LPS Control Group Research Articles

Fasciola hepaticaFh15 promote survival in a mouse septic shock model and downregulates inflammatory cytokines

Abstract Parasitic helminths and helminth-derived molecules, possess an inherent anti-inflammatory capacity. Fasciola hepatica has been reported to decrease in vivo particular Th1 immune responses caused by concurrent bacterial infections. Studies have shown that some Fasciola hepatica’s excretory-secretory products has the capacity to reduce phagocytic activity, affect cell recruitment, and induce activation of alternative activated macrophages. We hypothesized that recombinant F. hepatica FABP (Fh15) may be an excellent target for the development of new anti-inflammatory drugs against sepsis. For this, we developed a mouse model of septic shock to determine the capacity of Fh15 to suppress the cytokine storm and prevent the mortality rate of mice exposed to lethal doses of LPS. BALB/c mice were allotted into two experimental groups of 5 animals each, which received an IP injection with 12mg/kg body wt. of LPS from E. coli 0111: B4. One hour after LPS injection one group received a single IP injection with 100 μg Fh15 and the other group received two injections of 100 μg Fh15 two hours apart. Control groups received IP injection with LPS, Fh15 or PBS alone. Animals were monitored for mortality for 72 hrs. Blood samples were collected from all animals at baseline and by the time they were about to die or survived the LPS lethal challenge. As expected, all animals from LPS-control group succumbed between 12 to 24h following the LPS challenge. In contrast, between 40 to 100% of animals that received the Fh15 treatment survived to the LPS challenge. This notable increase in the survival rate was found associated to significant suppression in the production of pro-inflammatory cytokines such as IL-12, TNF-a and IFN-g, which were measured in serum by BioPlex.

Mechanism of TLR4 mediated immune effect in transfusion-induced acute lung injury based on Slit2/Robo4 signaling pathway

BackgroundTransfusion-related acute lung injury (TRALI) is the infusion of blood or blood system. ObjectiveTo explore the mechanism of TLR4-mediated T cell immune effect in TRALI. MethodsIn this animal study, a mouse model of LPS-induced TRALI was established. Sixty adult C57/BL6 mice (wild-type, WT) were randomly divided into 5 groups: 1) normal WT type, 2) LPS control group of WT type lipopolysaccharide, 3) WT type TRALI group (LPS + MHC-I mAb), 4) (TLR4 antibody) lipopolysaccharide LPS control group, 5) (TLR4 antibody) TRALI group (LPS + MHC-I mAb). Mice were injected with LPS (0.1 mg/kg) and MHC-I mAb (2 mg/kg) into the tail vein. H&E staining was performed to detect pathological features. The myeloperoxidase (MPO) activity and the level of inflammatory cytokines in lung tissue homogenate supernatant were measured. Blood, spleen single-cell suspension, and bronchoalveolar lavage fluid were collected to detect the ratio of Treg and Th17 cells by flow cytometry. RT-PCR and WB were used to detect mRNA or protein expression. ResultsTLR4 mAb treatment alleviated the pathogenesis of LPS-induced TRALI in vivo, the MPO activity, and the level of proinflammatory factors in lung tissues. TLR4 exerted its function by changing of Treg/Th17 ratio via the SLIT2/ROBO4 signaling pathway and downregulating CDH5 and SETSIP. ConclusionTLR4 mediates immune response in the LPS-induced TRALI model through the SLIT2/ROBO4 signaling pathway.

Pretreatment of nafithromycin attenuates inflammatory response in murine lipopolysaccharide induced acute lung injury

Acute respiratory distress syndrome following an acute lung injury (ALI) is a life threatening inflammatory condition predominantly characterized by vascular protein leakage, neutrophil recruitment and overexpression of proinflammatory cytokines. Pulmonary and systemic bacterial infections are the major cause of ALI wherein the bacterial cell components play a crucial role. Macrolide/ketolide antibiotics are reported to possess immunomodulatory activity; as a result improved survival has been noted in pneumonia patients. Hence immunomodulatory activity of nafithromycin, a novel lactone ketolide antibacterial agent was assessed in the murine LPS induced ALI model. Vehicle, nafithromycin (100 mg/kg), azithromycin (600 mg/kg) and dexamethasone (20 mg/kg) were administered orally, 1 h prior to LPS challenge and bronchoalveolar lavage (BAL) fluid was collected thereafter at 18, 24 and 48 h to determine the total cell count, total protein, myeloperoxidase (MPO), tumor necrosis factor (TNF)-α and interleukin (IL)-6. Results from the current study showed that pretreatment with nafithromycin significantly reduced the total cell count, total protein, MPO, TNF-α and IL-6 levels in BAL fluid compared to LPS control group. Histopathological evaluations also suggest significant reduction in neutrophil infiltration by nafithromycin. Dexamethasone, a positive reference standard as expected exhibited potent anti-inflammatory activity. The immunomodulatory effect of nafithromycin at dose of 100 mg/kg was comparable to azithromycin dosed at 600 mg/kg. As a result of immunomodulatory activity, nafithromycin is expected to provide additional clinical benefits by resolving the secondary complications associated with severe pneumonia and thereby improving survival in such patients.

