LPS-induced Cytokine Research Articles

Blocking osteopontin expression attenuates neuroinflammation and mitigates LPS-induced depressive-like behavior in mice

IntroductionNeuroinflammation plays an important role in the development of major depressive disorder (MDD). Osteopontin (OPN) is one of the key molecules involved in neuroinflammation. We demonstrate here for the first time a key role of OPN in lipopolysaccharide (LPS)-induced depressive-like behavioral syndrome. MethodsSystemic administration of LPS (5 mg/kg) mimics distinct depressive-like behavior, which could significantly upregulate OPN expression in microglia/macrophage in the hippocampus. The neurobehavioral assessments, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Western blot, immunofluorescent staining, flow cytometry cell staining and Golgi staining were performed. ResultsSimilar to fluoxetine treatment (the positive control), OPN knockdown with shRNA lentivirus markedly reversed LPS-induced depressive-like behavior. Moreover, knockdown of OPN suppressed LPS-induced proinflammatory cytokine expression, microglial activation, dendritic spines loss, as well as unregulated PSD-95 and BDNF in the hippocampus. ConclusionWe demonstrated that targeting OPN expression in microglia/macrophage might help to rescue LPS-induced depressive-like behavior. The underlying mechanism may relate to the modulation of neuroinflammation, BDNF signaling and synaptic structural complexity.

4-Methylesculetin ameliorates LPS-induced depression-like behavior through the inhibition of NLRP3 inflammasome.

The pathophysiology of depression is heavily dependent on inflammation. Evidence suggests that the etiology of depression is linked with NLRP3 inflammasome-induced inflammation. Therefore, blocking the activated NLRP3 inflammasome may be beneficial for treating depression. Due to the limitations of currently available antidepressants, it is necessary to develop novel, safe, and affordable drugs for the treatment of depression. A natural coumarin derivative named 4-methylesculetin (4-MESC) possesses anti-inflammatory properties. However, the role of 4-MESC as an antidepressant has not been elucidated. Therefore, in this study, we explored the antidepressant-like effects of 4-MESC and its underlying molecular mechanism through the modulation of the NLRP3 inflammasome. The docking and molecular dynamic simulation studies revealed that 4-MESC has a higher affinity for the NLRP3 PYD. Blood-brain barrier permeability was confirmed using the SwissADME pharmacokinetic tool. High doses (50mg/kg) of 4-MESC significantly reduced the immobility duration in the tail-suspension test (TST) and forced swim test (FST) without changing the overall locomotor activity in the female Swiss albino mice that were subjected to lipopolysaccharide (LPS). LPS-induced pro-inflammatory cytokines such as IL-6 and TNF-α were reduced in serum and brain tissues using 4-MESC. 4-MESC's neuroprotective effects are mediated by increased brain-derived neurotrophic factor (BDNF) and decreased cortisol levels. 4-MESC markedly reduced LPS-induced elevated levels of ROS and lipid peroxidation (malondialdehyde levels) and enhanced the superoxide dismutase (SOD) activity and glutathione levels, which revealed its anti-oxidant potential against oxidative stress. 4-MESC diminished the expression levels of NF-κBp65, IL-6, NLRP3, caspase-1, gasdermin D, and IL-1β in the hippocampus. These findings demonstrated that 4-MESC exhibited antidepressant-like effects by inhibiting the NLRP3 inflammasome. However, other antidepressant mechanisms might also be involved which require further studies.

Dual red and near-infrared light-emitting diode irradiation ameliorates LPS-induced otitis media in a rat model.

