Gasoline fume inhalation induces apoptosis, inflammation, and favors Th2 polarization in C57BL/6 mice.

Gasoline exposure has been widely reported in the literature as being toxic to human health. However, the exact underlying molecular mechanisms triggered by its inhalation have not been thoroughly investigated. We herein present a model of sub-chronic, static gasoline vapor inhalation in adult female C57BL/6 mice. Animals were exposed daily to either gasoline vapors (0.86 g/animal/90 min) or ambient air for 5 days/week over 7 consecutive weeks. At the end of the study period, toxic and molecular mechanisms underlying the inflammatory, oxidative, and apoptotic effects triggered by gasoline vapors, were examined in the lungs and liver of gasoline-exposed (GE) mice. Static gasoline exposure induced a significant increase (+21%) in lungs/body weight (BW) ratio in GE versus control (CON) mice along with a pulmonary inflammation attested by histological staining. The latter was consistent with increases in the transcript levels of proinflammatory cytokines [Interleukins (ILs) 4 and 6], respectively by ~ 6- and 4-fold in the lungs of GE mice compared to CON. Interestingly, IL-10 expression was also increased by ~ 10-fold in the lungs of GE mice suggesting an attempt to counterbalance the established inflammation. Moreover, the pulmonary expression of IL-12 and TNF-α was downregulated by 2- and 4-fold, respectively, suggesting the skewing toward Th2 phenotype. Additionally, GE mice showed a significant upregulation in Bax/Bcl-2 ratio, caspases 3, 8, and 9 with no change in JNK expression in the lungs, suggesting the activation of both intrinsic and extrinsic apoptotic pathways. Static gasoline exposure over seven consecutive weeks had a minor hepatic portal inflammation attested by H&E staining along with an increase in the hepatic expression of the mitochondrial complexes in GE mice. Therefore, tissue damage biomarkers highlight the health risks associated with vapor exposure and may present potential therapeutic targets for recovery from gasoline intoxication.

Cardiac Phosphodiesterases Are Differentially Increased in Diabetic Cardiomyopathy.

AIM: Diabetic cardiomyopathy (DCM) accomodates a spectrum of cardiac abnormalities. This study aims to investigate whether DCM is associated with changes in cyclic adenosine 3'-5' monophosphate (cAMP) signaling, particularly cyclic nucleotide phosphodiesterases (PDEs). MAIN METHODS: Type 1 diabetes (T1D) was induced in rats by streptozotocin (STZ, 65 mg/kg) injection. Myocardial remodeling, structure and function were evaluated by histology and echocardiography, respectively. We delineated the sequential changes affecting cAMP signaling and characterized the expression pattern of the predominant cardiac PDE isoforms (PDE 1-5) and ß-adrenergic (ß-AR) receptors at 4, 8 and 12 weeks following diabetes induction, by real-time quantitative PCR and Western blot. cAMP levels were measured by immunoassays. KEY FINDINGS: T1D-induced DCM was associated with cardiac remodeling, steatosis and fibrosis. Upregulation of ß1-AR receptor transcripts was noted in diabetic hearts at 4 weeks along with an increase in cAMP levels and an upregulation in the ejection fraction and fraction shortening. However, ß2-AR receptors expression remained unchanged regardless of the disease stage. Moreover, we noted an early and specific upregulation of cardiac PDE1A, PDE2A, PDE4B, PDE4D and PDE5A expression at week 4, followed by increases in PDE3A levels in diabetic hearts at week 8. However, DCM was not associated with changes in PDE4A gene expression irrespective of the disease stage. SIGNIFICANCE: We show for the first time differential and time-specific regulations in cardiac PDEs, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Evaluation of waterpipe smoke toxicity in C57BL/6 mice model.

Waterpipe smoking is a popular pastime worldwide with statistics pointing to an alarming increase in consumption. In the current paper, the evaluation of sub-chronic waterpipe smoke exposure was undertaken using C57BL/6 female mice using a dynamic exposure setting to emulate smoke exposure. Mice were daily subjected to either one (single exposure, SE) or two sessions (double exposure, DE) of waterpipe-generated smoke (two-apple flavor) for a period of two months. Although lungs histopathological examination pointed to a minor inflammation in smoke-exposed mice compared to control air-exposed (CON) group, the lung weights of the waterpipe-exposed mice were significantly higher (+72% in SE and +39% in DE) (p < 0.01) when compared to CON group. Moreover, changes in the protein expression of several proteins such as iNOS and JNK were noted in the lungs of smoke-exposed mice. However, no changes in p38 and EGFR protein levels were noted between the three groups of mice. Our results mainly showed a significant increase in urea serum levels (+28%) in SE mice along with renal pathological damage in both SE and DE mice compared to CON. Additionally, severe significant DNA damages (p < 0.05) were reported in the lungs, kidneys, bone marrow and liver of waterpipe-exposed animals, using MTS and COMET assays. These findings highlighted the significant risks posed by sub-chronic waterpipe smoke exposure in the selected animal model and the pressing need for future better management of waterpipe indoor consumption.

Ghrelin-induced multi-organ damage in mice fed obesogenic diet.