Nose-to-brain co-delivery of repurposed simvastatin and BDNF synergistically attenuates LPS-induced neuroinflammation

A therapeutic strategy that can combat the multifaceted nature of neuroinflammation pathology was investigated. Thus, we fabricated PEG-PdLLA polymersomes and evaluated the efficacy in co-delivery of simvastatin (Sim, as a repurposed anti-inflammatory agent) with brain derived neurotrophic factor (BDNF, as an exogeneous trophic factor supplementation). Using LPS model of neuroinflammation, intranasal administration of combination drug-loaded polymersomes (containing both Sim and BDNF; Sim-BDNF-Ps) markedly down-regulated brain levels of cytokines compared to free drug and single-drug-loaded polymersomes. Further, Sim-BDNF-Ps effectively replenished brain level of BDNF that was depleted following neuroinflammation, resulting in a 2-fold BDNF increase versus untreated LPS control group. We found out that the efficiency of the combination drug-loaded polymersomes to suppress microglia activation in brain regions followed the order: frontal cortex > striatum > hippocampus. Our findings indicated that Sim-BDNF-Ps could effectively inhibit microglial-mediated inflammation as well as potentially resolve the neurotoxic microenvironment that is often associated with neuroinflammation.

The Effects of Acorus calamus L. in Preventing Memory Loss, Anxiety, and Oxidative Stress on Lipopolysaccharide-induced Neuroinflammation Rat Models.

Objective:Several factors lead to memory loss, the most important of which is brain aging that is caused mostly by neuroinflammation and oxidative stress. The need of finding preventive treatments of memory impairment in elderly encouraged authors to assess the effect of Acorus calamus on memory loss, anxiety, and antioxidant indices on neuroinflammation rat models.Materials and Methods:Different fractions of A. calamus were prepared. The subject rats were grouped in 11 groups of 10 each. In the nine treated groups, the extract gavage began 1 week before intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) and continued for 2 weeks after the last injection of LPS. Behavioral tests, including passive avoidance and elevated plus-maze (EPM) tests, were run on days 24, 25, and 26 and the subjects were sacrificed on the day after the last behavioral test, and their hippocampus was isolated to measure the oxidative stress markers.Results:Assessment of oxidative stress markers in hippocampus samples revealed that the amounts of endogenous antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and total antioxidant activity) in the groups that received different fractions were less than their equivalent figures in LPS-control group, and levels of malondialdehyde (MDA) in treatment groups were less than MDA level in LPS-control group. Moreover, the treatment groups with different fractions of A. calamus revealed better performance compared to LPS-control group in shuttle-box test. In EPM test, the groups with different fractions revealed lower stress level in comparison with LPS-control group. The best performance in memory test and the lowest level of stress in EPM was observed in the group with aqueous fraction at 600 mg/kg dose, and the least figures of oxidative stress markers were of the group with aqueous fraction at 600 mg/kg dose.Conclusion:The oral administration of different fractions of A. calamus, especially aqueous fraction, prevented from memory deficits and stress through controlling oxidative stress and inflammation processes.