Objective: Otitis media (OM) is an infectious and inflammatory disease of the middle ear (ME) that often recurs and requires long-term antibiotic treatment. Light emitting diode (LED)-based devices have shown therapeutic efficacy in reducing inflammation. This study aimed to investigate the anti-inflammatory effects of red and near-infrared (NIR) LED irradiation on lipopolysaccharide (LPS)-induced OM in rats, human middle ear epithelial cells (HMEECs), and murine macrophage cells (RAW 264.7). Methods: An animal model was established by LPS injection (2.0mg/mL) into the ME of rats via the tympanic membrane. A red/NIR LED system was used to irradiate the rats (655/842nm, intensity: 102mW/m2, time: 30min/day for 3 days and cells (653/842nm, intensity: 49.4mW/m2, time: 3h) after LPS exposure. Hematoxylin and eosin staining was performed to examine pathomorphological changes in the tympanic cavity of the ME of the rats. Enzyme-linked immunosorbent assay, immunoblotting, and RT-qPCR analyses were used to determine the mRNA and protein expression levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Mitogen-activated protein kinases (MAPKs) signaling was examined to elucidate the molecular mechanism underlying the reduction of LPS-induced pro-inflammatory cytokines following LED irradiation. Results: The ME mucosal thickness and inflammatory cell deposits were increased by LPS injection, which were reduced by LED irradiation. The protein expression levels of IL-1β, IL-6, and TNF-α were significantly reduced in the LED-irradiated OM group. LED irradiation strongly inhibited the production of LPS-stimulated IL-1β, IL-6, and TNF-α in HMEECs and RAW 264.7 cells without cytotoxicity in vitro. Furthermore, the phosphorylation of ERK, p38, and JNK was inhibited by LED irradiation. Conclusion: This study demonstrated that red/NIR LED irradiation effectively suppressed inflammation caused by OM. Moreover, red/NIR LED irradiation reduced pro-inflammatory cytokine production in HMEECs and RAW 264.7 cells through the blockade of MAPK signaling.

Cinnamomum japonicum Siebold Branch Extracts Attenuate NO and ROS Production via the Inhibition of p38 and JNK Phosphorylation.

Cinnamomum japonicum (CJ) is widely distributed in Asian countries like Korea, China, and Japan. Modern pharmacological studies have demonstrated that it exhibits various biological activities, including antioxidant and anti-inflammatory effects. However, most studies have confirmed the efficacy of its water extract but not that of its other extracts. Therefore, in this study, Cinnamomum japonicum Siebold branches (CJB: 70% EtOH extract) were separated using hexane, chloroform, ethyl acetate (CJB3), butanol, and water. Then, their antioxidative activities and phenolic contents were measured. Results revealed that the antioxidant activities and phenolic contents of CJB3 were higher than those of the other extracts. Further, the inhibitory and anti-inflammatory effect of CJB3 on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production and LPS-activated macrophages, respectively, was determined. CJB3 suppressed oxidative stress in LPS-activated cells and dose-dependently decreased LPS-stimulated ROS production. CJB3 reduced oxidative stress and reversed the glutathione decrease in LPS-activated RAW264.7 cells. The inhibitory and reducing effect of CJB3 on LPS-induced nitric oxide (NO) production and inducible NO synthase protein and messenger RNA levels, respectively, was investigated. CJB3 inhibited LPS-induced cytokine production and p38 and c-Jun N-terminal kinase (JNK) phosphorylation but not extracellular signal-regulated kinase phosphorylation. Overall, the study results suggest that CJB3 may exert its anti-inflammatory effects via the suppression of p38, JNK, and c-Jun activation.

Effect of HSPA8 gene on the proliferation, apoptosis and immune function of HD11 cells

