Limitations of Limulus amebocyte lysate test for endotoxin control in raw materials for liposomal nanoformulations.

Aim: To evaluate the applicability of Limulus amebocyte lysate (LAL) assay for endotoxin determination in lipid compounding liposomal nanoformulations.Materials & methods: Spiked cholesterol, hydrogenated soy phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG 2000) samples with endotoxins, simulating contaminated samples or in-process contamination were analyzed by chromogenic LAL assay.Results: Recovery of spiked endotoxins was achieved from DSPE-PEG 2000 suspended in water, whereas recovery was not achieved from spiked cholesterol and hydrogenated soy phosphatidylcholine suspended in methanol, and from multilamellar vesicles. Conclusion: Endotoxins, when in contact with organic solvents, no longer react in the LAL assay as they do in aqueous media. This indicates limitations of the LAL assay for endotoxin control in raw materials for liposomal nanoformulations.

[Box: see text].

Nano-phytosome loaded Retama raetam extract/colistin: antibacterial, antioxidant activities and in vivo lipopolysaccharide-induced-neurotoxicity inhibition.

Antibiotics are misused nowadays, leading to the prevalence of antibiotic resistant bacterial strains; causing the world to move towards natural medicine. Retama raetam had wide medicinal use. In the present study, R. raetam ethanolic extract proved to be active against Pseudomonas aeruginosa with MIC values ranged from 15.62 to 250 µg/ml. Antioxidant analysis showed that the extract had high scavenging activity reached 92.40%. GC/MS analysis revealed that Sparteine and Tributyl acetylcitrate represent the extract major components. Furthermore, the combination between Retama raetam extract and colistin showed a synergistic effect. Moreover, nano-phytosome was designated and optimized to encapsulate Retama raetam extract/Colistin. Nano-phytosome characterized by particle size, Zeta potential, polydispersity index and Entrapment efficiency percentage of 16.92-32.85 nm, -30.40 mV, 0.26 and 89% respectively. The antibacterial activity of the prepared nano-phytosome formula against P. aeruginosa showed promising MIC, MBC, MIC index, and IZ diameter reaching 7.81, 15.62 µg/ml, 2, and 39 mm, respectively. While TEM examination of P. aeruginosa cells treated with nano-phytosome formula revealed cell wall breakage which led to cell death. Finally, P. aeruginosa LPS was used to induce neurodegenerative disease in rat model. Rats treated with nano-phytosome formula showed normal histoarchitecture organization and the cerebral cortex was partially restored compared to control groups.

Age and sex-dependent gut alterations in mice induced by neonatal immune activation with lipopolysaccharide.

Neonatal immune activation (NIA) through exposure to lipopolysaccharide (LPS) induces adult behavioral changes in rodents that resemble symptoms of developmental disorders, such as autism spectrum disorder. The neonatal timing of LPS exposure appears to play a crucial role in determining the nature and extent of long-term changes. This study aims to explore whether a 3-day LPS-NIA triggers sex- and age-related changes in gut function, potentially linking LPS-NIA to gastrointestinal dysfunction. Male and female Swiss mice received intraperitoneal injections of LPS or saline on postnatal days (PN) 3, 5, and 7. At PN35 (juvenile) and PN70 (adult), gut inflammation and oxidative stress were evaluated in addition to assessments of working memory, depressive-like symptoms, sociability, and repetitive behavior. Gut examination showed elevated C-X-C motif chemokine receptor 3 (CXCR3) in LPS-NIA mice, while MyD88 and Zonulin expressions were significantly higher only in adult LPS-NIA females. Interleukin (IL)-23 expression increased in juvenile and adult male and juvenile female LPS-NIA mice. Oxidative changes included decreased duodenal reduced glutathione (GSH) in juvenile females and ileal GSH in adult females exposed to LPS-NIA. Regarding behavioral alterations, adult LPS-NIA females exhibited depressive-like behavior. Working memory deficits were observed across all LPS-NIA groups. Only juvenile LPS-NIA females increased grooming, while rearing was higher in adult LPS-NIA mice of both sexes. The findings imply that LPS-NIA impacts intestinal barrier function and causes gut inflammatory alterations that are sex- and age-specific. These findings pave the way for exploring potential mechanisms that could contribute to LPS-induced gastrointestinal disturbances among individuals with ASD.