The effect of heparin on vascular endothelial cell injury induced by lipopolysaccharide in rats and the study of nuclear factor-κB, interleukin-1β, interleukin-6, tumor necrosis factor-α

Objective To investigate the effect of heparin on vascular endothelial cell injury induced by lipopolysaccharide in rats, and the changes of the levels of nuclear factor-κB (NF-κB), interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α. Methods 90 SD rats were randomly divided into three groups: LPS group, heparin group and control group, 30 rats in each group. Rats in the LPS group and heparin group were injected with 10 mg/kg by intraperitoneal injection of lipopolysaccharide solution to establish sepsis model. The heparin group was injected with 300 IU/kg heparin solution before 1H injection. Rats in control group were injected with NaCl solution by intraperitoneal injection in accordance with 10 mg/kg. The morphological changes of lung tissue in rats at 6 h were compared. The changes of NF-κB, IL-1β, IL-6, TNF-α in three groups were detected by enzyme linked immunosorbent assay. The pulmonary vascular permeability changes of rats were detected by Evans blue (EB). Results In Lipopolysaccharide group and heparin group 0 h to 6 h nf-kappa B significantly increased activity of lipopolysaccharide group by 0 h (2.51±0.49) U/L up to 6 h (18.85±2.97) U/L, heparin group by 0 h (2.39±0.34) U/L up to 6 h (3.37±0.82) U/L, lipopolysaccharide group nf-kappa B increased activity levels compared with heparin group is more significant (P=0.021); 6 to 12 h h two groups the nf-kappa B activity significantly decreased (P<0.05), of which the lipopolysaccharide group by 0 h (18.85±2.97) U/L fell to 6 h (13.47±2.19) U/L (P=0.022), heparin group by 0 h (3.37±0.82) U/L fell to 6 h (2.25±0.34) U/L (P=0.048), but it is still significantly higher than the control group, and 0 h level (P=0.000); 0 h three groups of blood beta, IL-6, IL-1β, TNF-α level difference was not significant (IL-1 beta: P=0.940; IL-6: P=0.626; TNF-α: P=0.948), and 0 h to 6 h, lipopolysaccharide group and heparin group rats each index in the blood rise rapidly (within the group, compared with 0 h, 6 h, lipopolysaccharide group, IL-1β: P=0.039; IL-6: P=0.018; TNF-α: P=0.000; heparin group, IL-1β: P=0.036; IL-6: P=0.036; TNF-α: P=0.012), the index level significantly dropped 12 h (comparison group, compared with 6 h, lipopolysaccharide group, IL-1 beta: P=0.042; IL-6: P=0.027; TNF-α: P=0.008; heparin group, IL-1β: P=0.047; IL-6: P=0.045; TNF-α: P=0.034). Lipopolysaccharide group 6 h and 12 h of the blood of rats beta, IL-6, IL-1 TNF-αlevel was significantly higher than that of heparin group (lipopolysaccharide group: heparin group, 6 h, IL-1β: P=0.031; IL-6: P=0.023; TNF-α: P=0.000; 12 h, IL-1β: P=0.046; IL-6: P=0.038; TNF-α: P=0.017). (3) the contents of rat lung EN heparin [(1.21±0.18) mg/ml], and significantly lower than the lipopolysaccharide group [(1.92±0.20) mg/ml] (t=-8.344, P=0.000). Conclusion The heparin can inhibit the damage of vascular endothelial cells by inhibiting the levels of NF-κB, IL-1β, IL-6 , TNF-α in sepsis rats, and thus play an anti-inflammatory role in sepsis. Key words: Heparin; Lipopolysaccharide; Sepsis; Vascular endothelial cell injury

Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cytokines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism

Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cytokines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism

Effects of allogeneic bone marrow mesenchymal stem cells on polarization of peritoneal macrophages in rats with sepsis