HSPA8 (Heat shock 70 kDa protein 8) is a molecular chaperone involved in a variety of cellular processes. This gene may affect the proliferation, apoptosis and immune function of chicken macrophages, but the specific mechanism remains unclear. The purpose of this study was to explore the effect of the HSPA8 gene on the proliferation, apoptosis and immune function of chicken macrophages. In this study, a chicken HSPA8 overexpression plasmid, interference fragment and corresponding controls were transfected into HD11 cells, and then the expression of the HSPA8 gene, cell proliferation, cell cycle, apoptosis rate and immune function of each group were detected. The results showed that transfection of the HSPA8 overexpression plasmid significantly upregulated the level of HSPA8 expression in HD11 cells compared with the control; significantly promoted the proliferation of HD11 cells and the expression of PCNA, CCND1 and CCNB3; decreased the number of cells in the G1 phase and increased the number of cells in the S phase; decreased the rate of apoptosis and upregulated the expression of Bcl-2; and promoted the expression of the LPS-induced cytokines IL-1β, IL-6 and TNF-α. Transfection of the HSPA8 interference fragment significantly downregulated the level of HSPA8 expression in HD11 cells; significantly inhibited the proliferation of HD11 cells and the expression of PCNA, CCND1 and CDK1; increased the number of cells in the G1 phase and decreased the number of cells in the S phase; increased the rate of apoptosis, downregulated the expression of Bcl-2 and upregulated the expression levels of Fas and FasL; and inhibited the expression of the LPS-induced cytokines IL-1β and NF-κB. The results suggested that HSPA8 promotes the proliferation of and inhibits the apoptosis of HD11 cells and has a proinflammatory effect.

Acute respiratory distress syndrome enhances tumor metastasis into lungs: Role of BRD4 in the tumor microenvironment.

Acute respiratory distress syndrome (ARDS) is associated with severe lung inflammation, edema, hypoxia, and high vascular permeability. The COVID-19-associated pandemic ARDS caused by SARS-CoV-2 has created dire global conditions and has been highly contagious. Chronic inflammatory disease enhances cancer cell proliferation, progression, and invasion. We investigated how acute lung inflammation activates the tumor microenvironment and enhances lung metastasis in LPS induced in vitro and in vivo models. Respiratory illness is mainly caused by cytokine storm, which further influences oxidative and nitrosative stress. The LPS-induced inflammatory cytokines made the conditions suitable for the tumor microenvironment in the lungs. In the present study, we observed that LPS induced the cytokine storm and promoted lung inflammation via BRD4, which further caused the nuclear translocation of p65 NF-κB and STAT3. The transcriptional activation additionally triggers the tumor microenvironment and lung metastasis. Thus, BRD4-regulated p65 and STAT3 transcriptional activity in ARDS enhances lung tumor metastasis. Moreover, LPS-induced ARDS might promote the tumor microenvironment and increase cancer metastasis into the lungs. Collectively, BRD4 plays a vital role in inflammation-mediated tumor metastasis and is found to be a diagnostic and molecular target in inflammation-associated cancers.

P037 Enhancing the efficacy of cannabidiol using protein nanoparticles to treat inflammatory bowel disease

Abstract Background Inflammatory bowel disease (IBD) is a global health burden and approximately 30% of the patients with severe IBD require surgery following treatments, leaving an unmet need of new and safe therapies. Cannabidiol (CBD) is one of the major non-psychoactive and water-insoluble constituents of medicinal cannabis, known for its anti-inflammatory properties in epilepsy. The compound is safe at high doses and doesn’t have the psychotropic effects of delta-9-tetrahydrocannabinol. Therefore, this study investigated the therapeutic potential of CBD in controlling intestinal inflammation. Methods CBD was loaded in the protein nanoparticles (PNP-CBD) and tested for its water solubility. Primary intestinal epithelial cells (IECs; organoids) and bone marrow-derived macrophages (BMDM) were isolated from C57BL/6 mice and treated with IL-1β and LPS, respectively. Stimulated cells were treated with ±CBD and checked for downstream signalling through inflammatory gene expression using qRT PCR. Conditioned media from LPS-treated BMDM was also harvested to treat mIECs to check link between macrophage activation and intestinal inflammation. Accordingly, we assessed the efficacy of CBD in suppressing intestinal inflammation using dextran sulfate sodium (DSS)-induced acute experimental colitis in the C57BL/6J mice. At the end, blood samples were collected for hematology and serum analyses, mesenteric lymph nodes for immune cell signatures, colon samples for histological analysis and qRT PCR analysis for inflammatory genes. Results CBD loading in the protein nanoparticles (PNP-CBD) increased its water solubility up to 60 times. In the mIECs, CBD did not have any effects on cytokine-induced inflammation; however, it significantly dampened LPS-induced BMDM activation and inflammatory cytokine production. The results also showed that inhibition of CB2 but not CB1 receptor can completely abolish the anti-inflammatory potential of CBD in the BMDM. Transferring conditioned media from BMDM to mIECs showed macrophage-specific targeting alleviated inflammation in the mIECs. These have also been observed in the acute DSS colitis model where not CBD alone but PNP-CBD improved disease severity index including body weight loss, diarrhoea, shorter colon length, and inflammatory gene expression in the colon. PNP-CBD also reduced histological colitis and increased goblet cell mucin production in the DSS-treated colon. Conclusion Encapsulation of CBD increased water solubility and efficacy in alleviating intestinal inflammation in ulcerative colitis. These data also demonstrated that CB2 receptor targeting of macrophage activation is the primary mechanism of CBD’s effect.

