Inflammatory parameters in Caco-2 cells: Effect of stimuli nature,concentration, combination and cell differentiation

Enterocytes regulate gut maintenance and defence by secreting and responding to inflammatory mediators and by modulating the intestinal epithelial permeability. In order to develop an in vitro model of the acute phase of intestinal inflammation, Caco-2 cells were exposed to the inflammatory mediators IL-1β, TNF-α, IFN-γ and LPS, and the importance of several experimental parameters, i.e. cell differentiation, stimulus nature, concentration and combination on the inflammatory response was assessed by measuring the production of IL-6, IL-8, PGE-2 and NO and by evaluating the monolayer permeability. A maximal increase in IL-8, IL-6 and PGE-2 production and monolayer permeability was observed when using the cytokines simultaneously at their highest level, but this relied mainly on IL-1β. The effects of TNF-α on IL-8 and IL-6 or NO production were stronger upon combination with IL-1β or IFN-γ, respectively, whereas cells were unaffected by the presence of LPS. Although NO production, induced by IFN-γ-containing combinations, was observed only in differentiated cells, general inflammatory response was higher in proliferating cells. The use of a mixture of IL-1β, TNF-α and IFN-γ thus accurately mimics intestinal inflammatory processes, but cell differentiation and stimuli combination are important parameters to take into account for in vitro studies on intestinal inflammation.     

Interleukin-23 mediates the pathogenesis of LPS/GalN-induced liver injury in mice

BackgroundInterleukin-23 (IL-23) is required for T helper 17 (Th17) cell responses and IL-17 production in hepatitis B virus infection. A previous study showed that the IL-23/IL-17 axis aggravates immune injury in patients with chronic hepatitis B virus infection. However, the role of IL-23 in acute liver injury remains unclear.ObjectiveThe purpose of this study was to determine the role of the inflammatory cytokine IL-23 in lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute liver injury in mice.MethodsSerum IL-23 from patients with chronic hepatitis B virus (CHB), acute-on-chronic liver failure (ACLF) and healthy individuals who served as healthy controls (HCs) was measured by ELISA. An IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody was administered intravenously at the time of challenge with LPS (10 μg/kg) and GalN (400 mg/kg) in C57BL/6 mice. Hepatic pathology and the expression of Th17-related cytokines, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and the stabilization factor Csf3 were assessed in liver tissue.ResultsSerum IL-23 was significantly upregulated in ACLF patients compared with CHB patients and HCs (P < 0.05 for both). Serum IL-23 was significantly upregulated in the non-survival group compared with the survival group of ACLF patients, which was consistent with LPS/GalN-induced acute hepatic injury in mice (P < 0.05 for both). Moreover, after treatment, serum IL-23 was downregulated in the survival group of ACLF patients (P < 0.001). Compared with LPS/GalN mice, mice treated with either an IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody showed less severe liver tissue histopathology and significant reductions in the expression of Th17-related inflammatory cytokine, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and stabilization factors Csf3 within the liver tissue compared with LPS/GalN mice (P < 0.05 for all).ConclusionHigh serum IL-23 was associated with mortality in ACLF patients and LPS/GalN-induced acute liver injury in mice. IL-23 neutralizing antibodies attenuated liver injury by reducing the expression of Th17-related inflammatory cytokines, neutrophil chemoattractants and stabilization factors within the liver tissue, which indicated that IL-23 likely functions upstream of Th17-related cytokine and chemokine expression to recruit inflammatory cells into the liver.     

Preventive effects of interleukin-6 in lipopolysaccharide/d-galactosamine induced acute liver injury via regulating inflammatory response in hepatic macrophages