Sexual dimorphism of hypothalamic serotonin release during systemic inflammation: Role of endothelin-1.

The hypothalamus receives serotonergic projections from the raphe nucleus in a sex-specific manner. During systemic inflammation, hypothalamic levels of serotonin (5-hydroxytryptamine [5-HT]) decrease in male rats. The present study evaluated the involvement of endothelin-1 (ET-1) in the febrile response, hypolocomotion, and changes in hypothalamic 5-HT levels during systemic inflammation in male and female rats. An intraperitoneal injection of lipopolysaccharide (LPS) induced a febrile response and hypolocomotion in both male and female rats. However, although LPS reduced hypothalamic levels of 5-HT and its metabolite 5-hydroxyindol acetic acid (5-HIAA) in male rats, it increased these levels in female rats. An intracerebroventricular injection of the endothelin-B receptor antagonist BQ788 significantly reduced LPS-induced fever and hypolocomotion and changes in hypothalamic 5-HT and 5-HIAA levels in both male and female rats. The i.c.v. administration of ET-1 induced a significant fever and hypolocomotion, but reduced the hypothalamic levels of 5-HT and 5-HIAA in both males and females. These results suggest an important sexual dimorphism during systemic inflammation regarding the release of 5-HT in the hypothalamus. Moreover, ET-1 arises as an important mediator involved in the changes in hypothalamic 5-HT levels in both male and female rats.

Applicability of mouse models for induction of severe acute lung injury.

Acute lung injury (ALI) is a significant clinical challenge associated with high morbidity and mortality. Worldwide, it affects approximately 200.000 individuals annually, with a staggering 40 % mortality rate in hospitalized cases and persistent complications in out-of-hospital cases. This review focuses on the key immunological pathways underlying bacterial ALI and the exploration of mouse models as tools for its induction. These models serve as indispensable platforms for unraveling the inflammatory cascades and biological responses inherent to ALI, while also facilitating the evaluation of novel therapeutic agents. However, their utility is not without challenges, mainly due to the stringent biosafety protocols required by the diverse bacterial virulence profiles. Simple and reproducible models of pulmonary bacterial infection are currently available, including intratracheal, intranasal, pleural and, intraperitoneal approaches. These models use endotoxins such as commercially available lipopolysaccharide (LPS) or live pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Streptococcus pneumoniae, all of which are implicated in the pathogenesis of ALI. Combining murine models of bacterial lung infection with in-depth studies of the underlying immunological mechanisms is a cornerstone in advancing the therapeutic landscape for acute bacterial lung injury.

Kinin B1 receptor and TLR4 interaction in inflammatory response.

OBJECTIVE: We aimed to broaden our understanding of a potential interaction between B1R and TLR4, considering earlier studies suggesting that lipopolysaccharide (LPS) may trigger B1R stimulation. METHODS: We assessed the impact of DBK and LPS on the membrane potential of thoracic aortas from C57BL/6, B1R, or TLR4 knockout mice. Additionally, we examined the staining patterns of these receptors in the thoracic aortas of C57BL/6 and in endothelial cells (HBMEC). RESULTS: DBK does not affect the resting membrane potential of aortic rings in C57BL/6 mice, but it hyperpolarizes preparations in B1KO and TLR4KO mice. The hyperpolarization mechanism in B1KO mice involves B2R, and the TLR4KO response is independent of cytoplasmic calcium influx but relies on potassium channels. Conversely, LPS hyperpolarizes thoracic aorta rings in both C57BL/6 and B1KO mice, with the response unaffected by a B1R antagonist. Interestingly, the absence of B1R alters the LPS response to potassium channels. These activities are independent of nitric oxide synthase (NOS). While exposure to DBK and LPS does not alter B1R and TLR4 mRNA expression, treatment with these agonists increases B1R staining in endothelial cells of thoracic aortic rings and modifies the staining pattern of B1R and TLR4 in endothelial cells. Proximity ligation assay suggests a interaction between the receptors. CONCLUSION: Our findings provide additional support for a putative connection between B1R and TLR4 signaling. Given the involvement of these receptors and their agonists in inflammation, it suggests that drugs and therapies targeting their effects could be promising therapeutic avenues worth exploring.

