Concentrations Of LPS Research Articles

The downregulation of tight junction proteins and pIgR in the colonic epithelium causes the susceptibility of EpCAM+/- mice to colitis and gut microbiota dysbiosis.

The genetic factors play important roles on the pathogenesis of inflammatory bowel disease (IBD). EpCAM is highly expressed in the intestinal epithelium. It is still unclear if the decrease or somatic mutation of EpCAM could cause IBD. The WT and EpCAM+/- mice were administrated with DSS intermittently for nearly 8 weeks. The colon, liver and feces were harvested to check the morphological and histological changes, the expression of inflammatory genes and the gut microbiota via H&E staining, immunofluorescence, qPCR, western blot and 16S rDNA sequence assays. The DSS administration induced more serious inflammation in the colon of EpCAM+/- mice than WT mice. Compared to DSS-induced WT mice, the transcriptional levels of IL-6, F4/80, Ly6g, Ly6d and Igha were significantly higher in the colon of DSS-induced EpCAM+/- mice. The protein levels of MMP7 and MMP8 and the activation of JNK, ERK1/2 and p38 were significantly increased in the colon of DSS-induced EpCAM+/- mice. The protein levels of CLDN1, CLDN2, CLDN3, CLDN7, OCLD, ZO-1 and pIgR were significantly decreased in the colon of DSS-induced EpCAM+/- mice. The serum concentration of LPS was significantly higher in the DSS-induced EpCAM+/- mice which caused the acute inflammation in the liver of them. The expression of Pigr was significantly reduced in the liver of DSS-induced EpCAM+/- mice. The ratio of Firmicutes/Bacteroidetes at the phylum level was higher in the gut microbiota of EpCAM+/- mice than WT mice. In conclusion, the heterozygous mutation of EpCAM increased the susceptibility to colitis, gut microbiota dysbiosis and liver injury.

Valoración de dos biomarcadores inmunológicos en sepsis bacteriana y shock séptico

IntroductionSepsis is a disorder that can progress to shock, with multiple organ failure and increased mortality, so the study of inflammation biomarkers can help prevent these complications. ObjectiveEstablish an association between serum concentrations of the biomarkers LPS and MyD88 with bacterial sepsis and progression to shock. MethodsA cross-sectional analytical study was carried out, using a cohort of septic adult patients with Gram-negative bacterial etiology and with progression to shock, of any gender and socioeconomic condition, in whom serum concentrations of the biomarkers LPS and MyD88 were established by enzyme immunoassay. ResultsLPS serum concentrations in patients with septic shock (n=50) were mean±SD: 31.86±11.4 ng/mL; and septic patients without progress to shock (n=75), the results were 24.7±13.6 ng/mL (p=0.010).; while those found for MyD88 in patients with septic shock (n=50) were mean±SD: 3267.2±2001.5 pg/mL and septic patients without progression to shock (n=75) were 2178.3±2948.9 pg/mL (p=0.004). ConclusionAn association was obtained between the serum levels of the biomarkers LPS and MyD88, both in patients with bacterial sepsis without progression to shock, and in those who progressed to this condition.

Effects of Dandelion Extract on Promoting Production Performance and Reducing Mammary Oxidative Stress in Dairy Cows Fed High-Concentrate Diet.

