system out into the abdominal cavity after intravenous injection. RESULTS: We found that radicicol significantly decreased CLP-induced leukocyte rolling and adhesion in colonic venules. Inhibition of Hsp90 reduced colonic levels of myeloperoxidase by 24% in septic animals. Moreover, radicicol significantly decreased CLP-provoked formation of CXC chemokines but had no significant effect on tumor necrosis factor-α levels in the colon. Notably, Hsp90 inhibition significantly attenuated intestinal tissue injury evoked by CLP. Lastly, it was found that radicicol reduced sepsis-induced intestinal leakage by 43%. CONCLUSION: Our novel findings suggest that targeting Hsp90 protects against intestinal inflammation and leakage and might be a useful strategy to ameliorate intestinal failure in polymicrobial sepsis.

Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion.

Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47(phox) and p67(phox), as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative respiratory bursts and through promotion of epithelial barrier integrity for luminal confinement of enteric bacteria.

Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury.

BACKGROUND: Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo. METHODS: C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically. RESULTS: Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment. CONCLUSIONS: These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.

The B-cell superantigen Finegoldia magna protein L causes pulmonary inflammation by a mechanism dependent on MyD88 but not B cells or immunoglobulins.

OBJECTIVE AND DESIGN: To determine whether Finegoldia magna protein L (PL) causes lung inflammation and, if so, whether the response is dependent on its immunoglobulin (Ig)-binding B-cell superantigenic property. MATERIAL: Pulmonary inflammatory reactions were analyzed at various time points after intratracheal administration of PL to various strains of mice. RESULTS: PL caused peribronchial and perivascular inflammation that peaked at 18-24 h. Polymorphonuclear cells (PMNs) began to accumulate in bronchoalveolar lavage fluid (BALF) of PL-challenged mice by 4 h and accounted for >90% of leukocytes by 18-24 h. Inflammation was marked by the appearance of MIP-2, KC, TNF-α, and IL-6 in the BALF with peak levels attained 4 h after PL administration. PL-induced pulmonary inflammation was associated with increased airway hyper-reactivity following inhalation of methacholine. The inflammatory reaction was unabated in mice lacking B cells and immunoglobulins. In contrast, PL-induced inflammation was abrogated in MyD88-deficient mice. PL-induced responses required alveolar macrophages. CONCLUSIONS: These results strongly suggest that PL-induced lung inflammation is dependent on an innate MyD88-dependent pathway rather than the Ig-binding properties of this microbial B cell superantigen. We propose that this pulmonary inflammatory reaction is caused by the interaction of PL with a Toll-like receptor expressed on alveolar macrophages.

Inhibitory effects and mechanisms of the anti-covid-19 traditional Chinese prescription, Keguan-1, on acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Keguan-1, a new traditional Chinese medicine (TCM) prescription contained seven Chinese herbs, is developed to treat coronavirus disease 19 (COVID-19). The first internationally registered COVID-19 randomised clinical trial on integrated therapy demonstrated that Keguan-1 significantly reduced the incidence of ARDS and inhibited the severe progression of COVID-19. AIM OF THE STUDY: To investigate the protective mechanism of Keguan-1 on ARDS, a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model was used to simulate the pathological state of ARDS in patients with COVID-19, focusing on its effect and mechanism on ALI. MATERIALS AND METHODS: Mice were challenged with LPS (2 mg/kg) by intratracheal instillation (i.t.) and were orally administered Keguan-1 (low dose, 1.25 g/kg; medium dose, 2.5 g/kg; high dose, 5 g/kg) after 2 h. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h and 24 h after i.t. administration of LPS. The levels of inflammatory factors tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, keratinocyte-derived chemokine (KC or mCXCL1), macrophage inflammatory protein 2 (MIP2 or mCXCL2), angiotensin II (Ang II), and endothelial cell junction-associated proteins were analysed using ELISA or western blotting. RESULTS: Keguan-1 improved the survival rate, respiratory condition, and pathological lung injury; decreased the production of proinflammatory factors (TNF-α, IL-6, IL-1ß, KC, and MIP2) in BALF and the number of neutrophils in the lung tissues; and ameliorated inflammatory injury in the lung tissues of the mice with LPS-induced ALI. Keguan-1 also reduced the expression of Ang II and the adhesion molecule ICAM-1; increased tight junction proteins (JAM-1 and claudin-5) and VE-cadherin expression; and alleviated pulmonary vascular endothelial injury in LPS-induced ALI. CONCLUSION: These results demonstrate that Keguan-1 can improve LPS-induced ALI by reducing inflammation and pulmonary vascular endothelial injury, providing scientific support for the clinical treatment of patients with COVID-19. Moreover, it also provides a theoretical basis and technical support for the scientific use of TCMs in emerging infectious diseases.

Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection.

Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1(-/-) mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B(4) (LTB(4)), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1(-/-) mice after infection. E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1(-/-) mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1(-/-) mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB(4).

Inflammation and matrix remodeling during repair of ventilator-induced lung injury.

High-pressure ventilation triggers different inflammatory and matrix remodeling responses within the lung. Although some of them may cause injury, the involvement of these mediators in repair is largely unknown. To identify mechanisms of repair after ventilator-induced lung injury (VILI), mice were randomly assigned to baseline conditions (no ventilation), injury [90 min of high-pressure ventilation without positive end-expiratory pressure (PEEP)], repair (injury followed by 4 h of low-pressure ventilation with PEEP), and ventilated controls (low-pressure ventilation with PEEP for 90 and 330 min). Histological injury and lung permeability increased during injury, but were partially reverted in the repair group. This was accompanied by a proinflammatory response, together with increases in TNF-α and IFN-γ, which returned to baseline during repair, and a decrease in IL-10. However, macrophage inflammatory protein-2 (MIP-2) and matrix metalloproteinases (MMP)-2 and -9 increased after injury and persisted in being elevated during repair. Mortality in the repair phase was 50%. Survivors showed increased cell proliferation, lower levels of collagen, and higher levels of MIP-2 and MMP-2. Pan-MMP or specific MMP-2 inhibition (but not MIP-2, TNF-α, or IL-4 inhibition) delayed epithelial repair in an in vitro wound model using murine or human alveolar cells cultured in the presence of bronchoalveolar lavage fluid from mice during the repair phase or from patients with acute respiratory distress syndrome, respectively. Similarly, MMP inhibition with doxycycline impaired lung repair after VILI in vivo. In conclusion, VILI can be reverted by normalizing ventilation pressures. An adequate inflammatory response and extracellular matrix remodeling are essential for recovery. MMP-2 could play a key role in epithelial repair after VILI and acute respiratory distress syndrome.

Protective and Detrimental Effects of Sodium Sulfide and Hydrogen Sulfide in Murine Ventilator-induced Lung Injury.

BACKGROUND: The antiinflammatory effects of hydrogen sulfide (H2S) and sodium sulfide (Na2S) treatment may prevent acute lung injury induced by high tidal volume (HVT) ventilation. However, lung protection may be limited by direct pulmonary toxicity associated with H2S inhalation. Therefore, the authors tested whether the inhalation of H2S or intravascular Na2S treatment can protect against ventilator-induced lung injury in mice. METHODS: Anesthetized mice continuously inhaled 0, 1, 5, or 60 ppm H2S or received a single bolus infusion of Na2S (0.55 mg/kg) or vehicle and were then subjected to HVT (40 ml/kg) ventilation lasting 4 h (n = 4-8 per group). RESULTS: HVT ventilation increased the concentrations of protein and interleukin-6 in bronchoalveolar lavage fluid, contributing to reduced respiratory compliance and impaired arterial oxygenation, and caused death from lung injury and pulmonary edema. Inhalation of 1 or 5 ppm H2S during HVT ventilation did not alter lung injury, but inhalation of 60 ppm H2S accelerated the development of ventilator-induced lung injury and enhanced the pulmonary expression of the chemoattractant CXCL-2 and the leukocyte adhesion molecules CD11b and L-selectin. In contrast, pretreatment with Na2S attenuated the expression of CXCL-2 and CD11b during HVT ventilation and reduced pulmonary edema. Moreover, Na2S enhanced the pulmonary expression of Nrf2-dependent antioxidant genes (NQO1, GPX2, and GST-A4) and prevented oxidative stress-induced depletion of glutathione in lung tissue. CONCLUSIONS: The data suggest that systemic intravascular treatment with Na2S represents a novel therapeutic strategy to prevent both ventilator-induced lung injury and pulmonary glutathione depletion by activating Nrf2-dependent antioxidant gene transcription.

