LPS Treatment Group Research Articles

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 < 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 < 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.

396 Implications of Repeated Subacute Lipopolysaccharide Exposure During Synchronized Estrous

Abstract Acute inflammation induced by chronic infection or a single intravenous administration of the bacterial endotoxin, lipopolysaccharide (LPS) induces immune responses capable of depressing female fertility. However, the effect of dose and repeated endotoxin exposure during the estrous cycle on immune response and reproductive performance has yet to be investigated. To investigate the effects of varying doses of LPS delivered at distinct stages of follicular maturation, thirty-six Ramboulliet ewes (68.2 ± 1.1 kg; age 3 to 7 yr) were stratified by weight and age and randomly assigned to vehicle (n = 12), low (n = 12) or high (n = 12) LPS-treated groups to evaluate febrile response, serum progesterone and cortisol concentrations, and reproductive performance. Ewes received subcutaneous injections of saline (CON), 1.5 µg/kg BW LPS (LOW), or 3.0 µg/kg BW LPS (HIGH) on d 5, 10, and 15 after onset of synchronized estrus. Estrous synchronization was determined by serum progesterone (P4) analysis. All ewes demonstrated similar P4 profiles, but HIGH-LPS ewes tended to have elevated values (P = 0.1). On treatment d 5 and 15, blood samples and rectal temperature were collected before and intermittently throughout the duration of the trial. Lipopolysaccharide increased rectal temperature of LOW and HIGH groups at 2, 3, and 6 h, respectively after initial LPS challenge (d 5) when compared with vehicle-treated controls (0.67, and 0.70 °C, P < 0.05), but no significance was observed at d 10 or 15. Serum concentrations of cortisol were similar among treatment groups at 0 h (P > 0.05). However, a rapid and sustained cortisol increase was demonstrated within 1 h of LPS exposure in both LPS-treatment groups compared with controls (P = 0.001). A similar pattern of cortisol release among treatment groups was demonstrated on d 15 (P < 0.05). While there was no significant difference in services per cycle by treatment, there was a marked difference (16.67%) in remarks during the second cycle for HIGH and LOW groups (P = 0.23) as compared with controls. Control ewes had d 100 pregnancy rate of 90.91%, while low and high LPS-treated groups had pregnancy rates of 91.67% (P = 0.96) and 83.33% (P = 0.59) respectively. The administration of LPS resulted in significant increases in serum cortisol in treated ewes, and serum progesterone was significantly increased in LOW LPS-treated ewes compared with CON or HIGH LPS-treated ewes. Thus, alterations of temperature, steroid hormone concentration, and time to estrus in response to LPS may ultimately implicate female fertility and prevent economic losses for producers.

A novel optical imaging probe for targeted visualization of NLRP3 inflammasomes in a mouse model of age-related macular degeneration.

Wet form of age-related macular degeneration (wet AMD) is a progressive vascular disease that mainly affects older adults and causes severe and irreversible vision loss. A key complication of wet AMD is choroidal neovascularization (CNV), which may be driven in part by NLRP3 inflammasomes that are associated with macrophages migration to CNV lesions. Since activated NLRP3 is correlated with CNV, visualizing NLRP3 inflammasomes and their associated macrophages is of great interest to monitor wet AMD progression and develop effective therapies against it. However, to the best of our knowledge, current ophthalmic imaging systems do not permit such targeted imaging. Therefore, in this study, we developed InflammaProbe-1, an optical imaging probe for targeted visualization of NLRP3 inflammasomes in CNV lesions. InflammaProbe-1 was synthesized by conjugating a clinically relevant fluorophore, Oregon Green® 488, to the selective NLRP3 inhibitor, CY-09. The ability of InflammaProbe-1 to target NLRP3 was assessed with an enzyme-linked immunosorbent assay by comparing its ability to inhibit NLRP3-mediated secretion of IL-1β to that of CY-09 in LPS-primed and nigericin-stimulated BMDMs. In vitro confocal imaging of NLRP3 was performed on InflammaProbe-1-stained BMDMs that had been induced to express NLRP3 with LPS. In vivo imaging of NLRP3 was conducted on mouse laser induced choroidal neovascularization (LCNV), a model of AMD, 6 h after an intraperitoneal injection of InflammaProbe-1 at 10 mg/kg on day 4 post-LCNV. InflammaProbe-1 was just as effective as CY-09 at inhibiting IL-1β secretion (p < 0.01 at 10 μM for both the InflammaProbe-1 and CY-09 groups relative to the control). InflammaProbe-1-stained BMDMs that had been induced to express NLRP3 showed significantly brighter fluorescence than untreated cells (p < 0.0001 for LPS treatment group and p < 0.001 for LPS and nigericin treatment group). Furthermore, in vivo molecular imaging of NLRP3 was achieved in mouse LCNV. We propose that InflammaProbe-1 may be a useful molecular imaging probe to monitor the onset, progression, and therapeutic response of AMD and other NLRP3-mediated diseases.