This study investigated the effects of rumen bypass dandelion extract on the lactation performance, immune index, and mammary oxidative stress of lactating dairy cows fed a high-concentrate diet. This study used a complete randomized block design, and initial milk production, somatic cell counts, and parities were set as block factors. Sixty Holstein cows with similar health conditions and lactating periods (70 ± 15 d) were divided into three groups with 20 replicates per group. The treatments included the LCD group (low-concentrate diet, concentrate-forage = 4:6), HCD group (high-concentrate group, concentrate-forage = 6:4), and DAE group (dandelion aqueous extract group, HCD group with 0.5% DAE). The experimental period was 35 d, and cows were fed three times in the morning, afternoon, and night with free access to water. The results showed the following: (1) Milk production in the HCD and DAE groups was significantly higher (p < 0.05) than that in the LCD group from WK4, and the milk quality differed during the experimental period. (2) The HCD group's pH values significantly differed (p < 0.01) from those of the LCD and DAE groups. (3) In WK2 and WK4 of the experimental period, the somatic cell counts of dairy cows in the HCD group were significantly higher (p < 0.05) than those in the DAE group. (4) The serum concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and protein carbonyl (PC) in the HCD group were significantly higher (p < 0.05) than those in the LCD group. The activity of catalase (CAT) in the LCD and DAE groups was stronger (p < 0.01) than that in the HCD group. (5) The correlation analysis revealed significantly positive correlations between the plasma LPS concentration and serum concentrations of 8-OHdG (p < 0.01), PC (p < 0.01), and malondialdehyde (MDA, p < 0.05) and significantly negative correlations (p < 0.01) between the plasma LPS concentration and activities of CAT and superoxide dismutase. (6) Compared with that in the HCD and DAE groups, the mRNA expression of α, β, and κ casein and acetyl CoA carboxylase in bovine mammary epithelial cells was significantly higher (p < 0.05) in the LCD group, and the mRNA expression of fatty acid synthetase and stearoyl CoA desaturase in the LCD group was significantly higher (p < 0.01) than that in the HCD group. (7) Compared with that in the LCD and HCD groups, the mRNA expression of Nrf2 was significantly higher (p < 0.01) in the DAE group, and the mRNA expression of cystine/glutamate transporter and NAD (P) H quinone oxidoreductase 1 in the DAE group was significantly higher (p < 0.05) than that in the HCD group. Overall, feeding a high-concentrate diet could increase the milk yield of dairy cows, but the milk quality, rumen homeostasis, and antioxidative capability were adversely affected. The supplementation of DAE in a high-concentrate diet enhanced antioxidative capability by activating the Nrf2 regulatory factor and improved rumen homeostasis and production performance.

Disturbances of Ruminal Microbiota and Liver Inflammation, Mediated by LPS and Histamine, in Dairy Cows Fed a High-Concentrate Diet.

The ecosystem of ruminal microbiota profoundly affects the health and milk production of dairy cows. High-concentrate diets are widely used in dairy farms and evoke a series of metabolic disorders. Several studies have reported the effects of high-concentrate diets on the ruminal microbiome, while the effect of changes in ruminal microbial flora, induced by high-concentrate diet feeding, on the liver of dairy cows has not been studied before. In this study, 12 mid-lactating Holstein Friesian cows (weight of 455 ± 28 kg; parities of 2.5 ± 0.5; starting milk yield of 31.59 ± 3.2 kg/d; DMI of 21.7 ± 1.1 kg/d; and a DIM at the start of the experiment of 135 ± 28 d) were fitted with ruminal fistulas, as well as with portal and hepatic vein catheters. All cows were randomly divided into 2 groups; then, they fed with low-concentrate diets (LC, concentrate: forage = 40:60) and high-concentrate diets (HC, concentrate: forage = 60:40) for 18 weeks. The forage sources were corn silage and alfalfa hay. After the cows of two groups were euthanized over two consecutive days, ruminal microbiota; the concentration of LPS in the rumen content; cecum content; the levels of blood and histamine in rumen fluid, blood, and the liver; the histopathological status of the rumen and cecum; and the inflammatory response of the liver were assessed in dairy cows under conditions of subacute ruminal acidosis (SARA). These conditions were caused by high-concentrate diet feeding. All data were analyzed using the independent t-test in SPSS. The results showed that high-concentrate diet feeding increased the concentration of LPS and histamine in the rumen and plasma of veins (p < 0.05). The abundance of Bacteroidetes at the phylum level, and of both Bacteroidetes and Saccharibacteria at the genus level, was decreased, while the abundance of Firmicutes at the phylum level and Oscillibacter at the genus level was increased by high-concentrate diet feeding. The decreased pH values of ruminal contents (LC = 6.02, HC = 5.90, p < 0.05) and the increased level of LPS in the rumen (LC = 4.921 × 105, HC = 7.855 × 105 EU/mL, p < 0.05) and cecum (LC = 11.960 × 105, HC = 13.115 × 105 EU/mL, p < 0.01) induced the histopathological destruction of the rumen and cecum, combined with the increased mRNA expression of IL-1β (p < 0.05). The histamine receptor H1R and the NF-κB signaling pathway were activated in the liver samples taken from the HC group. In conclusion, the elevated concentrations of LPS and histamine in the gut may be related to changes in the ruminal microbiota. LPS and histamine induced the inflammatory response in the ruminal epithelium, cecum epithelium, and liver. However, the cause-effect mechanism needs to be proved in future research. Our study offers a novel therapeutic strategy by manipulating ruminal microbiota and metabolism to decrease LPS and histamine release and to improve the health of dairy cows.

Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs

Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays.

Radix Sophorae Flavescentis of Sophora flavescens Aiton inhibits LPS-induced macrophage pro-inflammatory response via regulating CFHR2 expression

Ethnopharmacological relevanceLong-term chronic inflammation often leads to chronic diseases. Although Sophora flavescens has been shown to have anti-inflammatory properties, its detailed molecular mechanism is still unknown. Aim of studyThis study investigated the effect of Radix Sophorae Flavescentis on the LPS-induced inflammatory response in macrophages. Materials and methodsLPS was used to induce the peritoneal macrophages to simulate the inflammatory environment in vitro. Different concentrations of Radix Sophorae Flavescentis-containing (medicated) serum were used for intervention. The peritoneal macrophages were identified by using hematoxylin-eosin and immunofluorescence staining. ELISA was used to measure the TNF-α and IL-6 expression to determine the concentration of LPS. ELISA and Western blot (WB) were used to detect the PGE2 and CFHR2 expression in each group, respectively. The lentiviral vector for interference and overexpression of the CFHR2 gene was constructed, packaged, and transfected into LPS-induced macrophages. The transfection efficiency was verified by WB. Then, ELISA was used to detect the TNF-α, PGE2, and IL-6 expression. WB was used to detect the CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression. ResultsThe primary isolated cells were identified as macrophages. The LPS-treated macrophages exhibited significantly higher expression of PGE2 and CFHR2, and the inflammatory factors TNF-α and IL-6, as well as iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression compared with the control group (P < 0.05). The TNF-α, PGE2, and IL-6 levels, as well as CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression were considerably lower in the LPS-induced+10% medicated-serum group, LPS-induced+20% medicated-serum group, and shCFHR interference group compared with the LPS group (P < 0.05). ConclusionRadix Sophorae Flavescentis might mediate CFHR2 expression and play an important role in inhibiting the LPS-induced pro-inflammatory response of macrophages. Radix Sophorae Flavescentis could be a potential treatment for LPS-induced related inflammatory diseases.

Comparison of the gastrointestinal bacterial microbiota between dairy cows with and without mastitis.

Mastitis causes significant losses in the global dairy industry, and the health of animals has been linked to their intestinal microbiota. To better understand the relationship between gastrointestinal microbiota and mastitis in dairy cows, we collected blood, rumen fluid, and fecal samples from 23 dairy cows, including 13 cows with mastitis and 10 healthy cows. Using ELISA kit and high-throughput sequencing, we found that cows with mastitis had higher concentrations of TNF-α, IL-1, and LPS than healthy cows (p < 0.05), but no significant differences in microbiota abundance or diversity (p > 0.05). Principal coordinate analysis (PCOA) revealed significant differences in rumen microbial structure between the two groups (p < 0.05), with Moryella as the signature for rumen in cows with mastitis. In contrast, fecal microbial structure showed no significant differences (p > 0.05), with Aeriscardovia, Lactococcus, and Bacillus as the signature for feces in healthy cows. Furthermore, the results showed distinct microbial interaction patterns in the rumen and feces of cows with mastitis compared to healthy cows. Additionally, we observed correlations between the microbiota in both the rumen and feces of cows and blood inflammatory indicators. Our study sheds new light on the prevention of mastitis in dairy cows by highlighting the relationship between gastrointestinal microbiota and mastitis.

Supplementing Ryegrass Ameliorates Commercial Diet-Induced Gut Microbial Dysbiosis-Associated Spleen Dysfunctions by Gut-Microbiota-Spleen Axis.