Lipopolysaccharide/d-Galactosamine (LPS/d-Gal)-induced acute liver injury is characterized by significant inflammatory responses including TNF-α and interleukin-6 (IL-6) and is a widely applied experimental model for inflammation research. TNF-α is critical in the progression of LPS/d-Gal-induced liver injury. However, the role of IL-6 in this model is still unknown. In the present study, we aim to elucidate the involvement of IL-6 in the pathogenesis of acute liver injury induced by LPS/d-Gal in mice and its underlying mechanism. To induce acute liver injury, LPS (50 μg/kg body weight) and d-Gal (400 mg/kg body weight) were injected intraperitoneally in the C57BL/6 mice. The vehicle (saline) or a single dose of recombinant IL-6 (200 μg/kg body weight) was administered 2 h prior to LPS/d-Gal injection. Mice were sacrificed 2 h and 6 h after LPS/d-Gal injection. The results indicated that IL-6 treatment could protect mice from LPS/d-Gal-induced tissue damage, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation, as well as hepatocyte apoptosis and inflammation. Furthermore, in vitro study showed that IL-6 treatment could significantly suppress LPS-triggered expression of proinflammatory cytokines and chemokines, TNF-α, RANTES and MCP-1 in macrophages while promoting the expression of M2 markers, such as Arg-1 and Mrc-1 in macrophages. Taken together, these findings revealed a novel and unexpected role of IL-6 in ameliorating LPS/d-Gal-induced acute liver injury via regulating inflammatory responses in hepatic macrophages.     

Organ- and species-specific biological activity of rosmarinic acid

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1β, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regard to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis.     

Effects of gut microbiota on in vivo metabolism and tissue accumulation of cytochrome P450 3A metabolized drug: Midazolam

The link between drug-metabolizing enzymes and gut microbiota is well established. In particular, hepatic cytochrome P450 (CYP) 3A activities are presumed to be affected by gut microbiota. However, there is no direct evidence that the gut microbiota affects CYP3A metabolism or the clearance of clinically relevant drugs in vivo. Our purpose was to evaluate the effects of gut microbiota on in vitro and in vivo drug metabolism and on the clearance of midazolam, which is a standard CYP3A metabolized drug. Hepatic Cyp3a activity and in vitro midazolam hydroxylase activity were compared using specific pathogen-free (SPF) and germ-free (GF) mice. In a pharmacokinetics (PK) study, SPF and GF mice were intraperitoneally injected with 60 mg/kg of midazolam, and plasma and tissue concentrations were measured. Hepatic Cyp3a activity and midazolam hydroxylase activity were significantly lower in GF mice than in SPF mice. Notably, in the PK study, the area under the plasma concentration–time curve from time zero to infinity and the elimination half-life were approximately four-fold higher in GF mice compared with SPF mice. Furthermore, the concentration of midazolam in the brain 180 min after administration was about 14-fold higher in GF mice compared with SPF mice. Together, our results demonstrated that the gut microbiota altered the metabolic ability of Cyp3a and the tissue accumulation of midazolam.     

Airway Smooth Muscle Sensitivity to Methacholine in Precision-Cut Lung Slices (PCLS) from Ovalbumin-induced Asthmatic Mice

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR) and reversible airway obstruction. Methacholine (MCh) is widely used in broncho-provocation test to evaluate airway resistance. For experimental investigation, ovalbumin-induced sensitization is frequently used in rodents (Ova-asthma). However, albeit the inflammatory histology and AHR in vivo, it remains unclear whether the MCh sensitivity of airway smooth muscle isolated from Ova-asthma is persistently changed. In this study, the contractions of airways in precision-cut lung slices (PCLS) from control, Ova-asthma, and IL-13 overexpressed transgenic mice (IL-13TG) were compared by analyzing the airway lumen space (AW). The airway resistance in vivo was measured using plethysmograph. AHR and increased inflammatory cells in BAL fluid were confirmed in Ova-asthma and IL-13TG mice. In the PCLS from all three groups, MCh concentration-dependent narrowing of airway lumen (ΔAW) was observed. In contrast to the AHR in vivo, the EC₅₀ of MCh for ΔAW from Ova-asthma and IL-13TG were not different from control, indicating unchanged sensitivity to MCh. Although the AW recovery upon MCh-washout showed sluggish tendency in Ova-asthma, the change was also statistically insignificant. Membrane depolarization-induced ΔAW by 60 mM K⁺ (60K-contraction) was larger in IL-13TG than control, whereas 60K-contraction of Ova-asthma was unaffected. Furthermore, serotonin-induced ΔAW of Ova-asthma was smaller than control and IL-13TG. Taken together, the AHR in Ova-asthma and IL-13TG are not reflected in the contractility of isolated airways from PCLS. The AHR of the model animals seems to require intrinsic agonists or inflammatory microenvironment that is washable during tissue preparation.     

