Fibroblast growth factor 21 attenuates ventilator-induced lung injury by inhibiting the NLRP3/caspase-1/GSDMD pyroptotic pathway.

BACKGROUND: Ventilator-induced lung injury (VILI) is caused by overdistension of the alveoli by the repetitive recruitment and derecruitment of alveolar units. This study aims to investigate the potential role and mechanism of fibroblast growth factor 21 (FGF21), a metabolic regulator secreted by the liver, in VILI development. METHODS: Serum FGF21 concentrations were determined in patients undergoing mechanical ventilation during general anesthesia and in a mouse VILI model. Lung injury was compared between FGF21-knockout (KO) mice and wild-type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect. RESULTS: Serum FGF21 levels in patients and mice with VILI were significantly higher than in those without VILI. Additionally, the increment of serum FGF21 in anesthesia patients was positively correlated with the duration of ventilation. VILI was aggravated in FGF21-KO mice compared with WT mice. Conversely, the administration of FGF21 alleviated VILI in both mouse and cell models. FGF21 reduced Caspase-1 activity, suppressed the mRNA levels of Nlrp3, Asc, Il-1ß, Il-18, Hmgb1 and Nf-κb, and decreased the protein levels of NLRP3, ASC, IL-1ß, IL-18, HMGB1 and the cleaved form of GSDMD. CONCLUSIONS: Our findings reveal that endogenous FGF21 signaling is triggered in response to VILI, which protects against VILI by inhibiting the NLRP3/Caspase-1/GSDMD pyroptosis pathway. These results suggest that boosting endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for the treatment of VILI during anesthesia or critical care.

Elevated angiotensin II induces platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner during sepsis.

Thrombocytopenia is independently related with increased mortality in severe septic patients. Renin-angiotensin system (RAS) is elevated in septic subjects; accumulating studies show that angiotensin II (Ang II) stimulate the intrinsic apoptosis pathway by promoting reactive oxygen species (ROS) production. However, the mechanisms underlying the relationship of platelet apoptosis and RAS system in sepsis have not been fully elucidated. The present study aimed to elucidate whether the RAS was involved in the pathogenesis of sepsis-associated thrombocytopenia and explore the underlying mechanisms. We found that elevated plasma Ang II was associated with decreased platelet count in both patients with sepsis and experimental animals exposed to lipopolysaccharide (LPS). Besides, Ang II treatment induced platelet apoptosis in a concentration-dependent manner in primary isolated platelets, which was blocked by angiotensin II type 1 receptor (AT1R) antagonist losartan, but not by angiotensin II type 2 receptor (AT2R) antagonist PD123319. Moreover, inhibiting AT1R by losartan attenuated LPS-induced platelet apoptosis and alleviated sepsis-associated thrombocytopenia. Furthermore, Ang II treatment induced oxidative stress level in a concentration-dependent manner in primary isolated platelets, which was partially reversed by the AT1R antagonist losartan. The present study demonstrated that elevated Ang II directly stimulated platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner in sepsis-associated thrombocytopenia. The results would helpful for understanding the role of RAS system in sepsis-associated thrombocytopenia.

Downregulation of R-Spondin1 Contributes to Mechanical Stretch-Induced Lung Injury.