The anti-inflammatory cytokine IL-22 exhibited a proinflammatory effect in the ovary aiding with the parturition of ovoviviparous black rockfish (Sebastes schlegelii)

IntroductionAs a unique type of immunological process, pregnancy and subsequent parturition are associated with a series of inflammatory events. As an anti-inflammatory cytokine, IL-22 has been proven to participate in the parturition process in mammals. However, less is known about ovoviviparous teleosts. To investigate the role of IL-22 in fish parturition, black rockfish, an ovoviviparous teleost, was used. This fish is unique in that it provides over 60% nutrition supply for over 50 thousand embryos at the same time though a placenta-like structure during pregnancy while delivering all fries in two hours.MethodsSequence alignment, phylogenetic tree analysis and homology modeling were performed on IL-22 and its receptor. In situ hybridization demonstrated ovarian localization of il22 and il22r1. The protein of rIL-22 was obtained through the prokaryotic expression. RNA-seq analysis was performed on black rockfish ovarian cells treated with rIL-22.ResultsIn situ hybridization results showed that both il22 and il22ra1 were localized in the perinatal follicle layer and embryonic envelope. The il22 expression level was significantly increased during parturition compared to before and after parturition (P&amp;lt;0.05). To further understand the mechanism of IL-22 in parturition, rIL-22 was obtained. The downstream variation genes in primary cultured ovarian cells in the perinatal period were analyzed according to the transcriptomic results. A total of 168 differentially expressed genes (DEGs) were identified in the rIL-22 group compared with the PBS control group (padj &amp;lt; 0.05 and |log2FoldChange| 1). Of those, 134 DEGs were identified in the LPS and rIL-22 treatment groups, including chemokine, cytokine, and PG synthesis. 34 DEGs were mainly identified in immune-related pathways and reorganization of the cytoskeleton responded only to rIL-22 stimulation but not LPS. KEGG pathways mainly included immune response, PG synthesis, cell death and angiogenesis (p value &amp;lt; 0.05).DiscussionTaken together, our results indicated that IL-22 plays an important role in the parturition of black rockfish by upregulating PG synthesis and increasing chemokine, proinflammatory factor, as well as PG synthetase levels.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

Inhibition of Netosis with PAD Inhibitor Attenuates Endotoxin Shock Induced Systemic Inflammation.

Neutrophils play a pivotal role in innate immunity by releasing neutrophils extracellular traps (NETs). Excessive NETs are detrimental to the local tissue and further exacerbate inflammation. Protein arginine deiminases (PAD) mediate histone citrullination and NET formation that, in turn, exacerbate endotoxin shock damages. In this study, we further investigated the molecular mechanism underlying PAD and NETs in endotoxic stress in mice. The control group mice were injected with solvent, the LPS endotoxic shock group mice were intraperitoneally injected with LPS at 35 mg/kg only, while the LPS and PAD inhibitor YW3-56 treatment group mice were injected with YW3-56 at 10 mg/kg prior to the LPS injection. YW3-56 significantly prolonged the survival time of the LPS-treated mice. NETs, cfDNA, and inflammatory factors were detected by ELISA in serum, paitoneal cavity, and lung at 24 h after LPS administration. Lung injuries were detected by immunostaining, and lung tissue transcriptomes were analyzed by RNA-seq at 24 h after LPS administration. We found that YW3-56 altered neutrophil tissue homeostasis, inhibited NET formation, and significantly decreased cytokines (IL-6, TNFα and IL-1β) levels, cytokines gene expression, and lung tissue injury. In summary, NET formation inhibition offers a new avenue to manage inflammatory damages under endotoxic stress.