Objective: To explore the effects of allogeneic bone marrow mesenchymal stem cells (BMSCs) on polarization of peritoneal macrophages isolated from rats with sepsis induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) BMSCs were isolated, cultured and purified from 5 SD rats with whole bone marrow adherent method. The third passage of cells were collected for morphologic observation, detection of expressions of stem cell surface markers CD29, CD44, CD45, and CD90 with flow cytometer, and identification of osteogenic and adipogenic differentiation. (2) Another 45 SD rats were divided into sham injury group (SI, n=5), LPS control group (LC, n=20), and BMSCs-treated group (BT, n=20) according to the random number table. Rats in groups LC and BT were injected with LPS (5 mg/kg) via tail vein to induce sepsis; rats in group SI were injected with the same amount of normal saline to simulate the damage. At post injury hour (PIH) 1, rats in group BT were given 1 mL BMSCs (2×10(6)/mL) via tail vein injection; rats in another two groups were injected with equal volume of phosphate buffer saline. Five rats in group SI at PIH 24 and in groups LC and BT at PIH 6, 12, 24, and 48 were sacrificed to harvest lung tissue for pathological observation with HE staining. In addition, rats in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48 were simultaneously performed with intraperitoneal injection of low-glucose DMEM. Then peritoneal fluid was harvested to culture peritoneal macrophages. Flow cytometer was used to assess the positive expression of cell makers of macrophages including CD68 (making gate), CD11c, and CD206 in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48. Data were processed with one-way analysis of variance and LSD test. Results: (1) The third passage of cells showed uniform fiber-like shape similar to fibroblasts. These cells showed positive expressions of CD29, CD44, CD90 and weak positive expression of CD45. They were able to differentiate into osteoblasts and adipocytes. These cells were identified as BMSCs. (2) At PIH 24, the structure of pulmonary alveoli of rats in group SI was clear and complete with no congestion or inflammatory cell infiltration. At PIH 6, the structure of pulmonary alveoli of rats in groups LC and BT was clear with a small amount of inflammatory cell infiltration, slight congestion and pulmonary interstitial thickening. At PIH 12, the inflammatory responses in lung tissue of rats in group LC were more severe than those in group BT with a large amount of inflammatory cell infiltration, serious congestion, and obvious pulmonary interstitial thickening. The pathological results of rats in group BT at PIH 12 was consistent with the results at PIH 6. At PIH 24, the pathological results of rats in groups LC and BT were similar to the results at PIH 12. At PIH 48, the structure of pulmonary alveoli tissue of rats in group LC was still severely disrupted, with a large number of inflammatory cell infiltration and congestion in lung tissue, but pulmonary interstitial thickening was slightly alleviated than before. The condition of rats in group BT nearly recovered to that in group SI. (3) At PIH 24, the positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(83±10)%] was close to that in group BT [(87±7)%, P>0.05], and they were both significantly higher than the rate in group SI [(55±12)%, with P values below 0.01]. The positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(59±11)%] at PIH 48 was close to that in group SI at PIH 24 (P>0.05), and they were both significantly higher than the rate in group BT [(20±11)%] at PIH 48 (with P values below 0.01). At PIH 24, the positive expression percentages of CD206 in peritoneal macrophages of rats were similar among the three groups (with P values above 0.05). The positive expression percentage of CD206 in peritoneal macrophages of rats in group SI at PIH 24 was close to that in group BT at PIH 48 (P>0.05), and they were both significantly lower than the percentage in group LC at PIH 48 (with P values below 0.01). Conclusions: BMSCs can reduce the pathological inflammatory responses in the lung of rats with sepsis and inhibit peritoneal macrophages from polarizing into M1 phenotype, whereas they can not promote macrophages to polarize into M2 phenotype.

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte-derived dendritic cells.

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro-inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte-derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF-α, -β and -γ were significantly upregulated in the H5N1 group. Pro-inflammatory cytokines (IL-1β, TNF-α and IL-18) were highly upregulated in early mid (IL-1), and late (IL-6) phases of H5N1 virus infection. IL-8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus-induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV-infected cells.

Protective effects of the specific cyclooxygenase-2 inhibitor on microglia activation-mediated neuronal damage

Objective To investigate the protective effect of NS398, a specific cyclooxygenase-2 inhibitor, on microglial activation-mediated neuronal damage. Methods The microglia of neonatal SD rat were isolated and cultured in the medium containing 1μg/ml of LPS. The morphological changes of microglial were observed. The IL-1β and TNF-α levels were detected by Western blot in LPS group (group L) and control group (group C). The hippocampal neurons of neonatal SD rats were cultured in the control medium (group C), LPS-activated microglial conditioned medium (group L) and LPS-activated microglial conditioned medium with NS398 (group N ), respectively.The survival rate of neurons were detected by MTT. The respiration of hippocampus neurons was determined by detecting the ratio of lactic acid/pyruvic acid. Results LPS-induced microglial activation was characterized by the morphological change and increased secretion of IL-1β and TNF-α.The survival rate of neurons cultured by microglia activated conditioned medium was 64.37%, while the group N was 80.25%(P<0.01). In group L, the ratio of lactic acid/pyruvic acid (27.34±8.53) was significantly higher than that of group N (20.32±6.05, P<0.01) and group C (14.95±4.72, P<0.01). Conclusion NS398, a specific cyclooxygenase-2 inhibitor, has a protective effect on rat hippocampus neuron damaged by activated microglia. Key words: Cyclooxygenase 2 inhibitors; Microglia; Hippocampus; Neurons

Causes of immune dysfunction in hyperbilirubinemia model rats.