OBJECTIVES: The R-spondin family attenuates tissue damage via tightening endothelium and preventing vascular leakage. This study aims to investigate whether R-spondins protect against mechanical stretch-induced endothelial dysfunction and lung injury and to reveal the underlying mechanisms. DESIGN: Randomized controlled study. SETTING: University research laboratory. SUBJECTS: Patients scheduled to undergo surgery with mechanical ventilation support. Adult male Institute of Cancer Research mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Patients underwent a surgical procedure with mechanical ventilation support of 3 hours or more. Mice were subjected to mechanical ventilation (6 or 30 mL/kg) for 0.5-4 hours. Another group of mice were intraperitoneally injected with 1 mg/kg lipopolysaccharide, and 12 hours later subjected to mechanical ventilation (10 mL/kg) for 4 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 4 hours. MEASUREMENTS AND MAIN RESULTS: R-spondin1 were downregulated in both surgical patients and experimental animals exposed to mechanical ventilation. Intratracheal instillation of R-spondin1 attenuated, whereas knockdown of pulmonary R-spondin1 exacerbated ventilator-induced lung injury and mechanical stretch-induced lung vascular endothelial cell apoptosis. The antiapoptotic effect of R-spondin1 was mediated through the leucine-rich repeat containing G-protein coupled receptor 5 in cyclic stretched mouse lung vascular endothelial cells. We identified apoptosis-stimulating protein of p53 2 as the intracellular signaling protein interacted with leucine-rich repeat containing G-protein coupled receptor 5. R-spondin1 treatment decreased the interaction of apoptosis-stimulating protein of p53 2 with p53 while increased the binding of apoptosis-stimulating protein of p53 2 to leucine-rich repeat containing G-protein coupled receptor 5, therefore resulting in inactivation of p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. CONCLUSIONS: Mechanical ventilation leads to down-regulation of R-spondin1. R-spondin1 may enhance the interaction of leucine-rich repeat containing G-protein coupled receptor 5 and apoptosis-stimulating protein of p53 2, thus inactivating p53-mediated proapoptotic pathway in cyclic stretched mouse lung vascular endothelial cells. R-spondin1 may have clinical benefit in alleviating mechanical ventilator-induced lung injury.

NLRP3 Inflammasome Activation Contributes to Mechanical Stretch-Induced Endothelial-Mesenchymal Transition and Pulmonary Fibrosis.

OBJECTIVES: Mechanical ventilation can induce lung fibrosis. This study aimed to investigate whether ventilator-induced lung fibrosis was associated with endothelial-mesenchymal transition and to uncover the underlying mechanisms. DESIGN: Randomized, controlled animal study and cell culture study. SETTING: University research laboratory. SUBJECTS: Adult male Institute of Cancer Research, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) knockout and wild-type mice. Primary cultured mouse lung vascular endothelial cells. INTERVENTIONS: Institute of Cancer Research, NLRP3 knockout and wild-type mice were subjected to mechanical ventilation (20 mL/kg) for 2 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 24 hours. MEASUREMENTS AND MAIN RESULTS: Mice subjected to mechanical ventilation exhibited increases in collagen deposition, hydroxyproline and type I collagen contents, and transforming growth factor-ß1 in lung tissues. Ventilation-induced lung fibrosis was associated with increased expression of mesenchymal markers (α smooth muscle actin and vimentin), as well as decreased expression of endothelial markers (vascular endothelial-cadherin and CD31). Double immunofluorescence staining showed the colocalization of CD31/α smooth muscle actin, CD31/vimentin, and CD31/fibroblast-specific protein-1 in lung tissues, indicating endothelial-mesenchymal transition formation. Mechanical ventilation also induced NLRP3 inflammasome activation in lung tissues. In vitro direct mechanical stretch of primary mouse lung vascular endothelial cells resulted in similar NLRP3 activation and endothelial-mesenchymal transition formation, which were prevented by NLRP3 knockdown. Furthermore, mechanical stretch-induced endothelial-mesenchymal transition and pulmonary fibrosis were ameliorated in NLRP3-deficient mice as compared to wild-type littermates. CONCLUSIONS: Mechanical stretch may promote endothelial-mesenchymal transition and pulmonary fibrosis through a NLRP3-dependent pathway. The inhibition of endothelial-mesenchymal transition by NLRP3 inactivation may be a viable therapeutic strategy against pulmonary fibrosis associated with mechanical ventilation.

Salidroside Attenuates Ventilation Induced Lung Injury via SIRT1-Dependent Inhibition of NLRP3 Inflammasome.