Glaucocalyxin A attenuates carbon tetrachloride-induced liver fibrosis and improves the associated gut microbiota imbalance

Liver fibrosis refers to the pathophysiological process of dysplasia on the connective tissue of the liver, caused by a variety of pathogenic factors. Glaucocalyxin A (GLA) has anticoagulation, antibacterial, anti-inflammation, antioxidant and antitumour properties. However, whether GLA ameliorates liver fibrosis or not is still unclear. In this study, a liver fibrosis model was established using male C57BL/6 mice. The mice were treated with 5 and 10 mg/kg GLA via intraperitoneal injection, respectively. The ones that were treated with 5 mg/kg OCA were used as the positive control group. The levels of liver function, liver fibrosis biomarkers and liver pathological changes were then evaluated. We also explored the effects of GLA on inflammatory response and liver cell apoptosis. In addition, we investigated the gut microbiota mechanisms of GLA on liver fibrosis. The results from this study that GLA could significantly decrease the level of liver function (AST, ALT, TBA) and liver fibrosis (HA, LN, PC-III, IV-C). On the other hand, a significant decrease in inflammation levels (IL-1β, TNF-α) were also noted. GLA also improves CCl4-induced pathological liver injuries and collagen deposition, in addition to decreasing apoptosis levels. In addition, an increase in the ratio of Bacteroidetes and Firmicutes in liver disease was also observed. GLA also improves the gut microbiota. In conclusion, GLA attenuates CCl4-induced liver fibrosis and improves the associated gut microbiota imbalance.     

Palmatine attenuates d-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice

Palmatine is an isoquinoline alkaloid from Coptis chinensis, an herbal medicine used to treat various inflammatory diseases such as gastritis, edema and dermatitis. The present study examined the cytoprotective properties of palmatine on d(+)-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were intraperitoneally given GalN (700 mg/kg)/LPS (10 μg/kg). Palmatine (25, 50, 100, and 200 mg/kg) was administered 1 h before GalN/LPS. GalN/LPS increased the mortality and serum aminotransferase activities. These increases were attenuated by palmatine. GalN/LPS increased hepatic lipid peroxidation and decreased the contents of reduced glutathione. Palmatine did not affect the lipid peroxidation and glutathione content. GalN/LPS increased the circulating levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and IL-10. Palmatine prevented the increase of serum TNF-α and augmented that of serum IL-10. GalN/LPS treatment also increased the levels of TNF-α, IL-6 and IL-10 mRNA expression in liver tissue. Palmatine decreased the TNF-α mRNA expression and increased the IL-10 mRNA expression. Palmatine attenuated the apoptosis of hepatocytes, as evidenced by the TUNEL method and capase-3 analysis. Our data suggest that palmatine alleviates GalN/LPS-induced liver injury by modulating the cytokine response and inhibiting apoptosis.     

Ex vivo testing of immune responses in precision-cut lung slices

The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon gamma (IFNgamma), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1alpha, TNFalpha, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFNgamma resulting in increased levels of TNFalpha, IL-12(p40), RANTES, and IL-1alpha. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances.     

Protective effect of phlorotannins from Eisenia bicyclis against lipopolysaccharide-stimulated inflammation in HepG2 cells

In this study, four bioactive phloroglucinol derivates including phloroglucinol (1), eckol (2), dioxinodehydroeckol (3), and dieckol (4) were isolated from Eisenia bicyclis and characterized by nuclear magnetic resonance (NMR) spectroscopic methods. Moreover, the anti-inflammatory activity of these compounds was investigated on human hepatoma cell line HepG2 cells stimulated by lipopolysaccharide (LPS). It was demonstrated that LPS can induce the production of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) as well as the expression of inflammatory mediators as cyclooxygenase-2 (COX-2), and inducible nitric oxide synthases (iNOS) from HepG2 cells. Among isolated compounds, compound (1) exhibited significant inhibition on LPS-stimulated inflammatory responses in HepG2 cells without any cytotoxicity. Herein, compound (1) suppresses the production of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α and the expression of COX-2 and iNOS. Thus, these results indicated that phlorotannins isolated from E. bicyclis, especially compound (1), can be used as a beneficial source for preventing and treating inflammation response.     