OBJECTIVE AND DESIGN: Ghrelin has a key role in modulating energy metabolism and weight gain. The present study aimed at studying the potential role of ghrelin in the development and/or exacerbation of organ damage in a mouse model of diet-induced obesity. OBJECTIVE AND DESIGN: Adult mice were fed one of two diets for 20 weeks: standard high carbohydrate (HC) or high-fat high-sugar (HFHS). Starting week 17, the animals were given regular intraperitoneal ghrelin (160 µg/kg) or saline injections Abdominal fat, serum creatinine, and glucose levels, as well as kidney, liver and heart weight and pathology were assessed. RESULTS: Ghrelin-injected mice showed significant organ damage, which was more exacerbated in HFHS-fed animals. While the HFHS diet was associated with significant liver damage, ghrelin administration did not reverse it. Interestingly, ghrelin administration induced moderate kidney damage and significantly affected the heart by increasing perivascular and myocardium fibrosis, steatosis as well as inflammation. Moreover, serum creatinine levels were higher in the animal group injected with ghrelin. CONCLUSION: Ghrelin administration was associated with increased functional and structural organ damage, regardless of diet. The present study provides novel evidence of multi-organ physiologic alterations secondary to ghrelin administration.

Chemical Composition, Antioxidant and Anti-inflammatory Activity Evaluation of the Lebanese Propolis Extract.

BACKGROUND: Propolis is a resinous substance produced by bees and known to possess antioxidant, antimicrobial, antiproliferative and anti-inflammatory activities. OBJECTIVE: This study is aimed at evaluating the in vivo and in vitro anti-inflammatory potential of the Crude Ethanolic Extract (CE) of Lebanese propolis and its Ethyl Acetate Fraction (EAF). METHOD: Chemical content of propolis was characterized using high-performance liquid chromatography and LC-MS/MS. COX-2 and iNOS protein expression, nitric oxide (NO) and prostaglandin (PGE2) release in LPS-activated RAW monocytes were achieved respectively by western blot and spectrophotometry. Antioxidant activity was evaluated by DPPH free radical scavenging assay. Measurement of paw thickness in carrageenan-induced paw edema in mice and pathologic assessment of inflammation in paw sections were used to judge the anti-inflammatory properties of propolis. RESULTS: Pathology analysis revealed in the treated group significant reduction of immune cell infiltration and edema. Both extract and ethyl acetate fraction showed significant anti-inflammatory and antioxidant effects in LPS-treated RAW cells characterized by the inhibition of COX-2 and iNOS protein expression, as well as PGE2 and NO release. Chemical analysis of the crude extract and its ethyl acetate fraction identified 28 different compounds of which two phenolic acids and nine other flavonoids were also quantified. Ferulic acid, caffeic acid, chrysin, galangin, quercetin, and pinocembrin were among the most representative compounds. CONCLUSION: Lebanese propolis is rich in a various amount of flavonoids which showed promising antiinflammatory and antioxidant properties. Additionally, chemical analysis showed unique chemical compositions with the potential of identifying ingredients with interesting anti-inflammatory activities.

Acute Exposure to Cigarette Smoking Followed by Myocardial Infarction Aggravates Renal Damage in an In Vivo Mouse Model.

Cigarette smoking (S) is a risk factor for progressive chronic kidney disease, renal dysfunction, and renal failure. In this study, the effect of smoking on kidney function was investigated in a mouse model of myocardial infarction (MI) using 4 groups: control (C), smoking (S), MI, and S+MI. Histological analysis of S+MI group showed alterations in kidney structure including swelling of the proximal convoluted tubules (PCTs), thinning of the epithelial lining, focal loss of the brush border of PCTs, and patchy glomerular retraction. Molecular analysis revealed that nephrin expression was significantly reduced in the S+MI group, whereas sodium-hydrogen exchanger-1 (NHE-1) was significantly increased, suggesting altered glomerular filtration and kidney functions. Moreover, S+MI group, but not S alone, showed a significant increase in the expression of connective tissue growth factor (CTGF) and fibrotic proteins fibronectin (FN) and α-smooth muscle actin (SMA), in comparison to controls, in addition to a significant increase in mRNA levels of IL-6 and TNF-α inflammatory markers. Finally, reactive oxygen species (ROS) production was significantly accentuated in S+MI group concomitant with a significant increase in NOX-4 protein levels. In conclusion, smoking aggravates murine acute renal damage caused by MI at the structural and molecular levels by exacerbating renal dysfunction.

An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and ß-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and ß-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and ß-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

Cafeteria diet-fed mice is a pertinent model of obesity-induced organ damage: a potential role of inflammation.

OBJECTIVE: This study is aimed at evaluating the effects of a cafeteria diet (obesity) mouse model on early multi-organ functional, structural, endocrine and biochemical alterations. MATERIALS AND METHODS: Multi-organ damage is assessed using clinical, biochemical, pathological, and inflammatory parameters in 30 mice fed one of the three diets for 15 weeks: standard chow diet (SC), high fat (HF), or "Cafeteria diet" (CAF) (standard SC and a choice of highly palatable human cafeteria foods: chocolate, biscuits, and peanut butter). RESULTS: CAF diet was associated with an increase in body weight, energy intake, and serum cholesterol levels compared to the other diets, as well as higher insulin levels and lower glucose tolerance. Additionally, consumption of the CAF diet was associated with significantly higher weight gain, abdominal fat, and serum IL-6 levels, as well as more damage in the heart (coronary perivascular fibrosis and steatosis), kidney (chronic interstitial inflammation and glomerular sclerosis), and liver (liver weight, portal fibrosis, apoptosis, and steatosis) compared to the HF diet. CONCLUSION: Functional and structural damage in CAF were higher than HF of similar macronutrient composition. This study provides a novel dietary model in mice that mimics multi-organ physiologic alterations in humans secondary to obesity.