To explore the causes of immune dysfunction in neonatal rats with hyperbilirubinemia. A total of 60 newborn SD rats were equally randomized into normal saline (NS) group, LPS control group, bilirubin control group, low-dose group and high-dose group. After anesthesia, 0.1 mL NS was given to the NS and LPS control group and different doses of bilirubin for the other groups; 1 h later, the NS and bilirubin control group received the intraperitoneal injection of 0.05 mL NS and 1mg/kg LPS for the other groups. After 5 or 24 hours of model establishment, spleens were collected for detecting the expression levels of MyD88 and p-TAK1 protein and the spleen cells apoptosis by immunohistochemmistry and TUNEL method. After 24 hours of model establishment, serum inflammatory factors levels and T cell subsets distribution were determined by ELISA and flow cytometry. In contrast to low-dose bilirubin, high-dose bilirubin could induce spleen cells apoptosis in coordination with LPS. After 5 hours of model establishment, compared with NS group, MyD88 expression level in low-dose group elevated while p-TAK1 level in high-dose group reduced (P<0.05). In high-dose group, inflammotory factors levels and CD8(+) T cells percentage were all higher than LPS control and NS group (P<0.05), while CD4(+) T cells percentage was lower than NS group (P<0.05). High-concentration plasma bilirubin in coordination with LPS could inhibit NF-κB signal pathways activation and aggravate inflammatory reaction, thus caused immunosuppression with inflammation cascade, which resulted in the immune dysfunction.

Fermented Rhizoma Atractylodis Macrocephalae alleviates high fat diet-induced obesity in association with regulation of intestinal permeability and microbiota in rats.

Accumulating evidence suggests the anti-inflammatory and anti-obesity activities of Rhizoma Atractylodis Macrocephalae (RAM). Here, we evaluated the anti-obesity impact of unfermented (URAM) versus fermented RAM (FRAM) using both in vitro and in vivo models. Both URAM and FRAM exhibited marked anti-inflammatory, anti-adipogenic, and anti-obesity activities, and modulation of the gut microbial distribution. However, FRAM, compared to URAM, resulted in more efficient suppression of NO production and normalization of transepithelial electrical resistance in LPS-treated RAW 264.7 and HCT 116 cells, respectively. Compared to URAM, FRAM more effectively reduced the adipose tissue weight; ameliorated the serum triglyceride and aspartate transaminase levels; restored the serum HDL level and intestinal epithelial barrier function in the LPS control group. The relative abundance of Bifidobacterium and Akkermansia as well as Bacteriodetes/Firmicutes ratio in the gut of the LPS control group was significantly enhanced by both URAM and FRAM. However, FRAM, but not URAM, resulted in a significant increase in the distribution of Bacteriodetes and Lactobacillus in the gut of the HFD + LPS group. Our results suggest that FRAM with probiotics can exert a greater anti-obesity effect than URAM, which is probably mediated at least in part via regulation of the intestinal microbiota and gut permeability.

Effect of adipose tissue-derived mesenchymal stem cell treatment on oxidative stress and inflammatory response following Escherichia coli lipopolysaccharide

Stem cells are the focus of extensive experimental and clinical research with the aim of replacing lost or damaged tissues. This paper studies the effect of systemic stem cell injections on the redox status of the brain and liver in a model of systemic inflammatory illness. Mice were treated with intraperitoneal (i.p.) injection of adipose tissue-derived mesenchymal stem cell (AT-MSCs) suspension, or saline at time of lipopolysaccharide (LPS; 200 mg/kg, i.p.) administration and euthanized 4 h later. Results show that the administration of LPS increased the oxidative stress in the brain and liver tissues. Malondialdehyde (MDA) increased by 53 and 68.9 %, reduced glutathione (GSH) decreased by 44.4 and 50.6 %, and nitric oxide increased by 47.7 and 87.2 % in the brain and liver, respectively. Total antioxidant capacity (TAC) decreased by 37.8 and 76.2 %, catalase activity decreased by 58.2 and 29.2 %, and paraoxonase 1 (PON1) activity decreased by 53.1 and 22.5 % in the brain and liver, respectively. In addition, nuclear factor kappa B (NF-κB) and the monocyte chemoattractant protein-1 (MCP-1) increased in the brain and liver, respectively, after endotoxin injection. Mice treated with AT-MSCs showed unchanged MDA, increased brain GSH (45.5 %), TAC (171.1 %), catalase activity (58.4 %), and PON1 activity (110.4 %). In the liver, MDA and catalase activity were unchanged, but TAC decreased by −36.7 % and PON1 activity decreased by −22.8 %. NF-κB decreased in the liver, while MCP-1 decreased in the brain and liver by AT-MSCs treatment. AT-MSCs reduced inflammatory cell infiltration and necrotic damage in the liver and markedly decreased the number of degenerated neurons in the cortex and striatum in LPS-treated mice. Stem cell treatment was associated with lower tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), and caspase-3 immunoreactivity in the brain (cortex and striatum) and liver tissues compared to that in the LPS control group. Thus, the systemic administration of AT-MSCs suspension in a model of mild systemic illness modulates the cellular oxidative response and exerts anti-inflammatory effect. AT-MSCs prevent tissue injury via decreased nitric oxide generation due to iNOS expression and decreased activation of caspase-3, TNF-α, and MCP-1.