BACKGROUND: Salidroside (SDS) is the main effective ingredient of Rhodiola rosea L with a variety of pharmacologic properties. We aim to investigate the effects of SDS on ventilation induced lung injury (VILI) and explore the possible underlying molecular mechanism. METHODS: Lung injury was induced in male ICR mice via mechanical ventilation (30 ml/kg) for 4h. The mice were divided in four groups:(1) Control group; (2) Ventilation group; (3) SDS group; (4) Ventilation with SDS group. SDS (50 mg/kg) was injected intraperitoneally 1h before operation. Mouse lung vascular endothelial cells (MLVECs) were subjected to cyclic stretch for 4h. RESULTS: It was found that SDS attenuated VILI as shown in HE staining, cell count and protein content levels in BAL fluid, W/D and Evans blue dye leakage into the lung tissue. SDS treatment inhibited the activation of NLRP3 inflammasome and subsequent caspase-1 cleavage as well as interleukin (IL)-1ß secretion both in vivo and in vitro. Moreover, SDS administration up-regulated SIRT1 expression. Importantly, knockdown of SIRT1 reversed the inhibitory effect of SDS on NLRP3 inflammasome activation. CONCLUSIONS: Taken together, these findings indicate that SDS may confer protection against ventilation induced lung injury via SIRT1-de-pendent inhibition of NLRP3 inflammasome activation.

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-ß1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-ß1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Chronic restraint stress aggravated arthritic joint swell of rats through regulating nitric oxide production.

Stress-related hormone norepinephrine (NE) displayed diverse effects on immune system including macrophages, which influenced many kinds of inflammatory diseases. Nitric oxide (NO) from activated macrophages played an important role in inflammatory diseases. In this study, we investigated under chronic restraint stress how NE influenced the joint swell of Complete Freund's Adjuvant (CFA)-induced arthritis of rats and whether NE regulated macrophage's production of NO through influencing phosphorylation of protein kinases C (PKC). The results showed chronic restraint stress exacerbated paw swell of rats with arthritis. Inhibitor of inducible nitric oxide synthase, S-methylisothiourea (SMT), and 6-hydroxydopamine (6-OHDA) could counteract the effect of restraint stress on arthritis. NE, NO and endotoxin in plasma of rats underwent restraint were improved significantly. In vitro experiments, NE could promote macrophage to produce more NO and iNOS when macrophage was activated by lipopolysaccharide (LPS). This effect could be inhibited by α adrenergic antagonist phentolamine. Nevertheless, through α receptor NE could promote the phosphorylation of PKC and PKC inhibitor staurosporine could counteract NE's enhancive effect on production of NO and iNOS of macrophages. This study revealed that NE could exacerbate arthritic joint swell through promoting NO production, which was in α receptor dependent way through enhancing phosphorylation of PKC for NE to enhance the iNOS expression of activated macrophage.

DRD1 downregulation contributes to mechanical stretch-induced lung endothelial barrier dysfunction.

Rationale: The lung-protective effects of dopamine and its role in the pathology of ventilator-induced lung injury (VILI) are emerging. However, the underlying mechanisms are still largely unknown. Objective: To investigate the contribution of dopamine receptor dysregulation in the pathogenesis of VILI and therapeutic potential of dopamine D1 receptor (DRD1) agonist in VILI. Methods: The role of dopamine receptors in mechanical stretch-induced endothelial barrier dysfunction and lung injury was studied in DRD1 knockout mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in lung samples from patients who underwent pulmonary lobectomy with mechanical ventilation for different time periods. Measurements and Main Results: DRD1 was downregulated in both surgical patients and mice exposed to mechanical ventilation. Prophylactic administration of dopamine or DRD1 agonist attenuated mechanical stretch-induced lung endothelial barrier dysfunction and lung injury. By contrast, pulmonary knockdown or global knockout of DRD1 exacerbated these effects. Prophylactic administration of dopamine attenuated mechanical stretch-induced α-tubulin deacetylation and subsequent endothelial hyperpermeability through DRD1 signaling. We identified that cyclic stretch-induced glycogen-synthase-kinase-3ß activation led to phosphorylation and activation of histone deacetylase 6 (HDAC6), which resulted in deacetylation of α-tubulin. Upon activation, DRD1 signaling attenuated mechanical stretch-induced α-tubulin deacetylation and subsequent lung endothelial barrier dysfunction through cAMP/exchange protein activated by cAMP (EPAC)-mediated inactivation of HDAC6. Conclusions: This work identifies a novel protective role for DRD1 against mechanical stretch-induced lung endothelial barrier dysfunction and lung injury. Further study of the mechanisms involving DRD1 in the regulation of microtubule stability and interference with DRD1/cAMP/EPAC/HDAC6 signaling may provide insight into therapeutic approaches for VILI.