Functional infrared thermography imaging can be used to assess the effectiveness of Maxicam Gel® in pre-emptively treating transient synovitis and lameness in horses.

Introduction: Diagnosing and treating lameness in horses is essential to improving their welfare. In equine orthopedic practice, infrared thermography (IRT) can indirectly detect soreness. Non-steroidal anti-inflammatory drugs can treat painful and inflammatory processes in horses. Using IRT, the efficacy of meloxicam (Maxicam Gel®) was evaluated in pre-treating transient synovitis in horses induced by a middle carpal joint injection of lipopolysaccharides (LPS) from E. coli 055:B5 at a dose of 10 endotoxin units. Methods: In a cross-over design, six healthy horses were randomly assigned to receive either 0.6 mg/kg of oral Maxicam Gel® (MAXVO) or a mock administration (control group, C) following a two-week washout period. IRT of the middle carpal joint, visual lameness assessment and joint circumference were recorded over time. Clinical and hematological evaluations were performed. Synovial fluid aspirates were analyzed for total nucleated cell count, total protein, and prostaglandin E2. A mixed effects analysis of variance was performed for repeated measures over time, followed by Tukey's test. A multinomial logistic regression was conducted to determine whether there is a relationship between a thermography temperature change and the lameness score. Results: There were no changes in joint circumference. The MAXVO group showed a lower rectal temperature 4 h after synovitis induction. The C group presented an increase in neutrophils and a decrease in total hemoglobin and hematocrit 8 h after induction. No changes were observed in the synovial fluid between groups. The horses that received meloxicam did not show clinically significant lameness at any time, while the C group showed an increase in lameness 2, 4, and 8 h after synovitis induction. Discussion: IRT indicated that the skin surface temperature of the middle carpal joint was lower in horses who received meloxicam, suggesting a reduction in the inflammatory process induced by LPS. It was observed that the maximum temperature peaks in the dorsopalmar and lateropalmar positions can be utilized to predict the severity of lameness, particularly when the temperature rises above 34°C. Horses pre-treated with meloxicam showed either reduced or no indication of mild to moderate pain and presented a lowehr thermographic temperature, which indicates the effectiveness of Maxicam Gel® as an anti-inflammatory.

Study of two glycosyltransferases related to polysaccharide biosynthesis in Rhodococcus jostii RHA1.

The bacterial genus Rhodococcus comprises organisms performing oleaginous behaviors under certain growth conditions and ratios of carbon and nitrogen availability. Rhodococci are outstanding producers of biofuel precursors, where lipid and glycogen metabolisms are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying catalytic steps redirecting sugar moieties to storage molecules. Here, we analyzed two GT4 glycosyl-transferases from Rhodococcus jostii (RjoGlgAb and RjoGlgAc) annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were produced in Escherichia coli cells, purified to homogeneity, and kinetically characterized. RjoGlgAb and RjoGlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, RjoGlgAb exhibited both activities with similar kinetic efficiency and preference for short-branched α-1,4-glucans. Curiously, RjoGlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure-to-function relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view.

The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

Dietary Hydroxytyrosol Supplementation on Growth Performance, Gut Morphometry, and Oxidative and Inflammatory Status in LPS-Challenged Broilers.