The type and composition of food strongly affect the variation and enrichment of the gut microbiota. The gut-microbiota-spleen axis has been developed, incorporating the spleen's function and maturation. However, how short-chain fatty-acid-producing gut microbiota can be considered to recover spleen function, particularly in spleens damaged by changed gut microbiota, is unknown in geese. Therefore, the gut microbial composition of the caecal chyme of geese was assessed by 16S rRNA microbial genes, and a Tax4Fun analysis identified the enrichment of KEGG orthologues involved in lipopolysaccharide production. The concentrations of LPS, reactive oxygen species, antioxidant/oxidant enzymes, and immunoglobulins were measured from serum samples and spleen tissues using ELISA kits. Quantitative reverse transcription PCR was employed to detect the Kelch-like-ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2), B cell and T cell targeting markers, and anti-inflammatory/inflammatory cytokines from the spleen tissues of geese. The SCFAs were determined from the caecal chyme of geese by using gas chromatography. In this study, ryegrass-enriched gut microbiota such as Eggerthellaceae, Oscillospiraceae, Rikenellaceae, and Lachnospiraceae attenuated commercial diet-induced gut microbial alterations and spleen dysfunctions in geese. Ryegrass significantly improved the SCFAs (acetic, butyric, propionic, isovaleric, and valeric acids), AMPK pathway-activated Nrf2 redox signaling cascades, B cells (B220, CD19, and IgD), and T cells (CD3, CD4, CD8, and IL-2, with an exception of IL-17 and TGF-β) to activate anti-inflammatory cytokines (IL-4 and IL-10) and immunoglobulins (IgA, IgG, and IgM) in geese. In conclusion, ryegrass-improved reprogramming of the gut microbiota restored the spleen functions by attenuating LPS-induced oxidative stress and systemic inflammation through the gut-microbiota-spleen axis in geese.

Lithium-loaded GelMA-Phosphate glass fibre constructs: Implications for astrocyte response.

Combinations of different biomaterials with their own advantages as well as functionalization with other components have long been implemented in tissue engineering to improve the performance of the overall material. Biomaterials, particularly hydrogel platforms, have shown great potential for delivering compounds such as drugs, growth factors, and neurotrophic factors, as well as cells, in neural tissue engineering applications. In central the nervous system, astrocyte reactivity and glial scar formation are significant and complex challenges to tackle for neural and functional recovery. GelMA hydrogel-based tissue constructs have been developed in this study and combined with two different formulations of phosphate glass fibers (PGFs) (with Fe3+ or Ti2+ oxide) to impose physical and mechanical cues for modulating astrocyte cell behavior. This study was also aimed at investigating the effects of lithium-loaded GelMA-PGFs hydrogels in alleviating astrocyte reactivity and glial scar formation offering novel perspectives for neural tissue engineering applications. The rationale behind introducing lithium is driven by its long-proven therapeutic benefits in mental disorders, and neuroprotective and pronounced anti-inflammatory properties. The optimal concentrations of lithium and LPS were determined in vitro on primary rat astrocytes. Furthermore, qPCR was conducted for gene expression analysis of GFAP and IL-6 markers on primary astrocytes cultured 3D into GelMA and GelMA-PGFs hydrogels with and without lithium and in vitro stimulated with LPS for astrocyte reactivity. The results suggest that the combination of bioactive phosphate-based glass fibers and lithium loading into GelMA structures may impact GFAP expression and early IL-6 expression. Furthermore, GelMA-PGFs (Fe) constructs have shown improved performance in modulating glial scarring over GFAP regulation.

LPS-TLR4 pathway exaggerates alcoholic hepatitis via provoking NETs formation

BackgroundIntrahepatic infiltration of neutrophils is a character of alcoholic hepatitis (AH) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. The gut-liver axis has been thought to play a critical role in many liver diseases also including AH. However, whether NETs appear in AH and play role in AH is still unsure. MethodsSerum samples from AH patients were collected and LPS and MPO-DNA were detected. WT, NE KO, and TLR4 KO mice were used to build the AH model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AH-related markers were detected. ResultsThe serum MPO-DNA and LPS concentration was increased in AH patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AH mice. NETs formation decreased with antibiotic intervention and restored with antibiotic intervention plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AH-related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with a combined treatment with antibiotics plus LPS. But the AH-related markers did show a difference in TLR4 KO mice when they received the same treatment. ConclusionIntestinal-derived LPS promotes NETs formation in AH through the TLR4 pathway and further accelerates the AH process by NETs.

Effects of Sophora alopecuroides in a High-Concentrate Diet on the Liver Immunity and Antioxidant Function of Lambs According to Transcriptome Analysis.

