New thiazolidinedione LPSF/GQ-2 inhibits NFκB and MAPK activation in LPS-induced acute lung inflammation.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are responsible for high mortality rates in critical patients. Despite >50 years of intensive research, there is no pharmacologically effective treatment to treat ALI. PPARs agonists, chemically named thiazolidinediones (TZDs) have emerged as potential drugs for the treatment of ALI and ARDS due to their anti-inflammatory efficacy. The present study aims to evaluate the potential anti-inflammatory effects of new TZDs derivatives, LPSF/GQ-2 and LPSF/RA-4, on ALI induced by LPS. BALB/c mice were divided into five groups: 1) Control; 2) LPS intranasal 25 µg; 3) LPSF/GQ-2 30 mg/kg + LPS; 4) LPSF/RA-4 20 mg/kg + LPS; and 5) DEXA 1 mg/Kg + LPS. BALF analyses revealed that LPSF/GQ-2 and LPSF/RA-4 reduced NO levels in BALF and inflammatory cell infiltration induced by LPS. MPO levels were also reduced by the LPSF/GQ-2 and LPSF/RA-4 pre-treatments. In contrast, histopathological analyses showed better tissue protection with LPSF/GQ-2 than DEXA and LPSF/RA-4 groups. Similarly, LPSF/GQ-2 reduced inflammatory markers (IL-1, iNOS, TNFα, IL-1ß, IL-6) better than LPSF/RA-4. The LPSF/GQ-2 anti-inflammatory action could be attributed to the inhibition of NFκB, ERK, p38, and PARP pathways. In contrast, LPSF/RA-4 had no effect on the expression of p38, JNK, NFκB. The present study indicates that LPSF/GQ-2 presents a potential therapeutic role as an anti-inflammatory drug for ALI.

Diethylcarbamazine attenuates the expression of pro-fibrogenic markers and hepatic stellate cells activation in carbon tetrachloride-induced liver fibrosis.

BACKGROUND AND AIM: While diethylcarbamazine citrate (DEC) displays important anti-inflammatory effects in experimental models of liver injury, the mechanisms of its action remain poorly understood. The aim of the present study was to investigate the fibrolytic potential of DEC. METHODS: Mice receive two injections of carbon tetrachloride (CCl4) per week for 8 weeks. DEC 50 mg/kg body weight was administered through drinking water during the last 12 days of liver injury. RESULTS: The expression of hepatic stellate cells (HSCs) activation markers, including smooth muscle α-actin (α-SMA), collagen I, transforming growth factor-ß 1 (TGF-ß1), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) was assessed. The influence of DEC on the intracellular MAPK pathways of the HSCs (JNK and p38 MAPK) was also estimated. DEC inhibited HSCs activation measured as the production of α-SMA and collagen I. In addition, it down regulated the production of TGF-ß1 and TIMP-1, and concomitantly increased MMP-2 activity. Furthermore, DEC significantly inhibited the activation of the JNK and p38 MAPK signaling pathways. CONCLUSIONS: In conclusion, DEC significantly attenuated the severity of CCl4-induced liver injury and the progression of liver fibrosis, exerting a potential fibrolytic effect in the CCl4-induced fibrosis model.

Diethylcarbamazine attenuates LPS-induced acute lung injury in mice by apoptosis of inflammatory cells.