Upregulation of Matrix Metalloproteinase-9 Protects against Sepsis-Induced Acute Lung Injury via Promoting the Release of Soluble Receptor for Advanced Glycation End Products.

Dysregulation of matrix metalloproteinase- (MMP-) 9 is implicated in the pathogenesis of acute lung injury (ALI). However, it remains controversial whether MMP-9 improves or deteriorates acute lung injury of different etiologies. The receptor for advanced glycation end products (RAGE) plays a critical role in the pathogenesis of acute lung injury. MMPs are known to mediate RAGE shedding and release of soluble RAGE (sRAGE), which can act as a decoy receptor by competitively inhibiting the binding of RAGE ligands to RAGE. Therefore, this study is aimed at clarifying whether and how pulmonary knockdown of MMP-9 affected sepsis-induced acute lung injury as well as the release of sRAGE in a murine cecal ligation and puncture (CLP) model. The analysis of GEO mouse sepsis datasets GSE15379, GSE52474, and GSE60088 revealed that the mRNA expression of MMP-9 was significantly upregulated in septic mouse lung tissues. Elevation of pulmonary MMP-9 mRNA and protein expressions was confirmed in CLP-induced mouse sepsis model. Intratracheal injection of MMP-9 siRNA resulted in an approximately 60% decrease in pulmonary MMP-9 expression. It was found that pulmonary knockdown of MMP-9 significantly increased mortality of sepsis and exacerbated sepsis-associated acute lung injury. Pulmonary MMP-9 knockdown also decreased sRAGE release and enhanced sepsis-induced activation of the RAGE/nuclear factor-κB (NF-κB) signaling pathway, meanwhile aggravating sepsis-induced oxidative stress and inflammation in lung tissues. In addition, administration of recombinant sRAGE protein suppressed the activation of the RAGE/NF-κB signaling pathway and ameliorated pulmonary oxidative stress, inflammation, and lung injury in CLP-induced septic mice. In conclusion, our data indicate that MMP-9-mediated RAGE shedding limits the severity of sepsis-associated pulmonary edema, inflammation, oxidative stress, and lung injury by suppressing the RAGE/NF-κB signaling pathway via the decoy receptor activities of sRAGE. MMP-9-mediated sRAGE production may serve as a self-limiting mechanism to control and resolve excessive inflammation and oxidative stress in the lung during sepsis.

Methanol extract of Phellodendri cortex alleviates lipopolysaccharide-induced acute airway inflammation in mice.

BACKGROUND AND AIM: The effects of methanol extract of Phellodendri cortex on acute airway inflammation induced by intranasal administration of lipopolysaccharide (LPS, 300mug/kg) were investigated in female BALB/c mice. MATERIALS AND METHODS: At 2 h after LPS exposure, mice were treated orally with methanol extract of Phellodendri cortex (100, 200 and 400 mg/kg). At the end of this study, bronchoalveolar lavage fluids (BALF) were collected and number of total cells, macrophages and neutrophils, protein concentration were analyzed. Tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein (MIP-2), IL-10 levels and nitric oxide (NO) production in BALF were also determined. RESULTS: Methanol extract of Phellodendri cortex dose-dependently alleviated LPS-induced acute airway inflammation via decreasing the infiltration of inflammatory cells and the release of inflammatory mediators. CONCLUSION: The relief of airway inflammation provides a possible therapeutic application of Phellodendri cortex for the treatment of infectious pulmonary diseases.