The purpose of this study was to determine the effects of Sophora alopecuroides (SA) on liver function, liver inflammatory factor levels, antioxidant indexes and transcriptome in sheep. Twenty-four 3-month-old healthy Dumont hybrid lambs weighing 25.73 ± 2.17 kg were randomly divided into three groups: C1 (the control group), fed a concentrate-to-forage ratio of 50:50; H2 (the high-concentration group), fed a concentrate-to-forage ratio of 70:30; and S3 (the SA group), fed a concentrate-to-forage ratio of 70:30 + 0.1% SA. The results showed that the rumen pH values of the C1 and S3 groups were significant or significantly higher than that of the H2 group (p < 0.05 or p < 0.01). The serum ALT, AST and LDH activities and the LPS and LBP concentrations in the sheep serum and liver in the H2 group were significantly or extremely significantly higher than those in the C1 and S3 groups (p < 0.01), and the IL-10 content and SOD, GPX-PX and T-AOC activities showed the opposite trend (p < 0.05 or p < 0.01). KEGG enrichment analysis showed that the differentially expressed genes were significantly enriched in the ECM-receptor interaction and focal adhesion pathways, which are closely related to immune and antioxidant functions (p-adjust < 0.1). In summary, SA could improve the immune and antioxidant functions of lamb livers under high-concentrate conditions and regulate the mechanism of damage on sheep livers, which is caused by high-concentrate diets and through the expression of related genes in the ECM/FAs pathway.

BUTYRATE-PRODUCING BACTERIA INCREASE GUT MICROBIOTA DIVERSITY TO DELAY INTESTINAL INFLAMMAGING

Abstract Intestinal flora dysbiosis is a key link in intestinal inflammaging, and the diversity of gut microbiota plays an important role in maintaining the entire intestinal microecology. Our previous study found that butyrate-producing bacteria reversed aging-induced intestinal inflammation and promoted the expression of intestinal anti-inflammatory factors in 24-month-old mice, but the mechanism was unclear. We found that butyrate-producing bacteria promote the expression of short-chain fatty acid (SCFA) receptors in the intestine. Monocarboxylate transporter 1 (MCT-1) has anti-inflammatory effects on intestinal inflammation. We speculate that butyrate-producing bacteria upregulate MCT-1 expression through the activation of SCFA receptors, thereby promoting gut stability and bacterial diversity. In further experiments, we established inflammation models using cell lines, colon organoids, mouse colonic epithelial cells, and aged mice (24 months old) given different concentrations of LPS and DSS, and then added butyrate-producing bacteria to verify that MCT-1 is involved in the resistance of butyrate-producing bacteria to intestinal inflammation. We found that bacteria up-regulated the expression of MCT-1 by activating SCFA receptors. From the results of 16s rRNA sequencing, it can be seen that the intestinal stability and bacterial diversity of aged mice were improved after the administration of butyrate-producing bacteria. The research results will provide an experimental basis for treating intestinal inflammaging by restoring intestinal stability and diversity of gut microbiota and will help to explore the clinical value of butyrate-producing bacteria, which has important translational significance.

KNOCKDOWN OF CIRC_0114428 ALLEVIATES LPS-INDUCED HK2 CELL APOPTOSIS AND INFLAMMATION INJURY VIA TARGETING MIR-215-5P/TRAF6/NF-ΚB AXIS IN SEPTIC ACUTE KIDNEY INJURY.

Background: Sepsis is a systemic inflammatory disease that can cause multiple organ damage. Circular RNAs (circRNAs) have been reported to play a regulatory role in sepsis-induced acute kidney injury (AKI); however, the role of circ_0114428 has not been studied. Methods: In this study, HK2 cells were treated with different concentrations of LPS to induce cell damage, and then the expressions of circ_0114428, microRNA-215-5p (miR-215-5p), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot examined the Bax and cleaved-Caspase-3 proteins. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) assay. In addition, cell apoptosis was detected by flow cytometry, and the levels of inflammatory factors were detected by enzyme-linked immunosorbent assay. Results: After LPS treatment with different concentrations, we found that LPS at 10 μg/mL had the best effect on HK2 cells. Circ_0114428 was highly expressed in sepsis-AKI patients and LPS-treated HK2 cells. Knockdown of circ_0114428 restored the effects of LPS treatment on proliferation, apoptosis, and inflammatory response of HK2 cells. MiR-215-5p was a target of circ_0114428, and TRAF6 was a downstream target of miR-215-5p. Circ_0114428 regulated TRAF6 expression by sponging miR-215-5p in LPS-treated HK2 cells. Circ_0114428 regulated LPS-induced NF-κB signaling in HK2 cells by targeting miR-215-5p/TRAF6 axis. Conclusion: Circ_0114428 knockdown abolished the cell proliferation, apoptosis, and inflammatory damage in LPS-induced HK2 cells by targeting miR-215-5p/TRAF6/NF-κB.