New insights into the regulation mechanism of Litopenaeus vannamei hepatopancreas after lipopolysaccharide challenge using transcriptome analyses

Litopenaeus vannamei (L. vannamei) is the most economically valuable cultured shrimp in the world, while Gram-negative bacteria infection causes huge economic losses to shrimp culture. In this study, we performed transcriptome sequencing of the hepatopancreas in L. vannamei after lipopolysaccharide (LPS, the cell wall component of Gram-negative bacteria) injection to investigate the response of shrimp under Gram-negative bacteria invasion. A total of 306 differentially expressed genes (DEGs) (70 up- and 236 down-regulated) were identified in the LPS treatment group (L group) when compared to their expression levels in the control group (C group). The oxidoreductase activity (GO:0016491) in the molecular function category was enriched in the LPS-responsive DEGs in GO annotation, and the metabolism of xenobiotics by cytochrome P450 (ko00980) was the most enriched pathway in KEGG annotation. The transcriptome profiling revealed that the toll like receptor, C-type lectin receptor, and β-1,3-glucan binding protein were involved in the recognition of LPS during its early invasion stage. Although LPS could reduce the metabolic ability of exogenous substances, induce inflammation and reduce antioxidant capacity, L. vannamei could maintain its homeostasis by improving immunity, enhancing anti-stress ability and reducing apoptosis. Our research provides the first transcriptome profiling for the L. vannamei hepatopancreas after LPS injection. These results could offer a valuable reference on the mechanism of shrimp against Gram-negative bacteria and could provide guidance for shrimp farming.

Remimazolam Protects Against LPS-Induced Endotoxicity Improving Survival of Endotoxemia Mice.

Remimazolam is a new benzodiazepine of sedative drugs with an ultra-short-acting anesthetic effect, commonly used for critically ill patients (especially septic patients) in intensive care units (ICUs). Although some anesthetics have been reported to show certain anti-inflammatory effects, the role of remimazolam in inflammation is still remained unknown. Here, we studied the effects of remimazolam on macrophage in response to LPS both in vivo and in vitro. Interestingly, compared with LPS treatment group, remimazolam remarkably improved survival rate of endotoxemia mice and decreased the release of LPS-induced inflammatory mediators (such as TNF-α, IL-6, and IL-1β). We further found that remimazolam not only inhibited the activation of MAPK signal pathway at 15 min after LPS treatment but also disturbed Rab5a related TLR4 expression at cell surface in response to LPS at a later time. Such evidence suggests that remimazolam might be beneficial to septic patients who are suffering from uncontrolled inflammatory responses.

MicroSPECT Imaging-Guided Treatment of Idiopathic Pulmonary Fibrosis in Mice with a Vimentin-Targeting 99mTc-Labeled N-Acetylglucosamine-Polyethyleneimine.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease with poor prognosis. Evidence has shown that vimentin is a key regulator of lung fibrogenesis. 99mTc-labeled N-acetylglucosamine-polyethyleneimine (NAG-PEI), a vimentin-targeting radiotracer, was used for the early diagnosis of IPF, and NAG-PEI was also used as a therapeutic small interfering RNA (siRNA) delivery vector for the treatment of IPF in this study. Single-photon emission-computed tomography (SPECT) imaging of bleomycin (BM)- and silica-induced IPF mice with 99mTc-labeled NAG-PEI was performed to visualize pulmonary fibrosis and monitor the treatment efficiency of siRNA-loaded NAG-PEI, lipopolysaccharide (LPS, a tolerogenic adjuvant), or zymosan (ZYM, an immunostimulant). The lung uptakes of 99mTc-NAG-PEI in the BM- and silica-induced IPF mice were clearly and directly correlated with IPF progression. The lung uptake of 99mTc-NAG-PEI in the NAG-PEI/TGF-β1-siRNA treatment group or LPS treatment group was evidently lower than that in the control group, while the lung uptake of 99mTc-NAG-PEI was significantly higher in the ZYM treatment group compared to that in the control group. These results demonstrate that NAG-PEI is a potent MicroSPECT imaging-guided theranostic platform for IPF diagnosis and therapy.

