Cholesterol Exacerbates the Pathophysiology of Non-Alcoholic Steatohepatitis by Upregulating Hypoxia-Inducible Factor 1 and Modulating Microcirculatory Dysfunction.

Cholesterol is a pivotal lipotoxic molecule that contributes to the progression of Non-Alcoholic Steatohepatitis NASH). Additionally, microcirculatory changes are critical components of Non-Alcoholic Fatty Liver Disease (NAFLD) pathogenesis. This study aimed to investigate the role of cholesterol as an insult that modulates microcirculatory damage in NAFLD and the underlying mechanisms. The experimental model was established in male C57BL/6 mice fed a high-fat high-carbohydrate (HFHC) diet for 39 weeks. Between weeks 31-39, 2% cholesterol was added to the HFHC diet in a subgroup of mice. Leukocyte recruitment and hepatic stellate cells (HSC) activation in microcirculation were assessed using intravital microscopy. The hepatic microvascular blood flow (HMBF) was measured using laser speckle flowmetry. High cholesterol levels exacerbated hepatomegaly, hepatic steatosis, inflammation, fibrosis, and leukocyte recruitment compared to the HFHC group. In addition, cholesterol decreased the HMBF-cholesterol-induced activation of HSC and increased HIF1A expression in the liver. Furthermore, cholesterol promoted a pro-inflammatory cytokine profile with a Th1-type immune response (IFN-γ/IL-4). These findings suggest cholesterol exacerbates NAFLD progression through microcirculatory dysfunction and HIF1A upregulation through hypoxia and inflammation. This study highlights the importance of cholesterol-induced lipotoxicity, which causes microcirculatory dysfunction associated with NAFLD pathology, thus reinforcing the potential of lipotoxicity and microcirculation as therapeutic targets for NAFLD.

Pro-resolving role of glucagon in lipopolysaccharide-induced mice lung neutrophilia.

Prior research demonstrated that glucagon has protective roles against inflammation, but its effect on the resolution of inflammation remains elusive. Using in vitro and in vivo approaches, this study aimed to investigate the pro-resolving potential of glucagon on pulmonary neutrophilic inflammation caused by lipopolysaccharide. Lipopolysaccharide induced an increase in the proportions of neutrophils positives to glucagon receptor (GcgR) in vitro. In addition, lipopolysaccharide induced an increase in the neutrophil accumulation and expression of GcgR by the inflammatory cells in the lungs, however, without altering glucagon levels. Intranasal treatment with glucagon, at the peak of neutrophilic inflammation, reduced the neutrophil number in the bronchoalveolar lavage (BAL), and lung tissue within 24 h. The reduction of neutrophilic inflammation provoked by glucagon was accompanied by neutrophilia in the blood, an increase in the apoptosis rate of neutrophils in the BAL, enhance in the pro-apoptotic Bax protein expression, and decrease in the anti-apoptotic Bcl-2 protein levels in the lung. Glucagon also induced a rise in the cleavage of caspase-3 in the lungs; however, it was not significant. Glucagon inhibited the levels of IL-1ß and TNF-α while increasing the content of pro-resolving mediators transforming growth factor (TGF-ß1) and PGE2 in the BAL and lung. Finally, glucagon inhibited lipopolysaccharide-induced airway hyper-reactivity, as evidenced by the reduction in lung elastance values in response to methacholine. In conclusion, glucagon-induced resolution of neutrophilic inflammation by promoting cessation of neutrophil migration and a rise of neutrophil apoptosis and the levels of pro-resolving mediators TGF-ß1 and PGE2.

Effect of the renin-angiotensin system on the exacerbation of adrenal glucocorticoid steroidogenesis in diabetic mice: Role of angiotensin-II type 2 receptor.