BACKGROUND: Acute lung injury (ALI) is characterized by extensive neutrophil infiltration, and apoptosis delay considered part of the pathogenesis of the condition. Despite great advances in treatment strategies, few effective therapies are known for ALI. Diethylcarbamazine (DEC) is used against lymphatic filariasis, a number of studies have described its anti-inflammatory activities and pro-apoptotic effect. These properties have been associated with nuclear factor kappa-B inactivation. The aim of the present study was to investigate the effect of DEC on ALI induced by lipopolysaccharide (LPS) in mice. METHODS: DEC effect was evaluated by histological and ultrastructural analysis, immunohistochemistry and western blot (WB). Also TUNEL assays were performed and as well as myeloperoxidase (MPO) levels and nitric oxide (NO) were measured. RESULTS: The results demonstrate that LPS induced histological and ultrastructural changes with tissue damage, intense cell infiltration and pulmonary edema, and also increased levels of MPO and NO. DEC reversed these effects, confirming its anti-inflammatory action. DEC pro-apoptotic activity was also evaluated. The expression of TUNEL-positive cells and caspase-3 was increased in DEC treated group. Furthermore, immunohistochemical and WB analysis showed that DEC increased the expression of pro-apoptotic proteins in both the intrinsic (Bax, cytochrome c and caspase-9) and the extrinsic pathways of apoptosis (Fas, FADD and caspase-8). Additionally, DEC reduced the expression of the anti-apoptotic protein Bcl-2. CONCLUSION: Our results suggest that DEC attenuates ALI through the prevention of inflammatory cells accumulation by stimulating apoptosis. DEC accelerates the resolution of inflammation and may be a potential pharmacological treatment for ALI.

Effects of metformin on inflammation and short-term memory in streptozotocin-induced diabetic mice.

The aim of the present study was to analyze the action of metformin on short-term memory, glial cell activation and neuroinflammation caused by experimental diabetic encephalopathy in C57BL/6 mice. Diabetes was induced by the intraperitoneal injection of a dose of 90mg/kg of streptozotocin on two successive days. Mice with blood glucose levels ≥200dl/ml were considered diabetic and were given metformin hydrochloride at doses of 100mg/kg and 200mg/kg (by gavage, twice daily) for 21 days. On the final day of treatment, the mice underwent a T-maze test. On the 22nd day of treatment all the animals were anesthetized and euthanized. Diabetic animals treated with metformin had a higher spatial memory score. The hippocampus of the diabetic animals presented reactive gliosis, neuronal loss, NF-kB signaling activation, and high levels of IL-1 and VEGF. In addition, the T-maze test scores of these animals were low. Treatment with metformin reduced the expression of GFAP, Iba-1 (astrocyte and microglial markers) and the inflammation markers (p-IKB, IL-1 and VEGF), while enhancing p-AMPK and eNOS levels and increasing neuronal survival (Fox-1 and NeuN). Treatment with metformin also improved the spatial memory scores of diabetic animals. In conclusion, the present study showed that metformin can significantly reduce neuroinflammation and can decrease the loss of neurons in the hippocampus of diabetic animals, which can subsequently promote improvements in spatial memory.

Effects of Sildenafil Citrate and Heparin Treatments on Placental Cell Morphology in a Murine Model of Pregnancy Loss.

Lipopolysaccharide (LPS) injections during pregnancy are well established as models for pregnancy complications, including fetal growth restriction (FGR), thrombophilia, preterm labor and abortion. Indeed, inflammation, as induced by LPS injection has been described as a pivotal factor in cases of miscarriage related to placental tissue damage. The phosphodiesterase-5 inhibitor sildenafil (Viagra®) is currently used to treat FGR cases in women, while low-molecular weight heparin (Fragmin®) is a standard treatment for recurrent miscarriage (RM). However, the pathways and cellular dynamics involved in RM are not completely understood. The aim of this study was to evaluate the protective effect of sildenafil and dalteparin in a mouse model of LPS-induced abortion. Histopathology, ultrastructural analysis and immunofluorescence for P-selectin were studied in two different placental cell types: trophoblast cells and labyrinth endothelial cells. Treatment with sildenafil either alone or in combination with heparin showed the best response against LPS-induced injury during pregnancy. In conclusion, our results support the use of these drugs as future therapeutic agents that may protect the placenta against inflammatory injury in RM events. Analyses of the ultrastructure and placental immunophysiology are important to understand the mechanism underlying RM. These findings may spark future studies and aid in the development of new therapies in cases of RM.

Involvement of AMPK, IKßα-NFκB and eNOS in the sildenafil anti-inflammatory mechanism in a demyelination model.