Flos Lonicera ameliorates obesity and associated endotoxemia in rats through modulation of gut permeability and intestinal microbiota.

Background and AimIncreasing evidence has indicated a close association of host-gut flora metabolic interaction with obesity. Flos Lonicera, a traditional herbal medicine, is used widely in eastern Asia for the treatment of various disorders. The aim of this study was to evaluate whether unfermented or fermented formulations of Flos Lonicera could exert a beneficial impact to combat obesity and related metabolic endotoxemia.MethodsObesity and metabolic endotoxemia were induced separately or together in rats through feeding a eight-week high fat diet either alone (HFD control group) or in combination with a single LPS stimulation (intraperitoneal injection, 0.75 mg/kg) (LPS control group). While, the mechanism of action of the Lonicera formulations was explored in vitro using RAW 264.7 and HCT 116 cell lines as models.ResultsIn cell-based studies, treatment with both unfermented Flos Lonicera (UFL) and fermented Flos Lonicera (FFL) formulations resulted in suppression of LPS-induced NO production and gene expression of vital proinflammatory cytokines (TNF-α, COX-2, and IL-6) in RAW 264.7 cells, reduced the gene expression of zonula occludens (ZO)-1 and claudin-1, and normalized trans epithelial electric resistance (TEER) and horseradish peroxidase (HRP) flux in LPS-treated HCT-116 cells. In an animal study, treatment of HFD as well as HFD+LPS groups with UFL or FFL resulted in a notable decrease in body and adipose tissue weights, ameliorated total cholesterol, HDL, triglyceride, aspartate transaminase and endotoxin levels in serum, reduced the urinary lactulose/mannitol ratio, and markedly alleviated lipid accumulation in liver. In addition, exposure of HFD as well as HFD+LPS groups with UFL or FFL resulted in significant alteration of the distribution of intestinal flora, especially affecting the population of Akkermansia spp. and ratio of Bacteroidetes and Firmicutes.ConclusionThis evidence collectively demonstrates that Flos Lonicera ameliorates obesity and related metabolic endotoxemia via regulating distribution of gut flora and gut permeability.

Sulfated Astragalus polysaccharide can regulate the inflammatory reaction induced by LPS in Caco2 cells

This study evaluates the effect of sulfated Astragalus polysaccharide (SAPS) on inflammatory reaction induced by LPS in Caco2 cells. Sulfated modification was conducted using the chlorosulfonic acid–pyridine method. Caco2 cells were cultured with 25, 50 and 100μg/mL SAPS or 100μg/mL Astragalus polysaccharide (APS) for 24h. Then, 1μg/mL LPS was added for the next 24h to trigger an inflammatory response. DMEM culture medium was used as a blank control. In present study, LPS stimulation significantly increased the mRNA expression of TNF-α, IL-1β, IL-8 and TLR4, and reduced the expression of ZO-1 and occludin. Compared with the LPS control group, APS (100μg/mL) or SAPS (100μg/mL) administration decreased the expression of TNF-α, IL-1β and IL-8. Moreover, 25μg/mL and 50μg/mL SAPS down-regulated TNF-α and IL-1β expression. APS administration (100μg/mL) up-regulated occludin expression, but did not affect ZO-1 expression. However, the expression of ZO-1 and occludin was up-regulated by lower dose SAPS administration (25μg/mL and 50μg/mL). Compared with the other groups, the expression of TLR4 was lower in the SAPS group at all concentrations of SAPS. These results suggest that SAPS was to be a more effective anti-inflammatory agent than APS in vitro.