Hydrogen-rich saline reduces lung injury induced by intestinal ischemia/reperfusion in rats.

OBJECTIVE: Hydrogen has been reported to selectively reduce the hydroxyl radical, the most cytotoxic of reactive oxygen species. In this study we investigated the effects of hydrogen-rich saline on the prevention of lung injury induced by intestinal ischemia/reperfusion (I/R) in rats. METHODS: Male Sprague-Dawley rats (n=30, 200-220g) were divided randomly into three experimental groups: sham operated, intestinal I/R plus saline treatment (5ml/kg, i.v.), and intestinal I/R plus hydrogen-rich saline treatment (5ml/kg, i.v.) groups. Intestinal I/R was produced by 90min of intestinal ischemia followed by a 4h of reperfusion. RESULTS: Hydrogen-rich saline treatment decreased the neutrophil infiltration, the lipid membrane peroxidation, NF-kappaB activation and the pro-inflammatory cytokine interleukin IL-1beta and TNF-alpha in the lung tissues compared with those in saline-treated rat. CONCLUSION: Hydrogen-rich saline attenuates lung injury induced by intestinal I/R.

[Effects of salidroside-pretreatment on neuroethology of rats after global cerebral ischemia-reperfusion].

OBJECTIVE: To study the effects of salidroside-pretreatment on changes of neuroethology in rats with global cerebral ischemia-reperfusion injury so as to investigate its probable mechanism. METHODS: Sixty SD male rats were randomly divided into sham-operated group, untreated group and salidroside-pretreated group. The rats in salidroside-pretreated group were intraperitoneally administered with salidroside for seven days. The dose of salidroside was 12 mg/(kg.d). Thirty minutes after the last administration, the acute global cerebral ischemia-reperfusion in rats of the untreated group and the salidroside-pretreated group was induced by using the modified Pulsinelli's 4-vessel occlusion method. Five rats in each group were killed to obtain their brains 24 hours after reperfusion. The water content in the right brain was measured by calculating the ratio of dry weight to wet weight of the right brain. Activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in hippocampus of the rats were measured. Then neurological severity scores (NSSs) of the other 15 rats in each group were observed respectively before and 6, 12, 24, 48 and 96 h after reperfusion. At the fifth day after reperfusion, the test of Morris water maze was carried out to examine the memories and learning abilities of the rats. RESULTS: The content of MDA, the activity of SOD, the NSS, the mean incubation period and the ratio of time in the second quadrant in the untreated group were significant different from those in the sham-operated group (P<0.05). Compared with the untreated group, the brain water content, the content of MDA and the NSS degraded, and the mean incubation period shortened in salidroside-pretreated group. The activity of SOD and the ratio of residence time in the second quadrant increased in salidroside-pretreated group as compared with the untreated group (P<0.05). CONCLUSION: Salidroside can reduce the degree of cerebral edema of rats with global cerebral ischemia-reperfusion injury, relieve the metabolism abnormity of free radical and improve the function of cognition.

Interleukin-35 Attenuates D-Galactosamine/Lipopolysaccharide-Induced Liver Injury via Enhancing Interleukin-10 Production in Kupffer Cells.

Interleukin (IL) -35 is an anti-inflammatory cytokine which exerts various beneficial effects on autoimmune diseases. However, whether IL-35 plays a role in endotoxin induced hepatitis demands clarification. This study aims to reveal the effect and mechanism of IL-35 on endotoxin induced liver injury. Acute hepatic injury was induced by D-galactosamine (D-GalN, 400 mg/kg) and lipopolysaccharide (LPS, 5 µg/kg) administration in mice. IL-35 treatment ameliorated D-GalN/LPS induced liver injury in a dose dependent manner as shown by histological examination, ALT determination and Caspase-3 activity assay. It also reduced production of pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, IL-1ß, and IL-6, and increased production of anti-inflammatory cytokines, IL-4, IL-10, and transforming growth factor (TGF)-ß. This hepato-protective effect was proved mainly mediated by Kupffer cells (KC) via gadolinium chloride depletion and cell adoptive transfer experiment. In addition, IL-35 emolliated the cytotoxicity of LPS-triggered KCs to hepatocytes, suppressed nitric oxide (NO) and TNF-α production, and elevated IL-10 production in LPS stimulated KCs. Furthermore, IL-35 could not exert hepato-protective effect in IL-10-deficient mice in vivo and it could not suppress LPS induced NO and TNF-α production in IL-10-deficient KCs in vitro. In conclusion, IL-35 protects endotoxin-induced acute liver injury, which mainly acts thought increasing IL-10 production in KCs. This finding demonstrates a role of IL-35 in anti-infectious immunity and provides a potential therapeutic target in treating fulminant hepatitis.