Effects of Glucose Levels on Inflammation and Amino Acid Utilization in Lipopolysaccharide-Induced Bovine Mammary Epithelial Cells.

Glucose and amino acids are important sources of nutrients in the synthetic milk of dairy cows, and understanding the fate of amino acids is essential to optimize the utilization of amino acids in milk protein synthesis, thereby reducing nutrient inefficiencies during lactation. The purpose of this study was to investigate the effects of LPS and different concentrations of glucose on (1) the expression of inflammatory factors and genes, (2) the glucose metabolism, and (3) amino acid utilization in BMECs. The results showed that there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose content in the inflammatory cytokine genes (IL-6 and TNF-α) and the inflammatory regulatory genes (CXCL2, CXCL8, and CCL5). With the addition of LPS, the HG + LPS group caused downregulated (p < 0.05) expression of IL-6 and TNF-α, compared with the LG + LPS group. Interestingly, compared with the LG + LPS group, the HG + LPS group upregulated (p < 0.05) the expression of CXCL2, CXCL8, and CCL5. LPS supplementation increased (p = 0.056) the consumption of glucose and GLUT1 gene expression (p < 0.05) and tended to increase (p = 0.084) the LDHA gene expression of BMECs under conditions of different concentrations of glucose culture. High glucose content increased (p < 0.001) the consumption of glucose and enhanced (p < 0.05) the GLUT1, HK1, HK2, and LDHA gene expression of BMECs with or without LPS incubation, and there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose concentrations in GLUT1 gene expression. In this study, LPS enhanced (p < 0.05) the consumption of amino acids such as tryptophan, leucine, isoleucine, methionine, valine, histidine, and glutamate, while high levels of glucose decreased (p < 0.01) consumption, except in the case of tyrosine. For histidine, leucine, isoleucine, and valine consumption, there was an interaction (LPS × glucose, p < 0.05) between LPS and glucose levels. Overall, these findings suggest that relatively high glucose concentrations may lessen the LPS-induced BMEC inflammatory response and reduce amino acid consumption, while low glucose concentrations may increase the demand for most amino acids through proinflammatory responses.

COMPARISION OF THE GLIO-PROTECTIVE EFFECTS OF BIOPOLYMER COATED ELECTROSPUN SCAFFOLDS

Gliosis is a condition that plays a negative role in various neurological pathologies. In this study, it was aimed to obtain and compare the glio-protective effects of electrospun fibrous scaffolds coated by different biopolymers. First, in vitro gliosis model was obtained by inducing U-87 MG cells with LPS and IFN-γ. The optimized model was applied to U-87 MG cells grown on polycaprolactone (PCL), hyaluronic acid (HA) coated, gelatin coated, and collagen coated PCL scaffolds. Immunofluorescent (IF) staining was performed with glial fibrillary acidic protein (GFAP) antibody to determine the level of gliosis. It was found that 5µg/mL LPS concentration induced gliosis and hyaluronic acid-coated PCL scaffolds have shown a protective effect on gliosis.

PSIV-4 Impact of Lipopolysaccharide Endotoxin on Growth and Viability of Ovarian Granulosa Cells