CB2R agonist GW405833 alleviates acute liver failure in mice via inhibiting HIF-1α-mediated reprogramming of glycometabolism and macrophage proliferation.

The inflammatory responses involving infiltration and activation of liver macrophages play a vital role in acute liver failure (ALF). In the liver of ALF mice, cannabinoid receptor 2 (CB2R) is significantly upregulated on macrophages, while CB2R agonist GW405833 (GW) could protect against cell death in acute liver damage. In this study, we investigated the molecular mechanisms underlying the protective effects of GW against ALF in vivo and in vitro from a perspective of macrophage glycometabolism. Mice were pretreated with GW (10 mg/kg, i.p.), then were injected with D-GalN (750 mg/kg, i.p.) and LPS (10 mg/kg, i.p.) to induce ALF. We verified the protective effects of GW pretreatment in ALF mice. Furthermore, GW pretreatment significantly reduced liver macrophage infiltration and M1 polarization, and inhibited the release of inflammatory factors TNF-α and IL-1β in ALF mice. These protective effects were eliminated by CB2R antagonist SR144528 or in CB2R-/- ALF mice. We used LPS-stimulated RAW264.7 cells as an in vitro M1 macrophage-centered model of inflammatory response, and demonstrated that pretreatment with GW (10 μM) significantly reduced glucose metabolism by inhibiting glycolysis, which inhibited LPS-induced macrophage proliferation and inflammatory cytokines release. We verified these results in a stable CB2R-/- RAW264.7 cell line. Moreover, we found that GW significantly inhibited the expression of hypoxia inducible factor 1α (HIF-1α). Using a stable HIF-1α-/- RAW264.7 cell line, we confirmed that GW reduced the release of inflammatory cytokines from macrophages and inhibited glycolysis by downregulating HIF-1α expression. In conclusion, activation of CB2Rs inhibits the proliferation of hepatic macrophages and release of inflammatory factors in ALF mice through downregulating HIF-1α to inhibit glycolysis.

Ochrathinols A and B, two pairs of sulfur-containing racemates from an Antarctic fungus Aspergillus ochraceopetaliformis SCSIO 05702 inhibit LPS-induced pro-inflammatory cytokines and NO production

Ochrathinols A and B ((±)-1 and (±)-2), two undescribed sulfur-containing racemates, and ochracids A and B (3 and 4), two unprecedented pyrrolizidine alkaloids, were isolated from an Antarctic soil-derived fungus Aspergillus ochraceopetaliformis SCSIO 05702. Their structures including absolute configurations were determined through extensive spectroscopic analysis, chiral-phase HPLC analysis, quantum ECD calculations, and X-ray single-crystal diffraction. Ochrathinols A and B are unprecedented sulfur natural products featuring a novel 3-methylhexahydro-2H-cyclopenta [b]thiophene core. Interestingly, ochrathinol A ((±)-1) outstandingly suppressed the release of LPS-induced IL-1β, IL-6, and TNF-α inflammatory cytokines with concentration of 10 μM and alleviated the unbalanced NAD+/NADH ratio caused by LPS in RAW264.7 macrophages.