CXCL2 benefits acute myeloid leukemia cells in hypoxia.

INTRODUCTION: Drug resistance and relapse of acute myeloid leukemia (AML) is still an important problem in the treatment of leukemia. Leukemia outbreak causes severe hypoxia in bone marrow (BM), remolding BM microenvironment (niche), and transforming hematopoietic stem cell (HSC) niche into leukemia stem cell (LSC) niche. AML cells and the microenvironment usually conduct "cross-talk" through cytokines to anchor resistant AML cells into LSC niche, thus supporting their survival. Therefore, this study was aimed to investigate the role of CXCL2 in the hypoxic AML niche. METHODS: AML hypoxic niche was simulated by hypoxic culture of THP-1 and HL-60 cells in vitro, thus to study the effects of CXCL2 on the proliferation and migration of AML cells. The expression of hypoxia-inducible factor-1α (HIF-1α) and the activation of survival-related kinases such as PIM2 and mTOR under CoCl2 -simulated hypoxic conditions were detected. The correlation between CXCL2 and the prognosis of AML with big data was verified. RESULTS: (a) CXCL2 promoted the proliferation and migration of AML cells. (b) CXCL2 up-regulated the expression of PIM2 by enhancing the transcriptional activity of HIF-1α. (c) CXCL2 activated mTOR in AML cells. (d) CXCL2 was associated with poor prognosis in AML. CONCLUSION: CXCL2 promotes survival, migration, and drug resistance pathway of AML cells in hypoxia and is associated with poor prognosis in AML. Therefore, CXCL2 can be considered as an important factor in promoting the development of AML cells in hypoxia.

DARC on RBC limits lung injury by balancing compartmental distribution of CXC chemokines.

The Duffy antigen receptor for chemokines (DARC) has a high affinity for CC and CXC chemokines. However, it lacks the ability to induce cell responses that are typical for classical chemokine receptors. The role of DARC in inflammatory conditions remains to be elucidated. We studied the role of DARC in a murine model of acute lung injury. We found that in Darc-gene-deficient (Darc(-/-)) mice, LPS-induced PMN migration into the alveolar space was elevated more than twofold. In contrast, PMN adhesion to endothelial cells and within the interstitial space was reduced in Darc(-/-) mice. Darc(-/-) mice also exhibited increased microvascular permeability. Elevated PMN migration in Darc(-/-) mice was associated with increased concentrations of two essential CXCR2 ligands, CXCL1 and CXCL2/3 in the alveolar space. In the blood, CXCL1 was mostly associated with RBC in WT mice and with plasma in Darc(-/-) mice. We found that DARC on RBC prevented excessive PMN migration into the alveolar space. In contrast, DARC on non-hematopoietic cells appeared to have only minor effects on leukocyte trafficking in this model. These findings show how DARC regulates lung inflammation by controlling the distribution and presentation of chemokines that bind CXCR2.

Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1(-/-) mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2(-/-) mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3(-/-) mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells.

The immunotoxicity of three generations of polyamidoamine (PAMAM) dendrimers (G-4, G-5 and G-6) was evaluated in mouse macrophage cells in vitro. Using the Alamar blue and MTT assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 > G-5 > G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. An assessment of intracellular ROS generation by the PAMAM dendrimers was performed by measuring the increased fluorescence as a result of intracellular oxidation of carboxy H2DCFDA to DCF both quantitatively using plate reader and qualitatively by confocal laser scanning microscopy. The inflammatory mediators macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6, (IL-6) were measured by the enzyme linked immunosorbant assay (ELISA) following exposure of mouse macrophage cells to PAMAM dendrimers. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation (maximum at approximately 4 h), TNF-alpha and IL-6 secretion (maximum at approximately 24 h), MIP-2 levels and cell death (approximately 72 h) was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point towards the mechanistic pathway of cell death upon exposure to PAMAM dendrimers.