Prior investigation shows an increase in the activity of both hypothalamus-pituitary-adrenal (HPA) axis and the renin-angiotensin system (RAS) in diabetic patients. Moreover, activation of angiotensin-II type 1 receptor (AT1) has been associated with adrenal steroidogenesis. This study investigates the role of RAS on the overproduction of corticosterone in diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice. Captopril (angiotensin-converting enzyme inhibitor), Olmesartan (AT1 receptor antagonist), CGP42112A (AT2 receptor agonist) or PD123319 (AT2 receptor antagonist) were administered daily for 14 consecutive days, starting 7 days post-alloxan. Plasma corticosterone was evaluated by ELISA, while adrenal gland expressions of AT1 receptor, AT2 receptor, adrenocorticotropic hormone receptor MC2R, pro-steroidogenic enzymes steroidogenic acute regulatory protein (StAR), and 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) were assessed using immunohistochemistry or western blot. Diabetic mice showed adrenal gland overexpression of AT1 receptor, MC2R, StAR, and 11ßHSD1 without altering AT2 receptor levels, all of which were sensitive to Captopril or Olmesartan treatment. In addition, PD123319 blocked the ability of Olmesartan to reduce plasma corticosterone levels in diabetic mice. Furthermore, CGP42112A significantly decreased circulating corticosterone levels in diabetic mice, without altering the overexpression of MC2R and StAR in the adrenal glands. Our findings revealed that inhibition of both angiotensin synthesis and AT1 receptor activity reduced the high production of corticosterone in diabetic mice via the reduction of MC2R signaling expression in the adrenal gland. Furthermore, the protective effect of Olmesartan on the overproduction of corticosterone by adrenals in diabetic mice depends on both AT1 receptor blockade and AT2 receptor activation.

Glucagon Reduces Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice.

Sepsis is one of the most common comorbidities observed in diabetic patients, associated with a deficient innate immune response. Recently, we have shown that glucagon possesses anti-inflammatory properties. In this study, we investigated if hyperglucagonemia triggered by diabetes might reduce the migration of neutrophils, increasing sepsis susceptibility. 21 days after diabetes induction by intravenous injection of alloxan, we induced moderate sepsis in Swiss-Webster mice through cecum ligation and puncture (CLP). The glucagon receptor (GcgR) antagonist des-his1-[Glu9]-glucagon amide was injected intraperitoneally 24h and 1h before CLP. We also tested the effect of glucagon on CXCL1/KC-induced neutrophil migration to the peritoneal cavity in mice. Neutrophil chemotaxis in vitro was tested using transwell plates, and the expression of total PKA and phospho-PKA was evaluated by western blot. GcgR antagonist restored neutrophil migration, reduced CFU numbers in the peritoneal cavity and improved survival rate of diabetic mice after CLP procedure, however, the treatment did no alter hyperglycemia, CXCL1/KC plasma levels and blood neutrophilia. In addition, glucagon inhibited CXCL1/KC-induced neutrophil migration to the peritoneal cavity of non-diabetic mice. Glucagon also decreased the chemotaxis of neutrophils triggered by CXCL1/KC, PAF, or fMLP in vitro. The inhibitory action of glucagon occurred in parallel with the reduction of CXCL1/KC-induced actin polymerization in neutrophils in vitro, but not CD11a and CD11b translocation to cell surface. The suppressor effect of glucagon on CXCL1/KC-induced neutrophil chemotaxis in vitro was reversed by pre-treatment with GcgR antagonist and adenylyl cyclase or PKA inhibitors. Glucagon also increased PKA phosphorylation directly in neutrophils in vitro. Furthermore, glucagon impaired zymosan-A-induced ROS production by neutrophils in vitro. Human neutrophil chemotaxis and adherence to endothelial cells in vitro were inhibited by glucagon treatment. According to our results, this inhibition was independent of CD11a and CD11b translocation to neutrophil surface or neutrophil release of CXCL8/IL-8. Altogether, our results suggest that glucagon may be involved in the reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. This work collaborates with better understanding of the increased susceptibility and worsening of sepsis in diabetics, which can contribute to the development of new effective therapeutic strategies for diabetic septic patients.

Resveratrol-Loaded Lipid-Core Nanocapsules Modulate Acute Lung Inflammation and Oxidative Imbalance Induced by LPS in Mice.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are inflammatory and oxidative imbalance lung conditions with no successful pharmacological therapy and a high mortality rate. Resveratrol (RSV) is a plant-derived stilbene that presents anti-inflammatory and antioxidant effects. However, its therapeutic application remains limited due to its poor bioavailability, which can be solved by the use of nanocarriers. Previously, we demonstrated that nanoencapsulated RSV (RSV-LNC) pre-treatment, performed 4 h before lipopolysaccharide (LPS) stimulation in mice, increased its anti-inflammatory properties. In this study, we evaluated the anti-inflammatory and antioxidant effects, and lung distribution of RSV-LNCs administered therapeutically (6 h post LPS exposure) in a lung injury mouse model. The results showed that RSV-LNCs posttreatment improved lung function and diminished pulmonary inflammation. Moreover, RSV-LNC treatment enhanced the antioxidant catalase level together with a decrease in the oxidative biomarker in mouse lungs, which was accompanied by an increase in pulmonary Nrf2 antioxidant expression. Finally, the presence of RSV in lung tissue was significantly detected when mice received RSV-LNCs but not when they received RSV in its free form. Together, our results confirm that RSV nanoencapsulation promotes an increase in RSV bioavailability, enhancing its therapeutic effects in an LPS-induced lung injury model.