Protein Biocargo and Anti-Inflammatory Effect of Tomato Fruit-Derived Nanovesicles Separated by Density Gradient Ultracentrifugation and Loaded with Curcumin

Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1β cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.

Leishmania LPG interacts with LRR5/LRR6 of macrophage TLR4 for parasite invasion and impairs the macrophage functions.

Visceral leishmaniasis (VL) is a severe form of leishmaniasis, primarily affecting the poor in developing countries. Although several studies have highlighted the importance of toll-like receptors (TLRs) in the pathophysiology of leishmaniasis, the role of specific TLRs and their binding partners involved in Leishmania donovani uptake are still elusive. To investigate the mechanism of L. donovani entry inside the macrophages, we found that the parasite lipophosphoglycan (LPG) interacted with the macrophage TLR4, leading to parasite uptake without any significant alteration of macrophage cell viability. Increased parasite numbers within macrophages markedly inhibited lipopolysachharide-induced pro-inflammatory cytokines gene expression. Silencing of macrophage-TLR4, or inhibition of parasite-LPG, significantly stemmed parasite infection in macrophages. Interestingly, we observed a significant enhancement of macrophage migration, and generation of reactive oxygen species (ROS) in the parasite-infected TLR4-silenced macrophages, whereas parasite infection in TLR4-overexpressed macrophages exhibited a notable reduction of macrophage migration and ROS generation. Moreover, mutations in the leucine-rich repeats (LRRs), particularly LRR5 and LRR6, significantly prevented TLR4 interaction with LPG, thus inhibiting cellular parasite entry. All these results suggest that parasite LPG recognition by the LRR5 and LRR6 of macrophage-TLR4 facilitated parasite entry, and impaired macrophage functions. Therefore, targeting LRR5/LRR6 interactions with LPG could provide a novel option to prevent VL.

Magnolia kobus Extract Inhibits Periodontitis-Inducing Mediators in Porphyromonas gingivalis Lipopolysaccharide-Activated RAW 264.7 Cells.

Periodontitis, a disease caused by inflammation of oral bacteria, contributes to the loss of alveolar bone and destruction of connective tissues. Porphyromonas gingivalis, a Gram-negative bacterium, is known to possess important pathogenic factors for periodontal disease. In this study, we investigated the anti-periodontitis effects of Magnolia kobus extract (MKE) and magnolin as a component of Magnolia kobus (MK) in murine macrophage RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Effects of MKE and magnolin on the mechanism of RAW 264.7 cellular inflammation were determined by analyzing nitric oxide (NO) production and Western blot protein expression (n = 3). MKE/magnolin inhibited NO production without affecting cell survival. MKE/magnolin treatment inhibited LPS-induced pro-inflammatory cytokines, expression levels of matrix metalloproteinases (MMPs such as MMP-1, 3, 8, 9, and 13), and protein levels of inflammatory mediators (such as TNF-α, IL-1β, and mPGES-1). MKE/magnolin also suppressed NF-κB activation by inhibiting the TLR4 signaling pathway. These findings suggest that MKE has a therapeutic effect on inflammatory periodontal disease caused by oral bacterium P. gingivalis and that magnolin is a major functional component in the anti-inflammatory effect of MKE.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/ml) and Triton X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4-6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Role of heat shock protein 70 in regulation of anti-inflammatory response to curcumin in 3T3-L1 adipocytes.