Abstract Lipopolysaccharide (LPS) endotoxin from Gram-negative bacteria has been demonstrated to negatively impact reproductive parameters leading to ovarian dysfunction. Ovarian granulosa cells express the toll-like receptor 4 complex necessary to elicit inflammatory immune responses in the presence of LPS. Quantifiable amounts of LPS have been detected in follicular fluid from slaughterhouse-derived ovaries with further deleterious effects on granulosa cell steroidogenesis. Therefore, the objective of this study was to investigate the effects of LPS on granulosa cell proliferation in vitro. We hypothesized that increasing concentrations of LPS would negatively impact granulosa cell proliferation dynamics. A human granulosa cell line (KGN) was plated at 60% confluency in 96-well tissue culture plates and treated with phosphate buffered saline-vehicle or LPS (0.01, 0.1, 1, or 10 µg/mL) in the presence or absence of follicle-stimulating hormone (FSH). Cells were incubated for 48 h at 37 ºC and 5% CO2 in an IncuCyte incubator that utilizes real-time automated microscopy to continuously capture images of cells and measure cell confluency. Individual well images were collected every 6 h. The study was performed in 3 independent experiments with 8 replicates per treatment group in each experiment. Cells were allowed to equilibrate in the IncuCyte for 6 h before image collection. Cell confluency for each treatment group was normalized to vehicle-treated control confluency at 6 h. A significant difference was observed at the initial 6 h timepoint, with 1 and 10 µg/mL LPS treatment groups demonstrating decreased cell confluency compared with vehicle-treated controls (P &amp;lt; 0.05). However, no significant difference was observed in 0.01 (P = 0.65) and 0.1 (P = 0.53) µg/mL LPS treatment groups compared with vehicle-treated controls at 6 h timepoint. Interestingly, the deleterious effects of LPS on confluency and cell growth parameters were mitigated with the addition of FSH. No observable differences were detected in the two largest LPS doses concurrently incubated with FSH, 1 and 10 µg/mL LPS (P = 0.55 and P = 0.62, respectively), compared with vehicle-treated controls. From 12 h until the end of the incubation period, cell confluency in 1 and 10 µg/mL LPS treatment groups remained significantly different compared with FSH-treated cells (P &amp;lt; 0.05). Comparably, no differences were detected among FSH-stimulated groups, regardless of LPS dose or time (P = 0.99). Results indicate that greater LPS concentrations (1 and 10 µg/mL) negatively impacts cell confluency and proliferation in vitro; however, FSH has the ability to protect granulosa cells against the detrimental effects that LPS has on cell confluency. These data suggest that FSH is critical in regulating cell growth and proliferation despite the action of inflammatory pathways induced by bacterial endotoxin.

Low-grade endotoxemia in acute pulmonary embolism: Links with prothrombotic plasma fibrin clot phenotype

BackgroundLipopolysaccharide (LPS) can traverse the intestinal barrier and enter bloodstream, causing endotoxemia and triggering inflammation. Increased circulating LPS was reported in arterial thromboembolism. We investigated whether increased LPS levels occur in acute pulmonary embolism (PE) and if it is associated with a prothrombotic state. MethodsWe studied 120 normotensive PE patients (aged 59 [48–68] years) on admission, after 5–7 days, and after a 3-month anticoagulation. Serum LPS levels, along with zonulin, a marker of gut permeability, endogenous thrombin potential (ETP), fibrin clot permeability (Ks), clot lysis time (CLT), fibrinolysis proteins, and platelet markers were assessed. ResultsMedian LPS concentration on admission was 70.5 (61.5–82) pg/mL (min-max, 34–134 pg/mL), in association with C-reactive protein (r = 0.22, p = 0.018), but not with fibrinogen, D-dimer or platelet markers. Patients with more severe PE had higher LPS levels compared with the remainder. Median zonulin level was 3.26 (2.74–4.08) ng/mL and correlated with LPS (r = 0.66, p < 0.0001). Patients with baseline LPS levels in the top quartile (≥82 pg/mL; n = 29) compared to lower quartiles had 18.6 % increased ETP, 14.5 % reduced Ks, and 25.3 % prolonged CLT, related to higher plasminogen activator inhibitor type 1 (PAI-1) levels. LPS decreased by 23.4 % after 5–7 days and by 40.4 % after 3-month anticoagulation together with reduced zonulin by 18.4 % and 22.3 %, respectively, compared to baseline (all p < 0.001). LPS levels were not related with fibrin characteristics and other variables assessed at 3 months. ConclusionsLow-grade endotoxemia is detectable in patients with acute PE and may contribute to increased thrombin generation and PAI-1-mediated hypofibrinolysis.

Investigating the Association Between Diet-Induced "Leaky Gut" and the Development of Prediabetes.

Chronic consumption of a high-calorie diet compromises the gut microbiota and the integrity of the intestinal wall, which causes translocation of bacterial lipopolysaccharides (LPS) into the blood. This elicits the secretion of pro-inflammatory cytokines, resulting in inflammation. However, how a high-fat high carbohydrate diet affects intestinal permeability and its possible role in the development of prediabetes have not been investigated. This study investigated the effects of HFHC diet-induced prediabetes on gut microbiota and intestinal permeability in male Sprague Dawley rats. The animals were randomly assigned into the non-prediabetic (NPD) and diet-induced prediabetic (PD) groups (n=6) for 20 weeks. Then, the fecal samples were analyzed to measure the gut microbiota level of Firmicutes, Bacteroidetes, and Proteobacteria in both animal groups. Blood glucose, plasma insulin, serum zonulin, plasma LPS, soluble CD14, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and intestinal fatty-acid binding protein (IFABP) concentrations were measured. The PD group had a reduction in the Firmicutes and an increase in Bacteroidetes and Proteobacteria levels compared to those in the NPD group. Blood glucose, insulin concentration, serum zonulin, and plasma sCD14 concentrations in the PD group increased significantly, while plasma LPS concentrations were similar to the NPD group. Concentrations of plasma TNF-α, IL-6, CRP, and IFABP, an intracellular protein expressed in the intestine, increased in PD compared to the NPD group. the study results cumulatively suggest that chronic consumption of the HFHC diet may be associated with the dysregulation of gut microbiota, leading to increased intestinal permeability.