Anti-Bronchospasmodic Effect of JME-173, a Novel Mexiletine Analog Endowed With Highly Attenuated Anesthetic Activity.

Local anesthetics (LAs), such as lidocaine and mexiletine, inhibit bronchoconstriction in asthmatics, but adverse effects limit their use for this specific clinical application. In this study, we describe the anti-spasmodic properties of the mexiletine analog 2-(2-aminopropoxy)-3,5-dimethyl, 4-Br-benzene (JME-173), which was synthesized and screened for inducing reduced activity on Na+ channels. The effectiveness of JME-173 was assessed using rat tracheal rings, a GH3 cell line and mouse cardiomyocytes to access changes in smooth muscle contraction, and Na+, and Ca++ionic currents, respectively. Bronchospasm and airway hyper-reactivity (AHR) were studied using whole-body barometric plethysmography in A/J mice. We observed that the potency of JME-173 was 653-fold lower than mexiletine in inhibiting Na+ currents, but 12-fold higher in inhibiting L-type Ca++ currents. JME-173 was also more potent than mexiletine in inhibiting tracheal contraction by carbachol, allergen, extracellular Ca++, or sodium orthovanadate provocations. The effect of JME-173 on carbachol-induced tracheal contraction remained unaltered under conditions of de-epithelized rings, ß2-receptor blockade or adenylate cyclase inhibition. When orally administered, JME-173 and theophylline inhibited methacholine-induced bronchospasm at time points of 1 and 3 h post-treatment, while only JME-173 remained active for at least 6 h. In addition, JME-173 also inhibited AHR in a mouse model of lipopolysaccharide (LPS)-induced lung inflammation. Thus, the mexiletine analog JME-173 shows highly attenuated activity on Na+ channels and optimized anti-spasmodic properties, in a mechanism that is at least in part mediated by regulation of Ca++ inflow toward the cytosol. Thus, JME-173 is a promising alternative for the treatment of clinical conditions marked by life-threatening bronchoconstriction.

Intranasal Flunisolide Suppresses Pathological Alterations Caused by Silica Particles in the Lungs of Mice.

Silicosis is an occupational disease triggered by the inhalation of fine particles of crystalline silica and characterized by inflammation and scarring in the form of nodular lesions in the lungs. In spite of the therapeutic arsenal currently available, there is no specific treatment for the disease. Flunisolide is a potent corticosteroid shown to be effective for controlling chronic lung inflammatory diseases. In this study, the effect of flunisolide on silica-induced lung pathological changes in mice was investigated. Swiss-Webster mice were injected intranasally with silica particles and further treated with flunisolide from day 21 to 27 post-silica challenge. Lung function was assessed by whole body invasive plethysmography. Granuloma formation was evaluated morphometrically, collagen deposition by Picrus sirius staining and quantitated by Sircol. Chemokines and cytokines were evaluated using enzyme-linked immunosorbent assay. The sensitivity of lung fibroblasts was also examined in in vitro assays. Silica challenge led to increased leukocyte numbers (mononuclear cells and neutrophils) as well as production of the chemokine KC/CXCL-1 and the cytokines TNF-α and TGF-ß in the bronchoalveolar lavage. These alterations paralleled to progressive granuloma formation, collagen deposition and impairment of lung function. Therapeutic administration of intranasal flunisolide inhibited granuloma and fibrotic responses, noted 28 days after silica challenge. The upregulation of MIP-1α/CCL-3 and MIP-2/CXCL-2 and the cytokines TNF-α and TGF-ß, as well as deposition of collagen and airway hyper-reactivity to methacholine were shown to be clearly sensitive to flunisolide, as compared to silica-challenge untreated mice. Additionally, flunisolide effectively suppressed the responses of proliferation and MCP-1/CCL-2 production from IL-13 stimulated lung fibroblasts from silica- or saline-challenged mice. In conclusion, we report that intranasal treatment with the corticosteroid flunisolide showed protective properties on pathological features triggered by silica particles in mice, suggesting that the compound may constitute a promising strategy for the treatment of silicosis.

Synthesis, Pharmacological Profile and Docking Studies of New Sulfonamides Designed as Phosphodiesterase-4 Inhibitors.