Resveratrol ameliorates lipopolysaccharide-induced epithelial mesenchymal transition and pulmonary fibrosis through suppression of oxidative stress and transforming growth factor-ß1 signaling.

BACKGROUND & AIMS: Fibrotic changes seem to be responsible for the high mortality rate observed in patients with acute respiratory distress syndrome (ARDS). The present study aimed to determine whether resveratrol, a natural antioxidant polyphenol, had anti-fibrotic effects in the murine model of lipopolysaccharide (LPS)-induced pulmonary fibrosis. METHODS: Fibrosis was assessed by determination of collagen deposition, hydroxyproline and type I collagen levels in lung tissues. Development of epithelial-mesenchymal transition (EMT) was identified by the loss of E-cadherin accompanying by the acquisition of α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-ß1 content, levels of phosphorylated Smad2/Smad3 and Smad4, malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, and catalase (CAT) activity in lung tissues were determined. RESULTS: LPS increased collagen deposition, hydroxyproline and type I collagen contents, and meanwhile induced EMT process, stimulated TGF-ß1 production and Smad activation in lung tissues on day 21 to day 28 after LPS administration. In addition, LPS treatment resulted in a rapid induction of oxidative stress as evidenced by increase of MDA and decreases of T-AOC, CAT and SOD activities as early as 7 days after LPS treatment, which was persistent for at least 4 weeks. In contrast, resveratrol treatment attenuated LPS-induced EMT and pulmonary fibrosis, meanwhile it suppressed LPS-induced oxidative stress, TGF-ß1 production and activation of Smad signaling pathway. CONCLUSIONS: Resveratrol may ameliorate LPS-induced EMT and pulmonary fibrosis through suppression of oxidative stress and TGF-ß1/Smad signaling pathway. Application of antioxidants may represent a useful adjuvant pharmacologic approach to reduce ARDS-associated pulmonary fibrosis.

Lung endothelial barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced mitochondrial oxidative damage via an Nrf2-dependent mechanism.

High-mobility group box 1 (HMGB1) contributes to lung vascular hyperpermeability during ventilator-induced lung injury. We aimed to determine whether the natural antioxidant resveratrol protected against HMGB1-induced endothelial hyperpermeability both in vitro and in vivo. We found that HMGB1 decreased vascular endothelial (VE)-cadherin expression and increased endothelial permeability, leading to mitochondrial oxidative damage in primary cultured mouse lung vascular endothelial cells (MLVECs). Both the mitochondrial superoxide dismutase 2 mimetic MnTBAP and resveratrol blocked HMGB1-induced mitochondrial oxidative damage, VE-cadherin downregulation, and endothelial hyperpermeability. In in vivo studies, anesthetized male ICR mice were ventilated for 4h using low tidal volume (6 ml/kg) or high tidal volume (HVT; 30 ml/kg) ventilation. The mice were injected intraperitoneally with resveratrol immediately before the onset of ventilation. We found that resveratrol attenuated HVT-associated lung vascular hyperpermeability and HMGB1 production. HVT caused a significant increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and Nrf2 target gene expression in lung tissues, which was further enhanced by resveratrol treatment. HMGB1 had no effect on Nrf2 activation, whereas resveratrol treatment activated the Nrf2 signaling pathway in HMGB1-treated MLVECs. Moreover, Nrf2 knockdown reversed the inhibitory effects of resveratrol on HMGB1-induced mitochondrial oxidative damage and endothelial hyperpermeability. The inhibitory effect of resveratrol on cyclic stretch-induced HMGB1 mRNA expression in primary cultured MLVECs was also abolished by Nrf2 knockdown. In summary, this study demonstrates that resveratrol protects against lung endothelial barrier dysfunction initiated by HVT. Lung endothelial barrier protection by resveratrol involves inhibition of mechanical stretch-induced HMGB1 release and HMGB1-induced mitochondrial oxidative damage. These protective effects of resveratrol might be mediated through an Nrf2-dependent mechanism.