Acute lung injury is reduced in fat-1 mice endogenously synthesizing n-3 fatty acids.

RATIONALE: Acute lung injury (ALI) remains an important cause of mortality in intensive care units. Inflammation is controlled by cytokines and eicosanoids derived from the n-6 fatty acid (FA) arachidonic acid (AA). The n-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and mediators derived from EPA and DHA possess reduced inflammatory potency. OBJECTIVES: To determine whether the ability of fat-1 mice to endogenously convert n-6 to n-3 FA, and thus generate an increased ratio of n-3 to n-6 FA, impacts experimental ALI. METHODS: We investigated ALI induced by intratracheal instillation of endotoxin in fat-1 and wild-type (WT) mice, assessing leukocyte numbers, protein concentration, and prostaglandin and cytokine levels in bronchoalveolar lavage fluid, as well as free FA in plasma, and lung ventilator compliance. Body temperature and motor activity of mice--markers of sickness behavior--were also recorded. MEASUREMENTS AND MAIN RESULTS: In ALI, fat-1 mice exhibited significantly reduced leukocyte invasion, protein leakage, and macrophage inflammatory protein-2 and thromboxane B(2) levels in lavage fluid compared with WT mice. Free AA levels were increased in the plasma of WT mice in response to endotoxin, whereas EPA and DHA were increased in the fat-1 group. Ventilator compliance was significantly improved in fat-1 mice. Body temperature and motor activity were decreased in ALI. fat-1 Mice recovered body temperature and motor activity faster. CONCLUSIONS: fat-1 Mice exhibited reduced features of ALI and sickness behavior. Increasing the availability of n-3 FA may thus be beneficial in critically ill patients with ALI.

[Paeoniflorin improves the permeability of cardiac microvascular endothelial cells by regulating Src/vascular endothelial-cadherin pathway].