Precision-cut liver slices as a model for the early onset of liver fibrosis to test antifibrotic drugs

Induction of fibrosis during prolonged culture of precision-cut liver slices (PCLS) was reported. In this study, the use of rat PCLS was investigated to further characterize the mechanism of early onset of fibrosis in this model and the effects of antifibrotic compounds. Rat PCLS were incubated for 48 h, viability was assessed by ATP and gene expression of PDGF-B and TGF-β1 and the fibrosis markers Hsp47, αSma and Pcol1A1 and collagen1 protein expressions were determined. The effects of the antifibrotic drugs imatinib, sorafenib and sunitinib, PDGF-pathway inhibitors, and perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone, TGFβ-pathway inhibitors, were determined. After 48 h of incubation, viability of the PCLS was maintained and gene expression of PDGF-B was increased while TGF-β1 was not changed. Hsp47, αSma and Pcol1A1 gene expressions were significantly elevated in PCLS after 48 h, which was further increased by PDGF-BB and TGF-β1. The increased gene expression of fibrosis markers was inhibited by all three PDGF-inhibitors, while TGFβ-inhibitors showed marginal effects. The protein expression of collagen 1 was inhibited by imatinib, perindopril, tetrandrine and pirfenidone. In conclusion, the increased gene expression of PDGF-B and the down-regulation of fibrosis markers by PDGF-pathway inhibitors, together with the absence of elevated TGF-β1 gene expression and the limited effect of the TGFβ-pathway inhibitors, indicated the predominance of the PDGF pathway in the early onset of fibrosis in PCLS. PCLS appear a useful model for research of the early onset of fibrosis and for testing of antifibrotic drugs acting on the PDGF pathway.     

Protective effect of linalool against lipopolysaccharide/d-galactosamine-induced liver injury in mice

Linalool, a natural compound of the essential oils, has been shown to have antinociceptive, antimicrobial, and anti-inflammatory properties. The aim of this study was to investigate the effects of linalool against lipopolysaccharide (LPS)/d-galactosamine (GalN)-induced liver injury in mice. Mice were administered with linalool 1 h before receiving LPS (50 μg/kg) and GalN (800 mg/kg). The results demonstrated that linalool had a protective effect on LPS/GalN-induced acute liver injury, as evidenced by the attenuation of hepatic pathological damage, malondialdehyde (MDA) content, MPO activity and serum ALT and AST levels. Linalool alleviated serum and hepatic TNF-α and IL-6 production, as well as hepatic iNOS and COX-2 expression by inhibiting NF-κB activation. Treatment of linalool increased bcl-2 expression and inhibited caspase-3 and caspase-8 expression. In addition, linalool increased Nrf2 and heme oxygenase-1 expression up-regulation by LPS/GalN. In conclusion, our results suggested that linalool was protected against LPS/GalN-induced liver injury through induction of antioxidant defense via Nrf2 activating and reduction inflammatory response via NF-κB inhibition.     

Crocin protects cardiomyocytes against LPS-Induced inflammation

BackgroundSepsis causes organ dysfunctions via elevation of oxidative stress and inflammation. Lipopolysaccharide (LPS) is the major surface molecule of most gram-negative bacteria and routinely used as a sepsis model in investigation studies. Crocin is an active compound of saffron which has different pharmacological properties such as anti-oxidant and anti-inflammatory. In this research, the protective effect of crocin was evaluated against LPS-induced toxicity in the embryonic cardiomyocyte cell line (H9c2).MethodsThe cells were pre-treated with different concentration of crocin (10, 20 and 40 μM) for 24 h, and then LPS was added (10 μg/ml) for another 24 h. Afterward, the percentage of cell viability and the levels of inflammatory cytokines (TNF-α, PGE₂, IL-1β, and IL-6), gene expression levels (TNF-α, COX-2, IL-1β, IL-6, and iNOS), and the level of nitric oxide (NO) and thiol were measured.ResultsOur results showed that LPS reduced cell viability, increased the levels of cytokines, gene-expression, nitric oxide, and thiol. Crocin attenuated the LPS-induced toxicity in H9c2 cells via reducing the levels of inflammatory factors (TNF-α, PGE₂, IL-1β, and IL-6, p < 0.001), gene expression (TNF-α, COX-2, IL-1β, IL-6, and iNOS, p < 0.001), and NO (p < 0.001), whereas increased the level of thiol content (p < 0.001).ConclusionThe observed results revealed that crocin has preventive effects on the LPS induced sepsis and its cardiac toxicity in-vitro model. Probably, these findings are related to anti-inflammatory and anti-oxidant properties of crocin. However, performing further animal studies are necessary to support the therapeutic effects of crocin in septic shock cardiac dysfunction.     