Anti-Inflammatory Effect Fraction of Bletilla striata and Its Protective Effect on LPS-Induced Acute Lung Injury

Bletilla striata is a well-known traditional Chinese herb with anti-inflammatory properties that is widely used in the treatment of lung conditions such as silicosis, tuberculosis, and pneumogastric hemorrhage. However, little information on the anti-inflammatory ingredients and their activities is available. In this study, an effect fraction of Bletilla striata (EFBS) was enriched, and its anti-inflammatory activities and underlying mechanisms were investigated. EFBS was enriched by polyamide column chromatography and characterized by HPLC; an LPS-induced acute lung injury model was used to evaluate the anti-inflammatory activities of EFBS. Meanwhile, the main anti-inflammation-contributing ingredients and possible molecular mechanism of anti-inflammatory activity in EFBS were verified by component-knockout method combined with LPS-induced RAW264.7 cell model. The EFBS mainly consisted of coelonin (15.88%), batatasin III (32.49%), 3′-O-methylbatatasin III (6.96%), and 3-hydroxy-5-methoxy bibenzyl (2.51%). Pretreatment with the EFBS (20 mg/kg and 60 mg/kg) for five days prior to the administration of LPS resulted in decreases in wet-to-dry lung weight ratio, neutrophil number, MPO activity, total protein concentration, NO level, and MDA level, as well as IL-1β, IL-6, MCP-1, and TNF-α concentrations in the bronchoalveolar lavage fluid. Western blot analysis demonstrated the increased expressions of iNOS, COX-2, and NF-κB p65 in the LPS treatment group, all of which were ameliorated by EFBS pretreatment. Histological examination confirmed the protective effect of the EFBS. Additionally, component-knockout assay confirmed that these four quantitative components contributed significantly to the anti-inflammatory effect of EFBS. Coelonin, batatasin III, 3′-O-methylbatatasin III and 3-hydroxy-5-methoxy bibenzyl were the main anti-inflammatory components of EFBS and could regulate the expression of downstream inflammatory cytokines by inhibiting p65 nuclear translocation. These findings uncover, in part, the molecular basis underlying the anti-inflammatory activity of Bletilla striata.

Integrative Analysis of MicroRNAs and mRNAs in LPS-Induced Macrophage Inflammation Based on Adipose Tissue Stem Cell Therapy.

Severe inflammation can lead to multiple organ dysfunction syndrome, which has high mortality. Adipose-derived stem cells have been shown to affect the inflammatory response of macrophages. However, the molecular mechanism of the anti-inflammatory capacity of adipose-derived stem cells (ADSCs) remains to be understood. In the present study, a macrophage inflammation model was established by LPS, and treated with different volumes of ADSC supernatant. Then, we investigated the key genes in the LPS group and treatment group by RT-PCR, RNA sequencing technology, and bioinformatics analysis. A total of 26 miRNAs and 11,882 mRNAs were differentially expressed between them. The expression of 15 of the miRNAs (9 upregulated and 6 downregulated) was confirmed by RT-PCR. GO and KEGG pathway analyses of the targets of the 9 significantly upregulated miRNAs showed that they were related to immune system process, inflammatory response, lipopolysaccharide, and TNF-α, NF-κB, Toll-like receptor, and MAPK signaling pathways. Moreover, a miRNA-mRNA network also revealed 8 important genes (Mapkapk2, Sepp1, Cers6, Snn, ZfP568, Ccdc93, Pofut1, Pik3cd). We finally confirmed the expression of these 8 targeted genes by performing the RT-PCR analysis. This study may provide a new understanding of the molecular mechanism of ADSCs in the inflammatory response related to multiple miRNAs and mRNAs.