This study assessed the effects of hydroxytyrosol (HT) on 8- to 20-day-old broilers challenged with lipopolysaccharide (LPS); 180 Cobb500™ male chicks were randomly assigned to 3 treatment groups, each comprising 10 replicates with 6 birds per replicate. Treatments included a control diet (CON), CON with LPS administration, and CON + LPS supplemented with 10 mg of HT/kg of feed. LPS was administered intraperitoneally on days 14, 16, 18, and 20. Body weight (BW), body weight gain (BWG), and the feed conversion ratio (FCR) were measured. On day 20, ten birds per treatment were slaughtered for analysis. Bursa, spleen, and liver were collected, and their respective relative weight was determined. The jejunum was destined for morphological analyses of villus height (VH), crypt depth (CD), and their ratio (VH:CD), and for mRNA expression of nuclear factor kappa B (NF-κB), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and interleukins 10 (IL-10), 1 beta (IL-1ß), and 8 (IL-8). HT improved BW, BWG, and FCR, and reduced crypt depth (CD) while increasing the VH:CD ratio in the jejunum. Moreover, HT downregulated mRNA expression of CAT, GPx, IL-10, and IL-1ß. In conclusion, HT enhances broiler growth performance, mitigates jejunal mucosa damage from LPS, and modulates antioxidant and immune responses.

In Vitro Astroglial Dysfunction Induced by Neurotoxins: Mimicking Astrocytic Metabolic Alterations of Alzheimer's Disease.

Astrocytes play fundamental roles in the maintenance of brain homeostasis. The dysfunction of these cells is widely associated with brain disorders, which are often characterized by variations in the astrocyte protein markers GFAP and S100B, in addition to alterations in some of its metabolic functions. To understand the role of astrocytes in neurodegeneration mechanisms, we induced some of these metabolic alterations, such as energy metabolism, using methylglyoxal (MG) or fluorocitrate (FC); and neuroinflammation, using lipopolysaccharide (LPS) and streptozotocin (STZ), which is used for inducing Alzheimer's disease (AD) in animal models. We showed that MG, LPS, STZ and FC similarly caused astrocyte dysfunction by increasing GFAP and reducing S100B secretion. In the context of AD, STZ caused an amyloid metabolism impairment verified by increases in Aß1-40 peptide content and decreases in the amyloid degradation enzymes, IDE and NEP. Our data contribute to the understanding of the role of astrocytes in brain injury mechanisms and suggest that STZ is suitable for use in vitro models for studying the role of astrocytes in AD.

The protective effects of naringenin, a citrus flavonoid, non-complexed or complexed with hydroxypropyl-ß-cyclodextrin against multiorgan damage caused by neonatal endotoxemia.

BACKGROUND: Endotoxemia is a severe and dangerous clinical syndrome that results in elevated morbidity, especially in intensive care units. Neonates are particularly susceptible to endotoxemia due to their immature immune systems. There are few effective treatments for neonatal endotoxemia. One group of compounds with potential in the treatment of neonatal inflammatory diseases such as endotoxemia is the flavonoids, mainly due to their antioxidant and anti-inflammatory properties. Among these, naringenin (NGN) is a citrus flavonoid which has already been reported to have anti-inflammatory, antioxidant, anti-nociceptive and anti-cancer effects. Unfortunately, its clinical application is limited by its low solubility and bioavailability. However, cyclodextrins (CDs) have been widely used to improve the solubility of nonpolar drugs and enhance the bioavailability of these natural products. OBJECTIVE: We, therefore, aimed to investigate the effects of NGN non-complexed and complexed with hydroxypropyl-ß-cyclodextrin (HPßCD) on neonatal endotoxemia injuries in a rodent model and describe the probable molecular mechanisms involved in NGN activities. METHOD: We used exposure to a bacterial lipopolysaccharide (LPS) to induce neonatal endotoxemia in the mice. RESULTS: It was found that NGN (100 mg/kg i.p.) exposure during the neonatal period reduced leukocyte migration and decreased pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) levels in the lungs, heart, kidneys or cerebral cortex. In addition, NGN upregulated IL-10 production in the lungs and kidneys of neonate mice. The administration of NGN also enhanced antioxidant enzyme catalase and SOD activity, reduced lipid peroxidation and protein carbonylation and increased the reduced sulfhydryl groups in an organ-dependent manner, attenuating the oxidative damage caused by LPS exposure. NGN decreased ERK1/2, p38MAPK and COX-2 activation in the lungs of neonate mice. Moreover, NGN complexed with HPßCD was able to increase the animal survival rate. CONCLUSION: NGN attenuated inflammatory and oxidative damage in the lungs, heart and kidneys caused by neonatal endotoxemia through the MAPK signaling pathways regulation. Our results show that NGN has beneficial effects against neonatal endotoxemia and could be useful in the treatment of neonatal inflammatory injuries.