Human β-defensin-3 alleviates the progression of atherosclerosis accelerated by Porphyromonas gingivalis lipopolysaccharide

Background and aimPorphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) is reported to be associated with the progression of atherosclerosis (AS). In this study, we explored the potential of human β-defensin-3 (hBD3), an antimicrobial peptide with immunomodulatory properties, to alleviate AS progression accelerated by P. gingivalis LPS and the mechanism underlying this effect.Materials and methodsApolipoprotein E-deficient mice were injected intraperitoneally with hBD3, P. gingivalis LPS, or hBD3 + P. gingivalis LPS. The aorta was assessed immunohistologically and mRNA levels of inflammatory cytokines were determined by quantitative PCR. Macrophages and vascular endothelial cells were stimulated in vitro to investigate the hBD3 target cells. Inflammatory cytokines in serum and cell culture supernatants were detected using cytometric bead arrays. Signaling pathways were investigated by Western blotting.ResultsIn P. gingivalis LPS-treated mice, hBD3 significantly reduced serum IL-6 and TNF-α levels and aortic expression of ICAM-1, IL-6, and MCP-1 (mRNA and protein). The area and severity of atherosclerotic lesions were also diminished, with less advanced plaque formation, more continuous and distinct elastic lamina, and more normal smooth muscle cells arranged along the tunica media layer. In vitro, hBD3 decreased TNF-α, IL-1β, IL-6 secretion and downregulated TNF-α, IL-1β, IL-6, IL-8, VCAM-1, and IL-10 mRNA levels in macrophages. hBD3 did not influence TNF-α, IL-6, and IL-8 levels in HUVECs culture supernatants. Furthermore, hBD3 suppressed P. gingivalis LPS-induced activation of the NF-κB, p38 and JNK pathways.ConclusionhBD3 alleviates AS progression accelerated by P. gingivalis LPS in apolipoprotein E-deficient mice by downregulating the cytokine expression in macrophages via the MAPK and NF-κB signaling pathways.     

Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS)

Occupational asthma can be induced by a number of chemicals at the workplace. Risk assessment of potential sensitizers is mostly performed in animal experiments. With increasing public demand for alternative methods, human precision-cut lung slices (PCLS) have been developed as an ex vivo model.Human PCLS were exposed to increasing concentrations of 20 industrial chemicals including 4 respiratory allergens, 11 contact allergens, and 5 non-sensitizing irritants. Local respiratory irritation was characterized and expressed as 75% (EC₂₅) and 50% (EC₅₀) cell viability with respect to controls. Dose–response curves of all chemicals except for phenol were generated. Local respiratory inflammation was quantified by measuring the production of cytokines and chemokines. TNF-α and IL-1α were increased significantly in human PCLS after exposure to the respiratory sensitizers trimellitic anhydride (TMA) and ammonium hexachloroplatinate (HClPt) at subtoxic concentrations, while contact sensitizers and non-sensitizing irritants failed to induce the release of these cytokines to the same extent. Interestingly, significant increases in T_H1/T_H2 cytokines could be detected only after exposure to HClPt at a subtoxic concentration.In conclusion, allergen-induced cytokines were observed but not considered as biomarkers for the differentiation between respiratory and contact sensitizers. Our preliminary results show an ex vivo model which might be used for prediction of chemical-induced toxicity, but is due to its complex three-dimensional structure not applicable for a simple screening of functional and behavior changes of certain cell populations such as dendritic cells and T-cells in response to allergens.     