Sildenafil (Viagra®) has recently been found to have a neuroprotective effect, which occurs through the inhibition of inflammation and demyelination in the cerebellum. However, the mechanism of action of sildenafil remains unknown. AMPK, the regulatory protein of the lipid and glucose metabolism, plays a protective role by activating the eNOS enzyme. The production of a nanomolar concentration of NO by eNOS has an anti-inflammatory effect through the cGMP signaling pathway and plays an important role in the regulation of the nuclear transcription factor (NFkB), preventing the expression of inflammatory genes. The present study investigated whether AMPK-eNOS-NO-cGMP-IКßα-NFkB is involved in the mechanism of action of sildenafil in a cuprizone-demyelination model. Neuroinflammation and demyelination induced by cuprizone in rodents have been widely used as a model of MS. In the present study, five male C57BL/6 mice (7-10 weeks old) were used. Over a four week period, the groups received: cuprizone (CPZ) 0.2% mixed in feed; CPZ in the diet, combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently (sild-T0) or 15 days (sild-T15) after the start of CPZ. Control animals received pure food and water. The cerebella of the mice were dissected and processed for immunohistochemistry, immunofluorescence (frozen), western blotting and dosage of cytokines (Elisa). CPZ induced an increase in the expression of GFAP, IL-1ß TNF-α, total NFkB and inactive AMPK, and prompt microglia activation. CPZ also induced a reduction of IKßα. The administration of sildenafil reduced the expression of the pro-inflammatory cytokines IL-1ß and TNF-α and increased the expression of the anti-inflammatory cytokine IL-10. In addition, the administration of sildenafil reduced expression of GFAP, NFkB, inactive AMPK and iNOS, and increased IKßα. Interestingly, sildenafil also reduced levels of NGF. In general, the sild-T0 group was more effective than sild-T15 in improving clinical status and promoting the control of neuroinflammation. The present study offers evidence that sildenafil has anti-inflammatory and neuroprotective effects, which are probably achieved through modulation of AMPK-IKßα-NFκB signaling. In addition, eNOS may play a role in the sildenafil neuroprotective mechanism, contributing to the activation of AMPK. However, other pathways such as MAPK-NFkB and the downstream proteins AMPK (AMPK-SIRT1-NFκB) should also be further investigated. An understanding of these mechanisms of action is critical for the clinical use of sildenafil to control neuroinflammation in neurodegenerative diseases such as MS.

Diethylcarbamazine reduces chronic inflammation and fibrosis in carbon tetrachloride- (CCl4-) induced liver injury in mice.

This study investigated the anti-inflammatory effects of DEC on the CCl4-induced hepatotoxicity in C57BL/6 mice. Chronic inflammation was induced by i.p. administration of CCl4 0.5 µL/g of body weight through two injections a week for 6 weeks. DEC (50 mg/kg) was administered by gavage for 12 days before finishing the CCl4 induction. Histological analyses of the DEC-treated group exhibited reduced inflammatory process and prevented liver necrosis and fibrosis. Immunohistochemical and immunofluorescence analyses of the DEC-treated group showed reduced COX-2, IL1ß, MDA, TGF-ß, and αSMA immunopositivity, besides exhibiting decreased IL1ß, COX-2, NFκB, IFNγ, and TGFß expressions in the western blot analysis. The DEC group enhanced significantly the IL-10 expression. The reduction of hepatic injury in the DEC-treated group was confirmed by the COX-2 and iNOS mRNA expression levels. Based on the results of the present study, DEC can be used as a potential anti-inflammatory drug for chronic hepatic inflammation.

Diethylcarbamazine inhibits NF-κB activation in acute lung injury induced by carrageenan in mice.