Prior investigations showed that increased levels of cyclic AMP down-regulate lung inflammatory changes, stimulating the interest in phosphodiesterase (PDE)4 as therapeutic target. Here, we described the synthesis, pharmacological profile and docking properties of a novel sulfonamide series (5 and 6a-k) designed as PDE4 inhibitors. Compounds were screened for their selectivity against the four isoforms of human PDE4 using an IMAP fluorescence polarized protocol. The effect on allergen- or LPS-induced lung inflammation and airway hyper-reactivity (AHR) was studied in A/J mice, while the xylazine/ketamine-induced anesthesia test was employed as a behavioral correlate of emesis in rodents. As compared to rolipram, the most promising screened compound, 6a (LASSBio-448) presented a better inhibitory index concerning PDE4D/PDE4A or PDE4D/PDE4B. Accordingly, docking analyses of the putative interactions of LASSBio-448 revealed similar poses in the active site of PDE4A and PDE4C, but slight unlike orientations in PDE4B and PDE4D. LASSBio-448 (100 mg/kg, oral), 1 h before provocation, inhibited allergen-induced eosinophil accumulation in BAL fluid and lung tissue samples. Under an interventional approach, LASSBio-448 reversed ongoing lung eosinophilic infiltration, mucus exacerbation, peribronchiolar fibrosis and AHR by allergen provocation, in a mechanism clearly associated with blockade of pro-inflammatory mediators such as IL-4, IL-5, IL-13 and eotaxin-2. LASSBio-448 (2.5 and 10 mg/kg) also prevented inflammation and AHR induced by LPS. Finally, the sulfonamide derivative was shown to be less pro-emetic than rolipram and cilomilast in the assay employed. These findings suggest that LASSBio-448 is a new PDE4 inhibitor with marked potential to prevent and reverse pivotal pathological features of diseases characterized by lung inflammation, such as asthma.

Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis.

Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice. These findings highlight the potential of TMX-306, emphasizing its value in drug development for lung diseases, and particularly silicosis.

IL-13 immunotoxin accelerates resolution of lung pathological changes triggered by silica particles in mice.

Instillation of silica into the lungs of rodents results in pathological changes that strongly mimic human silicosis, an occupational lung disease marked by restrictive airway obstruction, inflammation, and fibrosis. Because IL-13 is a pivotal proinflammatory and fibrogenic cytokine, we examined whether a recombinant immunotoxin comprised of human IL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE) might affect pathological features of experimental silicosis. Mice received a single intranasal instillation of silica particles and were treated with intranasal IL-13-PE every other day from days 21 to 27 postsilica. The sensitivity of putative cell targets to IL-13-PE was also assessed in in vitro settings. Upregulation of IL-13, its receptor subunits IL-13Rα1 and IL-13Rα2, and shared receptor IL-4Rα were associated with development of granulomatous lung inflammation triggered by silica. IL-13-PE inhibited silica-induced granuloma and fibrotic responses noted at 24 h and 15 d after the last treatment. Upregulation of TNF-α, TGF-ß, and chemokines, as well as increased collagen deposition and airway hyperreactivity to methacholine were all clearly sensitive to IL-13-PE. In addition, IL-13-PE inhibited both IL-13-induced proliferation of cultured lung fibroblasts from silicotic mice and silica-induced IL-8 generation from A549 cells. In conclusion, our findings show that therapeutic treatment with IL-13-PE can reverse important pathological features caused by inhalation of silica particles, suggesting that this recombinant immunotoxin is a promising molecular template in drug discovery for the treatment of silicosis.

Synthesis and pharmacological evaluation of novel phenyl sulfonamide derivatives designed as modulators of pulmonary inflammatory response.

In this paper we report the design, synthesis and pharmacological evaluation of a new series of phenyl sulfonamide derivatives 2a-h and 3-8 planned by structural modification on the anti-inflammatory prototype LASSBio-468 (1). Among the synthesized analogues, the tetrafluorophthalimide LASSBio-1439 (2e) stands out showing an in vitro anti-TNF-α effect similar to the standard thalidomide. The relevance of tetrafluorination of the phthalimide nucleus was also confirmed by the anti-inflammatory profile of 2e, through oral administration, in a murine model of pulmonary inflammation. The corresponding tetrafluorocarboxyamide metabolite LASSBio-1454 (15), generated from partial hydrolysis of the derivative 2e, presented a significant in vitro effect and a pronounced anti-inflammatory activity in vivo.