SC5b-9-induced pulmonary microvascular endothelial hyperpermeability participates in ventilator-induced lung injury.

Mechanical ventilation with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses, termed ventilator-induced lung injury (VILI). VILI is characterized by an influx of inflammatory cells, increased pulmonary permeability, and endothelial and epithelial cell death. But the underlying molecular mechanisms that regulate VILI remain unclear. The purpose of this study was to investigate the mechanisms that regulate pulmonary endothelial barrier in an animal model of VILI. These data suggest that SC5b-9, as the production of the complement activation, causes increase in rat pulmonary microvascular permeability by inducing activation of RhoA and subsequent phosphorylation of myosin light chain and contraction of endothelial cells, resulting in gap formation. In general, the complement-mediated increase in pulmonary microvascular permeability may participate in VILI.

STY39, a novel alpha-melanocyte-stimulating hormone analogue, attenuates bleomycin-induced pulmonary inflammation and fibrosis in mice.

Various anti-inflammatory agents have been used to treat acute or chronic lung injury-induced pulmonary fibrosis (PF). However, the efficacy of the available treatments is disappointing, and new therapies are urgently needed. In the current study, we investigated the effect of a novel α-melanocyte-stimulating hormone analog, STY39, on bleomycin (BLM)-induced pulmonary inflammation and fibrosis in mice. C57BL/6 mice received an intratracheal injection of BLM before being treated with STY39 (0.625, 1.25, or 2.5 mg/kg, i.p.) once a day for 14 consecutive days. Various parameters, reflecting the inflammatory reaction, metabolism of extracellular matrix, myofibroblast proliferation, and degree of fibrosis in the lung, were evaluated. We found that STY39 significantly improved the survival of mice with lethal BLM-induced lung injury, limited body weight loss and the increase in the lung index, reduced the mRNA expression of types I and III procollagen and the production of hydroxyproline in the lung, diminished myofibroblast proliferation, and ultimately reduced BLM-induced lung damage. Further investigation revealed that, in a dose-dependent manner, STY39 treatment inhibited leukocyte migration into the lung; reduced the production of TNF-α, IL-6, macrophage inflammatory protein 2, and transforming growth factor ß1 in the lung; and altered the ratio of matrix metalloproteinase 1 to tissue inhibitors of metalloproteinase 1. These findings suggest that STY39 attenuates BLM-induced experimental PF by limiting the inflammatory reaction through the inhibition of proinflammatory and profibrosis cytokines and by accelerating the metabolism of extracellular matrix. Therefore, STY39 may be an effective therapy for preventing PF.

Analgesic effects of gabapentin on mechanical hypersensitivity in a rat model of chronic pancreatitis.