Comparison of Biological Activities of Different Parts of Asparagus

Abstract Objectives Asparagus is a spring vegetable, a flowering perennial plant species belonging to the lily family. Technavio's analysts forecast the Global Asparagus Market to grow at a CAGR of 3.15% during the period 2018–2022. All parts of asparagus, including roots, are rich in bioactive chemicals. However, other parts except asparagus spears are discarded. This study compared the physiological activity of different parts of asparagus and tried to improve the utilization of other parts. Methods Asparagus was divided into root, spear (5 cm from the bottom, 25 cm from the top, respectively), leaf and stem in order and used after lyophilized. Total phenol content, antioxidant activity (DPPH radical elimination ability, ABTS radical elimination ability), α-amylase inhibitory activity, and the effects of nitric oxide (NO) production were measured. Results The total polyphenol content of asparagus was highest in the leaves (22.87 mg TAE/g) and lowest in 5 cm under part of the spear(3.03 mg TAE/g). DPPH radical scavenging effect (44.52% in 1 mg/mL, 92.84% in 5 mg/mL), and ABTS radical scavenging effect(15.58% in 1 mg/mL, 48.02% in 5 mg/1 mL) were the highest on the leaf. On 5 cm under part of the spear, α-amylase inhibitory activity (79.16%) was the higher than any other parts. NO generation was significantly hindered by LPS treatment group 5 cm under part of the spear (12.93 μM), 25 cm above the part of the spear (12.10 μM), root (11.68 μM), leaf(10.43 μM), and stem(9.70 μM) in order(P &amp;lt; 0.001). All the extracts were treated with the cells at concentrations of 100, 500 and 1000 µg/mL for one day. As a result, the survival rate of the macrophages was not significantly different from that of the control group. So it was confirmed that there is no cytotoxicity. Conclusions Thus, these results suggest that the another parts of asparagus may be highly valuable as a high-quality functional component as well as its-antioxidant, antiinflammatory activities. we believe that the results obtained would be helpful in the development of nutraceutics. Funding Sources This study was supported by 2019 Regional Specialized Technology Development Project, Rural Development Administration, Republic of Korea.

Effects and Mechanism of lncRNA CRNDE on Sepsis-Induced Acute Kidney Injury.

Objective To investigate the effects of lncRNA CRNDE on sepsis-associated acute kidney injury in the human kidney 2 cell line and explore the potential mechanisms. Methods HK-2 cells were treated with lipopolysaccharides to induce injuries. The expression of CRNDE and miR-146a in HK-2 cells were altered by a transient transfection assay. Cell apoptosis was detected by a flow cytometry assay, and the levels of inflammatory cytokines including TNF-α, IL-6, IL-8, and IL-1β were assessed by ELISA. Furthermore, western blot analysis was performed to detect the expression levels of TLR4/NF-κB pathway-related proteins. And a luciferase reporter gene assay was used to verify if miR-146a was the target of CRNDE. Results LPS treatment increased CRNDE expression in HK-2 cells. CRNDE overexpression enhanced cell injuries in HK-2 cells significantly increasing inflammatory cytokine levels, including TNF-α, IL-6, IL-8, and IL-1β, and cell apoptosis. In addition, CRNDE overexpression further activated the TLR4/NF-κB pathways in HK-2 cells. Inversely, opposite results were observed in the miR-146a mimic treatment group, and the miR-146a inhibitor could reverse the protein expression changes of TLR4/NF-κB in the si-CRNDE and LPS treatment group. Conclusion This study demonstrated that CRNDE overexpression could activate the TLR4/NF-κB signaling pathway by regulating miR-146a, which accelerated LPS-induced inflammation and apoptosis in HK-2 cells.

Deep proteome profiling reveals novel pathways associated with pro-inflammatory and alcohol-induced microglial activation phenotypes

Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including those related to alcohol abuse. Given the complexity of global-scale molecular changes that define microglial activation, accurate phenotypic classification in the context of alcohol exposure is still lacking. We employed an optimized method for deep, quantitative proteome profiling of primary microglia in order to characterize their response to acute exposure to alcohol (ethanol) as well as the pro-inflammatory driver and TLR4 agonist, LPS. From this analysis, 5,062 total proteins were identified where 4,857 and 4,928 of those proteins were quantifiable by label-free quantitation in ethanol and LPS treatment groups, respectively. This study highlights the subtle, yet significant proteomic changes that occur in ethanol-treated microglia, which do not align with the robust pro-inflammatory phenotype induced by TLR4 activation. Specifically, our results indicate inhibition of several upstream regulators associated with inflammation, opposing effects on pathways such as phagocytosis upon comparison to TLR4-mediated pro-inflammatory phenotype, and a potential metabolic shift associated with increased expression of proteins related to OXPHOS and lipid homeostasis. Data are available via ProteomeXchange with identifier PXD14466. SignificanceAlcohol abuse has a significant impact on the central nervous system, which includes the pathophysiological mechanisms resulting from glial cell activation. Microglia, in particular, are the resident immune cells of the brain and exhibit a broad range of activation phenotypes. The molecular changes that drive microglial activation phenotype are complex and have yet to be fully characterized in the context of alcohol exposure. Our study highlights the first and most comprehensive characterization of alcohol-induced proteomic changes in primary microglia to date and has shed light on novel immune-related and metabolic pathways that are altered due to alcohol exposure. The results from this study provide an important foundation for future work aimed to understand the complexity of alcohol-induced microglial activation in vivo and other translational models of acute and chronic alcohol exposure.

Effects of estrogen-related receptor alpha on inflammatory response in pulmonary microvascular endothelial cells

Objective To investigate the effect of estrogen-related receptor alpha(ERRα)on lipopolysaccharide-induced inflammatory response in rat pulmonary microvascular endothelial cells (PMVECs) and its mechanism. Methods PMVECs were cultured in vitro. When the cells were in the logarithmic growth phase, the cell were ransfected with lentivirus, and a stable low-expression ERRα cell line was constructed. The cells were divided into four groups: Ctr group (normal control group), Ctr+LPS group (normal cell+LPS treatment group), shERRα1 (shERRα1 gene knockdown group), and shERRα1+LPS group (shERRα1 gene knockdown +LPS treatment group). After 20 μg/mL LPS stimulated cells in the control group and shERRα1 group for 6, 12 and 24 h, cell counting kit-8 (cck-8) was used to detect the cell proliferation ability of each group, and enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of tumor necrosis factor alpha (TNF-α) and Interleukin-1β (IL-1β) in cell culture fluid. After 12 h LPS stimulation, the expression levels of ERRα and NF-κB related proteins (p-p65, p65, P-IKBα, IKBα) were measured by Western blot. Pairwise comparisons were performed with SNK-q test (two-tailed), and multiple-group comparisons were performed with one-way ANOVA. The non-parametric test of rank transformation was used when homogeneity of variance were not met. P value<0.05 was considered significantly different. Results Compared with the control group, ERRα expression in the shERRα group was significantly decreased (0.09±0.01 vs 0.15±0.01). At 6, 12 and 24 h after LPS stimulation, compared with the control group, the cell proliferation ability (%) of the shERRα1+LPS group was significantly reduced (99.68±4.53 vs 48.62±1.60) and the concentration of TNF-α (ng/mL) (15.76±3.38 vs 5 498.91±367.95) and IL-1β (ng/mL) (14.41±3.86 vs 6 014.92±277.33) in the cell culture supernatant were significantly increased. The change was most obvious after 12 h stimulation. Meanwhile the expression of p-p65 (0.30±0.50 vs 1.05±0.07) and p-IKBα (0.27±0.04 vs 0.77±0.06) were increased significantly, while the expression of IKBα (0.96±0.07 vs 0.14±0.04) was decreased significantly in the shERRα1+LPS group (all P<0.05). Conclusion ERRα gene attenuates LPS-induced inflammatory response in rat pulmonary microvascular endothelial cells by inhibiting NF-κB signaling pathway activation. Key words: Sepsis; Estrogen-related receptor alpha; NF-κB pathway; Gene knockdown

Socheongryongtang suppresses COPD-related changes in the pulmonary system through both cytokines and chemokines in a LPS COPD model