Probiotics modulate the microbiota–gut–brain axis and improve memory deficits in aged SAMP8 mice

ProBiotic-4 is a probiotic preparation composed of Bifidobacterium lactis, Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus acidophilus. This study aims to investigate the effects of ProBiotic-4 on the microbiota–gut–brain axis and cognitive deficits, and to explore the underlying molecular mechanism using senescence-accelerated mouse prone 8 (SAMP8) mice. ProBiotic-4 was orally administered to 9-month-old SAMP8 mice for 12 weeks. We observed that ProBiotic-4 significantly improved the memory deficits, cerebral neuronal and synaptic injuries, glial activation, and microbiota composition in the feces and brains of aged SAMP8 mice. ProBiotic-4 substantially attenuated aging-related disruption of the intestinal barrier and blood–brain barrier, decreased interleukin-6 and tumor necrosis factor-α at both mRNA and protein levels, reduced plasma and cerebral lipopolysaccharide (LPS) concentration, toll-like receptor 4 (TLR4) expression, and nuclear factor-κB (NF-κB) nuclear translocation in the brain. In addition, not only did ProBiotic-4 significantly decreased the levels of γ-H2AX, 8-hydroxydesoxyguanosine, and retinoic-acid-inducible gene-I (RIG-I), it also abrogated RIG-I multimerization in the brain. These findings suggest that targeting gut microbiota with probiotics may have a therapeutic potential for the deficits of the microbiota–gut–brain axis and cognitive function in aging, and that its mechanism is associated with inhibition of both TLR4-and RIG-I-mediated NF-κB signaling pathway and inflammatory responses.     

A supercritical CO₂ extract from seabuckthorn leaves inhibits pro-inflammatory mediators via inhibition of mitogen activated protein kinase p38 and transcription factor nuclear factor-κB

In the present study, we have demonstrated the anti-inflammatory properties of supercritical CO₂ extract of seabuckthorn leaves (SCE) on mouse alveolar macrophage cell line (MH-S), human peripheral blood mononuclear cells (hPBMCs) in-vitro and in-vivo. Treatment of MH-S cells with SCE (0.5–100 μg/ml) significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. It also inhibited the release of LPS-induced pro-inflammatory cytokines IL-6 and TNF-α, which was further confirmed by suppression of LPS induced TNF-α in hPBMCs by ELISPOT assay. In addition, western blot analysis demonstrated that SCE decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in MH-S cells. Furthermore, SCE treatment also reduced nuclear factor-κB (NF-κB) translocation in nucleus induced by LPS in MH-S cells. To elucidate the molecular mechanism for inhibition of pro-inflammatory mediators by SCE (100 μg/ml), we further studied the effect of SCE on LPS-induced p38 mitogen-activated protein kinase (MAPK). It was observed that the phosphorylation of p38 MAPK in LPS-stimulated MH-S cells was significantly inhibited by SCE, which was further proven by suppression of LPS induced CD40 expression. The in-vivo model of AIA mice also showed a significant reduction in the inflammation of paw edema. These data collectively suggest that SCE suppressed the LPS-induced production of NO, IL-6, and TNF-α and expression of CD40, iNOS and COX-2 proteins by inhibiting NF-κB activation and phosphorylation of p38 MAPK. Hence, the SCE has potent anti-inflammatory activity and might be useful in chronic inflammatory diseases.     

Effects of bacterial endotoxin (lipopolysaccharides) on survival and metabolism of cultured precision-cut rat liver slices.

The effect of bacterial endotoxin (lipopolysaccharides from Escherichia coli, LPS) on cellular metabolism and drug biotransformation was studied in precision-cut rat liver slices (PCLS). Xenobiotic metabolism by PCLS was assessed by measuring phase I (midazolam hydroxylation) and phase II (paracetamol conjugates) enzyme activities. Nitrites formation was used as an indirect way to assess LPS-mediated activation of nitric oxide synthase (iNOS, type 2). PCLS incubation with various LPS doses results in a dose-dependent formation of nitrites. Such a nitrite formation is decreased by dexamethasone. After incubation of PCLS for 24 h LPS addition did not increase the basal nitrite formation, indicating that cells are not responsive any more. Paracetamol conjugation was unaffected by LPS treatment but midazolam hydroxylation was reduced by more than 50%. Such a loss is not due to cell impairment since neither survival (LDH leakage) nor cellular metabolism (protein synthesis or ATP content) were modified by LPS. Indeed, under defined conditions, namely Williams' medium E and O(2)/CO(2) (95:5), PCLS maintained both ATP and GSH levels and the capacity of hepatocytes to synthesize proteins. In conclusion, the in vitro model of PCLS reproduces the inhibitory effect of LPS on a CYP3A-dependent activity, allowing a mechanistic approach to study cell-cell interactions.