Characterization of extracellular trap production and release by equine neutrophils in response to different stimuli.

This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets.

Lack of Annexin A1 Exacerbates Inflammatory Response in Acute Endometritis Model.

Annexin A1 (AnxA1) is a glucocorticoid-inducible protein and an important endogenous modulator of inflammation. However, its effect in the endometrial microenvironment is poorly explained. This study aimed to evaluate the role of endogenous AnxA1 in an endometritis mouse model induced by lipopolysaccharide (LPS). Female C57BL/6 wild-type (WT) and AnxA1-/- mice were divided into two groups: SHAM and LPS. To induce endometritis, mice received a vaginal infusion of 50 µL of LPS (1 mg/mL) dissolved in phosphate-buffered saline. After 24 h, the mice were euthanized, and blood and uteri samples were collected. The endometrium inflammatory scores were significantly increased in the LPS-treated group. AnxA1-/- mice from the LPS group demonstrated a significant increase in the number of degranulated mast cell levels compared to AnxA1-/- SHAM mice. The Western blotting analysis revealed that a lack of AnxA1 promoted the upregulation of NLRP3 and pro-IL-1ß in the acute endometritis animal model compared to WT LPS animals. LPS-induced endometritis increased the number of blood peripheral leukocytes in both WT and AnxA1-/- mice compared with SHAM group mice (p < 0.001). AnxA1-/- mice also showed increased plasma levels of IL-1ß (p < 0.01), IL-6, IL-10, IL-17, and TNF-α (p < 0.05) following LPS-induced endometritis. In conclusion, a lack of endogenous AnxA1 exacerbated the inflammatory response in an endometritis model via NLRP3 dysregulation, increased uterine mast cell activation, and plasma pro-inflammatory cytokine release.

Effects of Branched-Chain Amino Acids on the Inflammatory Response Induced by LPS in Caco-2 Cells.

Branched-chain amino acids (BCAA) are essential for maintaining intestinal mucosal integrity. However, only a few studies have explored the role of BCAA in the modulation of intestinal inflammation. In this study, we investigated in vitro effects of BCAA on the inflammatory response induced by lipopolysaccharide (LPS) (1 µg/mL) in Caco-2 cells. Caco-2 cells were assigned to six groups: control without BCAA (CTL0), normal BCAA (CTL; 0.8 mM leucine, 0.8 mM isoleucine, and 0.8 mM valine); leucine (LEU; 2 mM leucine), isoleucine (ISO; 2 mM isoleucine), valine (VAL; 2 mM valine), and high BCAA (LIV; 2 mM leucine, 2 mM isoleucine, and 2 mM valine). BCAA was added to the culture medium 24 h before LPS stimulation. Our results indicated that BCAA supplementation did not impair cell viability. The amino acids leucine and isoleucine attenuated the synthesis of IL-8 and JNK and NF-kB phosphorylation induced by LPS. Furthermore, neither BCAA supplementation nor LPS treatment modulated the activity of glutathione peroxidase or the intracellular reduced glutathione/oxidized glutathione ratio. Therefore, leucine and isoleucine exert anti-inflammatory effects in Caco-2 cells exposed to LPS by modulating JNK and NF-kB phosphorylation and IL-8 production. Further in vivo studies are required to validate these findings and gather valuable information for potential therapeutic or dietary interventions.

Quantification of Enteric Dysfunction in Cystic Fibrosis: Inter- and Intraindividual Variability.