Effects of quercetin on LPS-induced disseminated intravascular coagulation (DIC) in rabbits

IntroductionQuercetin is widely distributed in plants and has been reported to have effects of anti-inflammation and anti-thrombosis. In this study, we evaluated the protective effect of quercetin on LPS-induced experimental DIC in rabbits, and tried to clarify its mechanism against DIC. Materials and MethodsLPS-induced DIC model in rabbits was established through continuous infusion of 100 ug·kg-1·h-1 LPS for a period of 6h. Six groups were divided: quercetin-treated groups (0.5, 1.0, and 2.0mg·kg-1·h-1, respectively), LPS-control group, heparin-control group (100IU·kg-1·h-1), and saline-control group. APTT, PT, and plasma FIB level were measured, the plasma levels of ALT, BUN, and TNF-α were detected, and the activity of Protein C and ATIII was recorded. ResultsA continuous injection of LPS induced a gradual impairment of hemostatic parameters, a rise in plasma level of TNF-α, and damage in renal and hepatic function. The intravenous administration of quercetin significantly attenuated the increase of APTT, PT, ALT, BUN, and TNF-α, and the decrease of plasma FIB level and activity of Protein C and ATIII. ConclusionQuercetin may have a protective effect against LPS-induced DIC in rabbits through anti-inflammation and anticoagulation.

Use of DLE as treatment in murine endotoxic shock (41.54)

Abstract The LPS is a structural component of the external membrane of the Gram negative bacteria and is the most powerful of the inflammation inductors. High doses of LPS have as consequence a high production of inflammatory cytokines, which causes the endotoxic shock. The Dializable Leukocyte Extracts (DLE) is the dialyzable fraction with lower molecular weight to12 KDa which come from leukocytes lysates. In this work a model of endotoxic shock LPS was induced established in C57BL6 male 6 weeks of age, the dose for endotoxic shock induction was 130mg/kg mouse weight. The doses of 0.001ug and 0.0001ug mDLE showed protection against endotoxic shock death in 10% of the mice and the 0.01ug mDLE dose protected against of endotoxic shock death from 10 to 30% of the mice. Levels of TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, IL-10 and TGF-beta were measured in serum of mice endotoxic shock induced and treated with mDLE, the groups in which LPS was administrated and then treated with 0.01ug, 0.001ug or 0.0001ug of mDLE showed smaller levels of inflammatory cytokines and more higher levels of antiinflammatory cytokines than the LPS treated control group. The splenic index was measured in the endotoxic shock induced and mDLE treated groups, the group LPS and the 0.001ug of mDLE treated showed the higher splenic index in comparison with the LPS control group. So the DLE can be use as a potential therapeutic agent as adjuvant in the treatment of sepsis.

Effects of RNA interference on expression of tumor necrosis factor-alpha in lipopolysaccharide-activated mouse macrophages

To investigate the effects of siRNAs targeting mouse tumor necrosis factor (TNF)-alpha gene on the expression of TNF-alpha mRNA and protein in the LPS activated macrophages. Mouse macrophages of the line RAW264.7 were cultured. Three siRNA sequences targeting different sites of mouse TNF-alpha gene (siRNA 1 - 3) and a fluorescein-labeled double-stranded RNA (dsRNA) oligomer (siRNA 4), as negative control, were designed and chemically synthesized. All siRNAs were transfected into the mouse macrophages. 0, 3, 6, 9, 12, 15, and 18 hours after the transfection samples of supernatant were collected. Inverse microscopy was used to observe the efficacy of transfection. Then the macrophages were co-incubated with LPS for 9 hours. Macrophages stimulated by LPS only and macrophages without transfection and LPS stimulation were used as controls. Then samples of supernatant were collected. TNF-alpha protein expression was detected by ELISA. Real time PCR was used to detect the TNF-alpha mRNA expression. The transfection rate was 72% - 80%. The TNF-alpha mRNA expression levels of the LPS + siRNA2 and LPS + siRNA3 groups were 0.158 +/- 0.031 and 0.114 +/- 0.028 respectively, both significantly lower than that of the LPS control group (0.294 +/- 0.147, P < 0.05 and P < 0.01 respectively), however, the TNF-alpha mRNA expression level of the LPS + siRNA1 and LPS + siRNA4 groups were not significantly different from that of the LPS control group. The TNF-alpha mRNA expression inhibition rates of the LPS + siRNA and LPS + siRNA3 groups were 46.0% and 61.2% respectively. The TNF-alpha protein expression levels of the LPS + siRNA2 and LPS + siRNA3 group were (1358 +/- 348) pg/ml and (817 +/- 138) pg/ml, both significantly lower than that of the LPS control group [(2104 +/- 32) pg/ml, P < 0.05 and P < 0.01], and the TNF-alpha mRNA expression levels of the LPS + siRNA1 and LPS + siRNA4 groups were not significantly different from that of the LPS control group. LPS time-dependently increases the expression of TNF-alpha in macrophages. SiRNAs targeting TNF-alpha inhibit the expression levels of TNF-alpha mRNA and protein in macrophages treated with LPS.