Context Socheongryongtang is a traditional Korean medical prescription used to treat pulmonary diseases. Objective This study investigated the therapeutic mechanism of socheongryongtang for pulmonary diseases. Materials and methods Seventy BALB/c mice were used: control, 0.8 mg/kg/study LPS intranasal instillation, 1 mg/kg/day Spiriva oral administration for five days, two socheongryongtang groups (150 or 1500 mg/kg/day orally treatment for five days). To illuminate the anti-COPD mechanism, several factors were evaluated such as WBC and differential counts in BALF and IgE in serum, morphological changes, and changes of COPD-related cytokines (TNF-α, IFN-γ, TGF-β) and chemokines (CXCL1, CCL-2, CCR2) in the lung. In order to confirm the statistical significance, all results were compared under p < 0.01 and p < 0.05. Results LPS induced a high level of WBC, neutrophils and eosinophils in our in vivo study. Additionally, COPD related cytokines and chemokines such as TNF-α, IFN-γ, TGF-β, CXCL1, CCL-2 and CCR2 were induced by LPS. Compared to the LPS treatment group, socheongryongtang significantly controlled the level of WBC, neutrophils and eosinophils as well as the level of IgE. It effectively down-regulated the morphological changes, such as fibrosis near bronchoalveolar spaces, small airway destruction (emphysema), etc. It also inhibited the levels of COPD-related cytokines (TNF-α, IFN-γ, TGF-β) and chemokines (CXCL1, CCL-2, CCR2) compared to the LPS treatment group. In particular, socheongryongtang significantly down-regulated the levels of TNF-α, IFN-γ, and CCR2. Conclusions Socheongryongtang controlled COPD, but as it has been used as a prescription for respiratory disease, we should additionally evaluate the therapeutic effects against various pulmonary diseases.

Expression profile of circular RNA in LPS-treated murine peritoneal macrophages

Objective To analyze the expression profile of circular RNA (circRNA) in LPS-treated murine peritoneal macrophages (MPMs) and to investigate the effects of mmu_circ_0000790 (circ790) on the secretion of pro-inflammatory cytokines in MPMs following LPS stimulation. Methods MPMs were isolated from C57BL/6 male mice and then stimulated with (LPS treatment group) or without (blank control group) 1 mg/L of LPS for 12 hours. Supernatants of cell culture and cell pellets were collected from each group. Enzyme linked immunosorbent assay (ELISA) was used to measure the changes in the secretion of IL-1β, IL-6 and TNF-α in the supernatants. Expression profile of circRNA in macrophages from the two groups (n=3) was analyze by circRNA microarray. Some differentially expressed circRNAs were validated by real-time PCR (RT-PCR). Moreover, RT-PCR was also performed to detect the expression of circ790 in MPMs after LPS stimulation. Small interfering RNA (siRNA) oligo specific for circ790 was designed and synthesized. Then the synthesized siRNA oligo and normal control (NC) oligo were transfected into MPMs by Lipofectamine™2000. The transfected MPMs were treated with LPS. ELISA was used to detect the levels of IL-1β, IL-6 and TNF-α in the supernatants. Arraystar′s home-made microRNA target prediction software was used to predict circ790/microRNA. Results The concentrations of IL-1β , IL-6 and TNF-α in the LPS treatment group were significantly higher than those in the blank control group (P<0.01). These results indicated that the inflammatory model of MPMs was successfully constructed. Statistical differences in 34 differentially expressed circRNAs were found between the two groups (P<0.05). Among them, 12 circRNAs were up-regulated and the other 22 circRNAs were down-regulated in the LPS treatment group. RT-PCR results were generally consistent with the microarray data. The expression of circ790 was increased by (1.94±0.15), (3.18±0.13) and (4.21±0.22) folds as compared with that of the blank control group at 3, 6 and 12 h after LPS stimulation (P<0.05). After transfecting the MPMs with circ790-siRNA, the secretion of IL-6 was significantly decreased (P<0.05) without notable influence on the secretion of IL-1β and TNF-α. circ790 could function as a microRNA sponge to regulate the gene expression. Conclusion These data show a significantly altered circRNA expression profile in the LPS-treated MPMs. circ790 may be involved in the regulation of IL-6 secreted by macrophages. Key words: Circular RNA; LPS; Macrophage; Inflammatory response