OBJECTIVES: To test the utility of various biomarkers as indicators of gut dysfunction in cystic fibrosis (CF) and determine whether intraindividual variations in these measures are repeatable over short intervals and whether interindividual variations correlate with clinical outcomes. STUDY DESIGN: We performed a cross-sectional, limited longitudinal study of children with CF aged 1-21 years who provided blood and stool samples at 2 or 3 visits, 2 weeks and 3 months apart, which were assayed for markers of intestinal inflammation (fecal calprotectin [fCal], lipocalin-2 [fLcn2], neopterin), and permeability (plasma lipopolysaccharide [LPS] antibodies, LPS-binding protein) by enzyme immunoassays. Control specimens were obtained from children without CF who had undergone esophagogastroduodenoscopy and had no evidence of gut inflammation. RESULTS: Twenty-six of 29 participants with CF completed the study. Sixty-nine stools (57 case/12 control) and 76 plasmas (60 case/16 control) were analyzed. LPS antibody had reliable intraindividual stability. fCal, fLcn2, and neopterin were significantly greater in CF than in control samples. fCal was negatively correlated with 3-month interval change (Δ) in weight-for-age z-score, body mass index/weight-for-length z-score, and forced expiratory volume in 1 second. fLcn2 was negatively correlated with FEV1 but not with anthropometrics. No marker correlated with Δbody mass index/weight-for-length z-score or ΔFEV1. CONCLUSIONS: fLcn2 is elevated in people with CF and might predict worse interval pulmonary function. Expanded studies are warranted to test if fLcn2 correlates with changes in additional outcomes.

Evaluation of the anti-inflammatory effect of 1,4-dihydropyridine derivatives.

INTRODUCTION: Inflammation is a physiological event that protects the organism against different factors that lead to loss of tissue homeostasis. Dihydropyridine (DHP) derivatives are heterocyclic compounds known for their different biological activities, including anti-inflammatory activities. OBJECTIVE: To evaluate the anti-inflammatory activity of 1,4-dihydropyridine (1,4-DHP) derivatives using anti-inflammatory models in vitro, in RAW264.7 cells induced by lipopolysaccharide (LPS) and in vivo using the acute lung injury (ALI) model in mice. RESULTS: Fifteen compounds derived from 1,4-DHP were tested in RAW264.7 cells for their cytotoxic effect and cell viability. Thereafter, only the six compounds that showed the highest cell viability were tested for the production or inhibition of the pro-inflammatory cytokine interleukin 6 (IL-6). The best compound (compound 4) was tested for its anti-inflammatory effects in vitro and in vivo, showing inhibition of nitric oxide (NO), pro-inflammatory cytokines, increased phagocytic activity, and an increase in IL-10 in vitro. In in vivo tests, compound 4 also reduces the levels of NO, myeloperoxidase (MPO) activity, leukocyte migration, and exudation, as well as reducing the levels of tumor necrosis factor-alpha (TNF-α) and IL-6 and preventing the loss in the lung architecture. CONCLUSION: This compound showed important anti-inflammatory activity, with a significant ability to reduce the production of pro-inflammatory mediators and increase the phagocytic activity of macrophages and anti-inflammatory mediator secretion (IL-10). These findings led us to hypothesize that this compound can repolarize the macrophage response to an anti-inflammatory profile (M2). Moreover, it was also able to maintain its anti-inflammatory activity in vivo experiments.

Fibrillation of α-synuclein triggered by bacterial endotoxin and lipid vesicles is modulated by N-terminal acetylation and familial Parkinson's disease mutations.