OBJECTIVE: To investigate the effect and mechanism of paeoniflorin on the permeability of cardiac microvascular endothelial cells (CMECs) in sepsis. METHODS: Primary rat CMECs were isolated and cultured in vitro, and the cells in the logarithmic growth phase were used for experiments. Tetramethylazozolium colorimetry (MTT) was used to screen the safe and effective concentrations of paeoniflorin at 10, 20, and 40 µmol/L. The cells were divided into blank control group, lipopolysaccharide (LPS) group and low, medium and high concentration paeoniflorin pretreatment group. The cells in the blank control group were cultured in complete medium; the cells in the LPS group were challenged with LPS (1 mg/L) in complete medium; and the cells in the paeoniflorin pretreatment groups were pretreated with 10, 20, and 40 µmol/L paeoniflorin at 4 hours before LPS stimulation. The cells in each group were further cultured for 24 hours after LPS stimulation. The horseradish peroxidase (HRP) method was used to detect the permeability of rat CMECs. The enzyme-linked immunosorbent assay (ELISA) was used to detect the CXC chemokine ligand (CXCL1, CXCL2) levels in the cell supernatant. The real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of CXCL1 and CXCL2 in the cells. Western Blot was used to detect phosphorylated Src (p-Src), vascular endothelial-cadherin (VE-cadherin) and phosphorylated mitogen activated protein kinase (p-MAPK). RESULTS: Compared with the blank control group, the permeability of rat CMECs in the LPS group was significantly increased. The cell permeability was improved to some extent after paeoniflorin pretreatment at different concentrations, and the improvement was most obvious in the 40 µmol/L paeoniflorin group, with statistically significant difference as compared with the LPS group (A value: 1.61±0.07 vs. 2.13±0.06, P < 0.01). ELISA results showed that there were moderate amounts of CXCL1 and CXCL2 in the cell supernatant of rat CMECs in the blank control group. However, the secretion of CXCL1 and CXCL2 in the cell supernatant was increased significantly under the induction of LPS. After pretreatment with paeoniflorin at different concentrations, the secretion of CXCL1 and CXCL2 in the cell supernatant was significantly reduced. The most obvious inhibitory effect on CXCL1 was 40 µmol/L paeoniflorin, and the most obvious inhibition on CXCL2 was 20 µmol/L paeoniflorin, the differences were statistically significant as compared with the LPS group [CXCL1 (ng/L): 337.51±68.04 vs. 829.86±65.06, CXCL2 (ng/L): 4.48±0.11 vs. 9.41±0.70, both P < 0.01]. RT-qPCR results showed that the mRNA expressions of CXCL1 and CXCL2 in the rat CMECs were consistent with the ELISA results. LPS could increase mRNA expressions of CXCL1 and CXCL2 in the rat CMECs, and pretreatment with different concentrations of paeoniflorin could significantly reduce the mRNA expressions of CXCL1 and CXCL2. The 40 µmol/L paeoniflorin had the best inhibitory effect on CXCL1 mRNA expression, and the 20 µmol/L paeoniflorin had the best inhibitory effect on CXCL2 mRNA expression, the differences were statistically significant as compared with the LPS group [CXCL1 mRNA (2-ΔΔCt): 0.543±0.004 vs. 0.812±0.089, CXCL2 mRNA (2-ΔΔCt): 10.52±0.71 vs. 17.68±1.09, both P < 0.01]. Western Blot results showed that moderate amounts of p-Src, VE-cadherin and p-MAPK proteins were expressed in the rat CMECs in the blank control group. After LPS stimulation, the expressions of p-Src and p-MAPK proteins were increased significantly, while the expression of VE-cadherin protein was decreased significantly. After pretreatment with different concentrations of paeoniflorin, the expressions of p-Src and p-MAPK proteins in the cells were decreased to varying degrees, while the expression of VE-cadherin protein was increased, and 40 µmol/L paeoniflorin had the most obvious effect, the differences were statistically significant as compared with the LPS group [p-Src protein (p-Src/GAPDH): 1.02±0.09 vs. 1.29±0.05, p-MAPK proteins (p-MAPK/GAPDH): 0.24±0.02 vs. 0.62±0.02, VE-cadherin protein (VE-cadherin/GAPDH): 0.64±0.03 vs. 0.31±0.02, all P < 0.01]. CONCLUSIONS: Paeoniflorin can regulate the Src/VE-cadherin pathway in CMECs, inhibit the expression and secretion of inflammation-related proteins and chemokines, and improve the cell permeability of CMECs induced by LPS.

Herbal Combinational Medication of Glycyrrhiza glabra, Agastache rugosa Containing Glycyrrhizic Acid, Tilianin Inhibits Neutrophilic Lung Inflammation by Affecting CXCL2, Interleukin-17/STAT3 Signal Pathways in a Murine Model of COPD.

Chronic obstructive pulmonary disease (COPD) is caused by exposure to toxic particles, such as coal fly ash (CFA), diesel-exhaust particle (DEP), and cigarette smoke (CS), leading to chronic bronchitis, mucus production, and a subsequent lung dysfunction. This study, using a mouse model of COPD, aimed to evaluate the effect of herbal combinational medication of Glycyrrhiza glabra (GG), Agastache rugosa (AR) containing glycyrrhizic acid (GA), and tilianin (TN) as active ingredients. GA, a major active component of GG, possesses a range of pharmacological and biological activities including anti-inflammatory, anti-allergic, anti-oxidative. TN is a major flavonoid that is present in AR. It has been reported to have anti-inflammatory effects of potential utility as an anti-COPD agent. The COPD in the mice model was induced by a challenge with CFA and DEP. BALB/c mice received CFA and DEP alternately three times for 2 weeks to induce COPD. The herbal mixture of GG, AR, and TN significantly decreased the number of neutrophils in the lungs and bronchoalveolar lavage (BAL) fluid. It also significantly reduced the production of C-X-C motif chemokine ligand 2 (CXCL-2), IL-17A, CXCL-1, TNF-α, symmetric dimethylarginine (SDMA) in BALF and CXCL-2, IL-17A, CXCL-1, MUC5AC, transient receptor potential vanilloid-1 (TRPV1), IL-6, COX-2, NOS-II, and TNF-α mRNA expression in the lung tissue. Notably, a combination of GG and AR was more effective at regulating such therapeutic targets than GG or AR alone. The histolopathological lung injury was alleviated by treatment with the herbal mixture and their active ingredients (especially TN). In this study, the herbal combinational mixture more effectively inhibited neutrophilic airway inflammation by regulating the expression of inflammatory cytokines and CXCL-2 by blocking the IL-17/STAT3 pathway. Therefore, a herbal mixture of GG and AR may be a potential therapeutic agent to treat COPD.