Akkermansia muciniphila and its outer membrane protein Amuc_1100 prevent high-fat diet-induced nonalcoholic fatty liver disease in mice

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. A. muciniphila and its outer membrane protein Amuc_1100 ameliorate metabolic disorders, enteritis, depression, and other diseases in mice. The NAFLD mouse model was established by feeding a high-fat diet (HFD) for 10 weeks. To assess the effect of A. muciniphila and Amuc_1100 on NAFLD, we used atorvastatin, a common lipid-lowering drug, as a positive control. A. muciniphila and Amuc_1100 significantly reduced body weight and serum ALT and AST levels, and improved serum lipid levels in NAFLD mice, which had similar effects to Ator. In addition, A. muciniphila and Amuc_1100 decreased the concentration of LPS in the serum and upregulated the mRNA expression of the colonic tight junction proteins. In the liver, A. muciniphila and Amuc_1100 significantly reduced the mRNA expression levels of nodular receptor protein 3 (NLRP3) and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB), and the protein and mRNA expression levels inflammatory cytokines. At the genus level, Amuc_1100 treatment significantly reduced the abundance of Coriobacteriaceae_UCG-002 produced by the HFD. The abundances of Blautia, norank_f__Ruminococcaceae, Lachnoclostridium, GCA-900066575 and Lachnospiraceae_UCG-006 increased dramatically. Together, A. muciniphila and Amuc_1100 alleviate HFD-induced NAFLD by acting on the gut-liver axis and regulating gut microbes.

A Comparison between SARS-CoV-2 and Gram-Negative Bacteria-Induced Hyperinflammation and Sepsis.

Sepsis is a life-threatening condition caused by the body's overwhelming response to an infection, such as pneumonia or urinary tract infection. It occurs when the immune system releases cytokines into the bloodstream, triggering widespread inflammation. If not treated, it can lead to organ failure and death. Unfortunately, sepsis has a high mortality rate, with studies reporting rates ranging from 20% to over 50%, depending on the severity and promptness of treatment. According to the World Health Organization (WHO), the annual death toll in the world is about 11 million. One of the main toxins responsible for inflammation induction are lipopolysaccharides (LPS, endotoxin) from Gram-negative bacteria, which rank among the most potent immunostimulants found in nature. Antibiotics are consistently prescribed as a part of anti-sepsis-therapy. However, antibiotic therapy (i) is increasingly ineffective due to resistance development and (ii) most antibiotics are unable to bind and neutralize LPS, a prerequisite to inhibit the interaction of endotoxin with its cellular receptor complex, namely Toll-like receptor 4 (TLR4)/MD-2, responsible for the intracellular cascade leading to pro-inflammatory cytokine secretion. The pandemic virus SARS-CoV-2 has infected hundreds of millions of humans worldwide since its emergence in 2019. The COVID-19 (Coronavirus disease-19) caused by this virus is associated with high lethality, particularly for elderly and immunocompromised people. As of August 2023, nearly 7 million deaths were reported worldwide due to this disease. According to some reported studies, upregulation of TLR4 and the subsequent inflammatory signaling detected in COVID-19 patients "mimics bacterial sepsis". Furthermore, the immune response to SARS-CoV-2 was described by others as "mirror image of sepsis". Similarly, the cytokine profile in sera from severe COVID-19 patients was very similar to those suffering from the acute respiratory distress syndrome (ARDS) and sepsis. Finally, the severe COVID-19 infection is frequently accompanied by bacterial co-infections, as well as by the presence of significant LPS concentrations. In the present review, we will analyze similarities and differences between COVID-19 and sepsis at the pathophysiological, epidemiological, and molecular levels.