Diethylcarbamazine citrate (DEC) is widely used to treat lymphatic filariasis and Tropical Pulmonary Eosinophilia. A number of studies have reported a possible role in the host immune system, but exactly how DEC exerts this effect is still unknown. The present study reports the effects of DEC pretreatment on NF-κB regulation using the pleurisy model induced by carrageenan. Swiss male mice (Mus musculus) were divided into four experimental groups: control (SAL); carrageenan (CAR); diethylcarbamazine (DEC) and curcumin (CUR). The animals were pretreated with DEC (50mg/kg, v.o), CUR (50mg/kg, i.p) or distilled water for three consecutive days before pleurisy. One way analysis of variance (ANOVA) was performed by Tukey post-hoc test, and values were considered statistically significant when p<0.05. DEC pretreatment reduced tissue damage and the production of inflammatory markers, such as NO, iNOS, PGE2, COX-2, and PARP induced by carrageenan. Similarly, a known inhibitor of NF-κB pathway (curcumin) was also able to reduce these parameters. Like curcumin, DEC prevents NF-κB activation by reducing NF-κB p65 phosphorylation and IκBα degradation. DEC prevented NF-κB activation via p38 MAPK, but did not interfere in the ERK pathway in this experimental model. However, further studies should be developed to confirm this hypothesis. These findings suggest that DEC could be a promising drug for inflammatory disorders, especially in pulmonary diseases such as Acute Lung Inflammation, due its high anti-inflammatory potential which prevents NF-κB activation.

Diethylcarbamazine attenuates the development of carrageenan-induced lung injury in mice.

Diethylcarbamazine (DEC) is an antifilarial drug with potent anti-inflammatory properties as a result of its interference with the metabolism of arachidonic acid. The aim of the present study was to evaluate the anti-inflammatory activity of DEC in a mouse model of acute inflammation (carrageenan-induced pleurisy). The injection of carrageenan into the pleural cavity induced the accumulation of fluid containing a large number of polymorphonuclear cells (PMNs) as well as infiltration of PMNs in lung tissues and increased production of nitrite and tumor necrosis factor-α and increased expression of interleukin-1ß, cyclooxygenase (COX-2), and inducible nitric oxide synthase. Carrageenan also induced the expression of nuclear factor-κB. The oral administration of DEC (50 mg/Kg) three days prior to the carrageenan challenge led to a significant reduction in all inflammation markers. The present findings demonstrate that DEC is a potential drug for the treatment of acute lung inflammation.

Effect of diethylcarbamazine on chronic hepatic inflammation induced by alcohol in C57BL/6 mice.

Some pharmacological studies showed that diethylcarbamazine (DEC) interferes with the arachidonic acid metabolism, acting as an anti-inflammatory drug. The chronic alcohol consumption activates the hepatic inflammatory response associated to T-cell activation and overproduction of pro-inflammatory cytokines. The present work analyzed the anti-inflammatory effect of DEC on hepatic cells of alcoholic mice. Thirty-two male C57BL/6 mice were equally divided in the following groups: (a) control group (C), which received only water, (b) DEC-treated group, which received 50 mg/kg for 12 day (DEC50), (c) the alcoholic group (EtOH), submitted to only alcohol and (d) the alcohol-DEC treated group (EtOH50), submitted to alcohol plus DEC treatment after the induction of chronic alcoholism for 5 weeks. Biochemical analyses were performed and liver fragments were processed for light microscopy, transmission electron microscopy, immunohistochemical and western blot. The level of AST increased significantly in alcoholic group whereas a significant reduction of serum AST was detected in the EtOH50 group. Histological and ultrastructural analysis of alcoholic group showed evident hepatocellular damage, which was strikingly reduced in the alcoholic DEC-treated group. Immunohistochemistry results revealed highly expression of inflammatory markers as MDA, NF-κB, TNF-α, IL-6, VCAM and ICAM by the hepatic cells of the EtOH group; however no immunoreactivity for any of these cytokines was detected after DEC treatment. Western blot analyses showed increased MCP-1 and iNOS expression in EtOH group, which was significantly inhibited by DEC treatment. According to the present results, DEC can be a potential drug for the treatment of chronic inflammation induced by chronic alcoholism.