It has been hypothesized that --Parkinson's disease (PD) may be initiated in the gastrointestinal tract, before manifesting in the central nervous system. In this respect, it was demonstrated that lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, accelerates the in vitro formation of α-synuclein (aSyn) fibrils, whose intracellular deposits is a histological hallmark of the degeneration of dopaminergic neurons in PD. Herein, N-terminal acetylation and missense mutations of aSyn (A30P, A53T, E46K, H50Q and G51D) linked to rare, early-onset forms of familial PD were investigated regarding their effect on aSyn aggregation stimulated by either LPS or small unilamellar lipid vesicles (SUVs). Our findings indicated that LPS as well as SUVs induce the fibrillation of N-terminally acetylated wild-type aSyn (Ac-aSyn-WT) more remarkably than the non-acetylated protein, while the LPS-free protein alone did not undergo fibrillation under our assay conditions. In addition, with the exception of A30P, PD mutations increased the fibrillation of Ac-aSyn in the presence of LPS compared with Ac-aSyn-WT. The most pronounced effect of LPS was noticed for A53T, as observed when either Thioflavin-T or JC-1 were used as fluorescent probes for fibrils. Overall, our results suggest for the first time the existence of a synergy between LPS and PD mutations/N-terminal acetylation toward aSyn fibrillation.

Garlic prevents the oxidizing and inflammatory effects of sepsis induced by bacterial lipopolysaccharide at the systemic and aortic level in the rat. Role of trpv1.

Garlic (Allium sativum) possesses healing properties for diseases like systemic arterial hypertension, cancer and diabetes, among others. Its main component, allicin, binds to the Transient Receptor Potential Vanilloid Type 1 (TRPV1). In this study, we investigated TRPV1's involvement in the regulation of various molecules at the systemic and aortic levels in Wistar rats treated with bacterial lipopolysaccharide (LPS) and garlic to activate the receptor. The experimental groups were as follows: 1) Control, 2) LPS, 3) Garlic, and 4) LPS + Garlic. Using Uv-visible spectrophotometry and capillary zone electrophoresis, we measured the levels of nitric oxide (NO), biopterins BH2 and BH4, total antioxidant capacity (TAC) and oxidizing capacity (OXCA). We also analyzed molecules related to vascular homeostasis such as angiotensin Ang 1-7 and Ang II, as well as endothelin ET-1. In addition, we assessed the inflammatory response by determining the levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and galectin-3 (GTN-3). For cell damage assessment, we measured levels of malondialdehyde (MDA), malonate (MTO) and 8-hydroxy-2-deoxyguanosine (8HO2dG). The results showed that LPS influenced the NO pathway at both systemic and aortic levels by increasing OXCA and reducing TAC. It also disrupted vascular homeostasis by increasing Ang-II and ET-1, while decreasing Ang1-7 levels. IL-6, TNFα, GTN-3, as well as MDA, MTO, and 8HO2dG were significantly elevated compared to the control group. The expression of iNOS was increased, but TRPV1 remained unaffected by LPS. However, garlic treatment effectively mitigated the effects of LPS and significantly increased TRPV1 expression. Furthermore, LPS caused a significant decrease in calcitonin gene-related peptide (CGRP) in the aorta, which was counteracted by garlic treatment. Overall, TRPV1 appears to play a crucial role in regulating oxidative stress and the molecules involved in damage and inflammation induced by LPS. Thus, studying TRPV1, CGRP, and allicin may offer a potential strategy for mitigating inflammatory and oxidative stress in sepsis.

Long-term LPS systemic administration leads to memory impairment and disturbance in astrocytic homeostasis.

Dementia is the most prevalent neurodegenerative disorder, characterized by progressive loss of memory and cognitive function. Inflammation is a major aspect in the progression of brain disorders, and inflammatory events have been associated with accelerated deterioration of cognitive function. In the present work, we investigated the impact of low-grade repeated inflammation stimuli induced by lipopolysaccharide (LPS) in hippocampal function and spatial memory. Adult male Wistar rats received a weekly injection of LPS (500 ug/kg) for sixteen weeks, eliciting systemic inflammation. Animals submitted to LPS presented impaired spatial memory and neuroinflammation. While neuronal synaptic markers such as synaptophysin and PSD-95 were unaltered, critical aspects of astrocyte homeostatic functions, such as glutamate uptake and glutathione content, were reduced. Also, glucose uptake and astrocyte lactate transporters were altered, suggesting a disturbance in the astrocyte-neuron coupling. Our present work demonstrates that long-term repeated systemic inflammation can lead to memory impairment and hippocampal metabolic disorders, especially regarding astrocyte function.