Low-Level Laser Therapy (GaAsλ= 904 nm) Reduces Inflammatory Cell Migration in Mice with Lipopolysaccharide-Induced Peritonitis

This study was designed to study the effect of an infrared low-level laser (GaAs lambda = 904 nm, 4 mW) on inflammatory cell migration in lipopolysaccharide (LPS)-induced peritonitis in mice. It has been suggested that red wavelengths of low-level laser therapy (LLLT) can exert anti-inflammatory effects, but little is known about the anti-inflammatory effects of infrared lasers. Peritonitis is a potentially life-threatening inflammatory condition that may be suitable for studying anti-inflammatory effects of infrared lasers. Sixty male mice were randomly divided into five groups, and one group was given an intraperitoneal sterile saline injection. In the remaining four groups, peritonitis was induced by an intraperitoneal LPS injection. Animals in three of the LPS groups were irradiated at a single point over the peritoneum with doses of 3 J/cm(2), 7.5 J/cm(2), and 15 J/cm(2), respectively. The fourth group injected with LPS was an LPS-control group. At 6 hours after injection the groups irradiated with doses of 3 J/cm(2) and 7.5 J/cm(2) had a reduced number of neutrophil cells in the peritoneal cavity compared with the LPS-control group, and there were significant differences between the number of neutrophils in the peritoneal cavity between the LPS-control group and groups irradiated with doses of 3 J/cm(2) (-42%) and 7.5 J/cm(2) (-70%). In the group irradiated with 15 J/cm(2), neutrophil cell counts were lower than, but not significantly different from, LPS controls (-38%; p = 0.07). At 24 hours after injection, both neutrophil and total leukocyte cell counts were lower in all the irradiated groups than in the LPS controls. The 3-J/cm(2) exposure group showed the best results at 24 hours, with reductions of 77% in neutrophil and 49% in leukocyte counts. Low-level laser therapy (904 nm) can reduce inflammatory cell migration in mice with LPS-induced peritonitis in a dose-dependent manner.

GLC756 decreases TNF-α via an alpha2 and beta2 adrenoceptor related mechanism

GLC756, a polyvalent anti-glaucoma drug showed in an endotoxin-induced-uveitis model (EIU) in rats a significant tumor necrosis factor-α (TNF-α) decrease in serum, indicating an additional anti-inflammatory potential of this compound. The receptors on which GLC756 binds (D1, D2, D4, α-1, α-2, 5-HT1A, 5-HT2C, 5-HT1D, 5-HT2 A, β-1, and β-2) were suggested to play a role. In order to identify a receptor type mediating the TNF-α lowering response, GLC756 was combined with various counteracting compounds (CP). For EIU, 8 week old Lewis rats were intravenously injected at 160 μg lipopolysaccharide (LPS) from Salmonella typhimurium. Before EIU-induction animals received either one of the CP's or GLC756 alone, or GLC756 in combination with one of the CP's. TNF-α was determined in serum 2 h post EIU-induction. Treatment with CP's alone indicated that agonistic effects on β-2 adrenoceptors and antagonistic effects on α-2, 5-HT1A and 5-HT1D receptors resulted in statistically significant decreased TNF-α levels in comparison to the LPS-control group. In combination with GLC756, the counteracting CP's domitor (α-2 adrenoceptor agonist) and ICI 118551 (β-2 adrenoceptor antagonist) inhibited completely the TNF-α decreasing effect of GLC756. Counteracting the 5-HT1A receptor with the 5-HT1A agonist 8-OH-DPAT could not prevent the TNF-α decreasing effect of GLC756. In conclusion, the antagonistic effect on α-2 adrenoceptors and the agonistic effect on β-2 adrenoceptors were identified as mechanism for the TNF-α decreasing effect of GLC756.