Altered Irinotecan Pharmacokinetics During Inflammation in Mice

Irinotecan is an anti‐cancer drugprimarily used in the treatment of colorectal cancer and its primary metabolite, SN‐38, is an active compound that also causes intestinal toxicity. It is widelyknown that inflammation downregulates the expression of many drug metabolizingenzymes, resulting in changes in drug disposition. Our aim is to investigate the role of inflammation on pharmacokinetics of irinotecan and its metabolites, SN‐38 and SN‐38G. Mice (C57BL6, male) were treated with either saline or LPS(2mg/kg) I.P and Irinotecan (10 mg/kg) was administered 16 hours later via oralgavage. Blood samples were collected by snipping the tail at different time points from 0–24 h and the blood concentrations of CPT‐11, SN‐38, and SN‐38G were measured by LC‐MS/MS. The concentrations of SN‐38 were found to be significantly higher in LPS treatment group compared to saline group. Our findings show that the toxic metabolite SN‐38 blood concentrations were increased during inflammation, which suggests that patients with inflammation may have higher risk of adverse effects of irinotecan due to higher SN‐38 concentrations.

The Role of IL-17 in a Lipopolysaccharide-Induced Rhinitis Model.

PurposeLipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria and important for pro-inflammatory mediators. This study aimed to establish a rhinitis model using ovalbumin (OVA) and LPS in order to evaluate the role of interleukin (IL)-17 in the pathogenesis of an LPS-induced non-eosionophilic rhinitis model.MethodsMice were divided into 4 groups and each group consisted of 10 mice (negative control group, allergic rhinitis model group, 1-µg LPS treatment group, and 10-µg LPS treatment group). BALB/c mice were sensitized with OVA and 1 or 10 µg of LPS, and challenged intranasally with OVA. Multiple parameters of rhinitis were also evaluated to establish the LPS-induced rhinitis model. IL-17 knockout mice were used to check if the LPS-induced rhinitis model were dependent on IL-17. Eosinophil and neutrophil infiltration, and mRNA and protein expression profiles of cytokine in nasal mucosa or spleen cell culture were evaluated using molecular, biochemical, histopathological, and immunohistological methods.ResultsIn the LPS-induced rhinitis model, neutrophil infiltration increased in the nasal mucosa, and systemic and nasal IL-17 and interferon-gamma (IFN-γ) levels also increased as compared with the OVA-induced allergic rhinitis model. These findings were LPS-dose-dependent. In IL-17 knockout mice, those phenotypes (neutrophil infiltration, IL-17, and IFN-γ) were reversed, showing IL-17 dependency of LPS-induced rhinitis. The expression of vascular endothelial growth factor (VEGF), an important mediator for inflammation and angiogenesis, decreased in IL-17 knockout mice, showing the relationship between IL-17 and VEGF.ConclusionsThis study established an LPS-induced rhinitis model dependent on IL-17, characterized by neutrophil infiltration and increased expression of IL-17.

Modulation of the innate immune-related genes expression in H9N2 avian influenza virus-infected chicken macrophage-like cells (HD11) in response to Escherichia coli LPS stimulation

Macrophages play important roles in mediating virus-induced innate immune responses and are thought to be involved in the pathogenesis of bacterial superinfections. The innate immune response initiated by both low pathogenicity AIV and bacterial superinfection in their avian host is not fully understood. We therefore determine the transcripts of innate immune-related genes following avian H9N2 AIV virus infection and E. coli LPS co-stimulation of avian macrophage-like cell line HD11 cells. More pronounced expression of pro-inflammatory cytokines (IL-6 and IL-1β) as well as the inflammatory chemokines (CXCLi1 and CXCLi2) was observed in virus infected plus LPS treated HD11 cells compared to H9N2 virus solely infected control. For two superinfection groups, the levels of genes examined in a prior H9N2 virus infection before secondary LPS treatment group were significantly higher as compared with simultaneous virus infection plus LPS stimulation group. Interestingly, similar high levels of IL-6 gene were observed between LPS sole stimulation group and two superinfection groups. Moreover, IL-10 and TGF-β3 mRNA levels in both superinfection groups were moderately upregulated compared to sole LPS stimulation group or virus alone infection group. Although TLR4 and MDA5 levels in virus alone infection group were significantly lower compared to that in both superinfection groups, TLR4 upregulation respond more rapid to virus sole infection compared to LPS plus virus superinfection. Collectively, innate immune-related genes respond more pronounced in LPS stimulation plus H9N2 virus infection HD11 cells compared to sole virus infection or LPS alone stimulation control cells.