Gabapentin, an anticonvulsant, is widely accepted as an alternative therapeutic agent for neuropathic pain and has proved to produce analgesic effects in a mouse model of visceral pain. However, it is unknown whether gabapentin is also analgesically effective in chronic pancreatitis. The aim of the present study was to investigate the role and underlying mechanisms of gabapentin in a rat model of chronic pancreatitis. Chronic pancreatitis induced by dibutyltin dichloride (DBTC) produced a marked increase in mechanical sensitivity of the abdomen after the establishment of the model. During the first day to the sixth day in the fourth week, Gabapentin was administered intraperitoneally daily at a dose of 100mg/kg. The behavioral test began 1h after drug administration. The analgesic effect of gabapentin was not evident with a single injection, but gabapentin significantly reduced the responsive frequencies to mechanical stimulation in rats with chronic pancreatitis from the third day to the end of the experiment. To explore the underlying mechanisms, the expression of alpha(2)delta-1 calcium channel subunit was examined in the thoracic spinal cord (T8-11). There was no significant change in alpha(2)delta-1 level of T8-11 following the first injection. But after the sixth injection, the alpha(2)delta-1 level of T8-11 in rats with chronic pancreatitis was declined. Taken together, the present study suggested that repeated administration of gabapentin daily could reduce mechanical hypersensitivity in the upper abdomen and produce an analgesic effect in a rat model of chronic pancreatitis. The down-regulation of alpha(2)delta-1 calcium channel subunit might be one of the mechanisms underlying the analgesic effect of gabapentin.

Protective effects of the free radical scavenger edaravone on acute pancreatitis-associated lung injury.

Impaired lung function is the primary contributor to most deaths associated with severe acute pancreatitis. It is widely accepted that oxidative stress plays a central role in the pathogenesis of pancreatitis and associated complications. Therefore, in the present study, we investigated whether therapeutic treatment with the free radical scavenger edaravone could protect rats against acute pancreatitis and the associated lung injury. Acute pancreatitis was induced by infusion of 1ml/kg of sodium taurocholate (3% solution) into the biliopancreatic duct. Edaravone (8mg/kg) was administered 1h and 13h after inducing pancreatitis, the severity of pancreatic and pulmonary injuries was evaluated 24h after inducing pancreatitis. Edaravone treatment significantly reduced the elevated malondialdehyde levels in rat lungs after acute pancreatitis, suggesting an important role for free radicals in acute pancreatitis-associated lung injury. In addition, edaravone showed significant protective effects against neutrophil infiltration and tissue injury in both pancreas and lung, as demonstrated by serum amylase levels, myeloperoxidase activity and histopathological analysis. Edaravone treatment also attenuated the elevated mRNA levels of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) in rat lungs after acute pancreatitis. In conclusion, edaravone protects rats against acute pancreatitis-associated lung injury, probably through its antioxidant and anti-inflammatory effects. Thus, edaravone shows promise as a treatment for lung injury in patients with acute pancreatitis.

Edaravone, a free radical scavenger, is effective on neuropathic pain in rats.

Recent studies indicate that reactive oxygen species (ROS) are involved in persistent pain, including neuropathic and inflammatory pain. Edaravone, a free radical scavenger, which is widely used clinically in Japan for acute cerebral infarction to prevent ischemia reperfusion injury, has been shown to inhibit inflammatory-induced pain in rats. However, it is unknown whether edaravone is effective on neuropathic pain. In the present study, we used the spinal nerve ligation (SNL)-induced neuropathic pain model of rats to investigate the role of edaravone in the generation or development of neuropathic pain. Edaravone was administrated intraperitoneally per day at a dose of 4 mg/kg. We found that preemptive treatment of edaravone had analgesic effects on SNL-induced chronic pain without inducing any behavioral side-effects or motor disturbances at the dose given. By contrast, when administered on the third day after SNL surgery, edaravone cannot reverse the established pain but only produced tenuous analgesic effects on the rats of neuropathic pain. To explore the underlying mechanisms, effects of edaravone on the excitability of dorsal root ganglion (DRG) neurons and activation of JNK in DRG were observed. We found that preemptive edaravone treatment can decrease the H(2)O(2)-induced depolarization in the acutely dissociated DRG neurons. Furthermore, we found that preemptive edaravone treatment can reduce the SNL-induced pJNK expression in the ipsilateral DRG. Taken together, the present study indicated that edaravone could prevent the development of SNL-induced neuropathic pain but had little effects on the established neuropathic pain. The inhibition of the signaling pathway of JNK cascade or suppression of the possible ROS-induced hyper-excitability of DRG neurons might be, at least in part, mechanisms underlying the effects of edaravone on SNL-induced neuropathic pain.