Partial protection against Helicobacter pylori in the absence of mast cells in mice.

The goal of this study is to evaluate the contribution of mast cells to Helicobacter pylori immunity in a model of vaccine-induced protection. Mast cell-deficient Kitl(Sl)/Kitl(Sl-d) and control mice were immunized with H. pylori sonicate plus cholera toxin and challenged with H. pylori, and the bacterial loads, inflammatory infiltrates, and cytokine responses were evaluated and compared at 1, 2, and 4 weeks postchallenge. In vitro stimulation assays were performed using bone marrow-derived mast cells, and recall assays were performed with spleen cells of immunized mast cell-deficient and wild-type mice. Bacterial clearance was observed by 2 weeks postchallenge in mast cell-deficient mice. The bacterial load was reduced by 4.0 log CFU in wild-type mice and by 1.5 log CFU in mast cell-deficient mice. Neutrophil numbers in the gastric mucosa of immune Kitl(Sl)/Kitl(Sl-d) mice were lower than those for immune wild-type mice (P < 0.05). Levels of gastric interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-alpha) were also significantly lower in immune Kitl(Sl)/Kitl(Sl-d) mice than in wild-type mice (P < 0.001). Immunized mast cell-deficient and wild-type mouse spleen cells produced IFN-gamma and IL-17 in response to H. pylori antigen stimulation. TNF-alpha and CXC chemokines were detected in mast cell supernatants after 24 h of stimulation with H. pylori antigen. The results indicate that mast cells are not essential for but do contribute to vaccine-induced immunity and that mast cells contribute to neutrophil recruitment and inflammation in response to H. pylori.

Augmented effect of early antibiotic treatment in mice with experimental lung infections due to sequentially adapted mucoid strains of Pseudomonas aeruginosa.

BACKGROUND: Effects of treatment with tobramycin initiated 1 or 24 h post-infection were investigated in a new version of a pulmonary infection model in mice. The model reflects the differentiated behaviour of Pseudomonas aeruginosa mucoid strains isolated from the lungs of one chronically infected cystic fibrosis (CF) patient at different time periods during chronic lung infection. METHODS: BALB/c mice were challenged with alginate-embedded mucoid clinical isolates isolated in 1988, 1997 or 2003. Mice were euthanized on day 1, 2 or 3 post-infection for estimation of quantitative bacteriology, histopathology, and measurement of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein 2 (MIP-2). RESULTS: There was a significant reduction of bacteria when comparing treatment initiated 1 h post-infection with treatment initiated after 24 h for isolates 1997 and 2003. Treatment initiated 1 h post-infection also resulted in a reduction of the pulmonary cytokines G-CSF, for all three isolates, and MIP-2, for isolates 1997 and 2003. Histological evaluation showed a shift from the acute-type inflammatory immune response to a chronic-type in mice infected with isolate 2003. CONCLUSIONS: A significant reduction in the number of bacteria was observed when initiating treatment 1 h post-infection compared with initiating treatment after 24 h, although the latest isolate avoided complete clearance. Early antibiotic treatment directed at the mucoid phenotype in mice also reduced the inflammation and, thereby, the lung tissue damage.