Curcumin is a well-known phytochemical with anti-inflammatory effects. Heat shock protein (HSP) 70, an intracellular chaperone, inhibits proinflammatory signaling activation. Although curcumin has been shown to induce HSP70 expression in various cell types, whether HSP70 mediates the anti-inflammatory effects of curcumin in mature adipocytes remains unclear. To assess the role of HSP70 in regulating the anti-inflammatory response to curcumin in adipocytes, fully differentiated 3T3-L1 adipocytes were treated with curcumin, lipopolysaccharide (LPS), and/or the HSP70 inhibitor pifithrin-μ (PFT-μ). The expression levels of HSP70 and proinflammatory cytokines were then measured. Curcumin upregulated HSP70 expression at both protein and mRNA levels and attenuated LPS-induced Il6, Ptx3, and Ccl2 mRNA upregulation. PFT-μ tended to exacerbate the LPS-induced upregulation of Il6, Ptx3, Ccl2, and Tnfa mRNA expression. However, on curcumin pretreatment, the tendency of PFT-μ to upregulate LPS-induced proinflammatory cytokine expression decreased or disappeared. These results indicate that HSP70 is involved in the regulation of inflammatory responses but may not be crucial for the anti-inflammatory effects of curcumin in 3T3-L1 adipocytes.

Vanillin prevents the growth of endometriotic lesions through anti-inflammatory and antioxidant pathways in a mouse model.

Endometriosis is an estrogen-dependent chronic inflammatory gynecological disease defined by the presence of endometrial glands and mesenchyme outside the uterine cavity, named ectopic endometrium. Recent studies showed that endometriosis is associated with hormone imbalance, inflammation and oxidative stress. As the main component of vanilla bean extract, vanillin is widely used as a flavoring agent in the food, pharmaceutical, and cosmetic industries. It is known for its anti-inflammatory, antibacterial, and antitumor properties, but its therapeutic efficacy in endometriosis has not been studied. In this study, we evaluated the roles of vanillin in this disease using an induced endometriotic mouse model. The results showed that vanillin significantly inhibited the growth of endometrial lesions. Compared with the control group, the weight and volume of lesions were reduced considerably in the vanillin-treated group, showing its fantastic ability to inhibit cell proliferation and promote apoptosis. In addition, in the treatment group, mRNA expression of the pro-inflammatory cytokines Tnfa, Infg, Il1b, and Il6 was reduced, the number of macrophages and neutrophils was decreased, and the NF-κB signaling pathway was inhibited, indicating that vanillin suppressed the inflammatory response in the ectopic endometrium. Besides, we found that the intensity of tissue reactive oxygen species (ROS) was significantly lower, and mitochondrial complex IV expression was reduced in the vanillin-treated group. Meanwhile, treatment of the immortalized human endometriotic epithelial cell line (11Z) with vanillin resulted in the downregulation of cyclin genes that drive the cell proliferation process, inhibited cell proliferation, promoted apoptosis, and downregulated the expression of LPS-induced inflammatory cytokines. Most importantly, our data showed that the vanillin treatment had only minimal effects on the eutopic endometrium with respect to the pregnancy process, indicating its safety to be used in treating endometriosis in adults. In conclusion, our data suggest that vanillin has potential therapeutic properties for endometriosis as a regulatory molecule of cell proliferation, apoptosis, inflammation, and oxidative stress.

Precision-cut rat placental slices as a model to study sex-dependent inflammatory response to LPS and Poly I:C.

Maternal inflammation in pregnancy represents a major hallmark of several pregnancy complications and a significant risk factor for neurodevelopmental and neuropsychiatric disorders in the offspring. As the interface between the mother and the fetus, the placenta plays a crucial role in fetal development and programming. Moreover, studies have suggested that the placenta responds to an inflammatory environment in a sex-biased fashion. However, placenta-mediated immunoregulatory mechanisms are still poorly understood. Therefore, we have developed a model of ex vivo precision-cut placental slices from the rat term placenta to study acute inflammatory response. Rat placental slices with a precise thickness of 200 µm were generated separately from male and female placentas. Inflammation was stimulated by exposing the slices to various concentrations of LPS or Poly I:C for 4 and 18 hours. Treatment of placental slices with LPS significantly induced the expression and release of proinflammatory cytokines TNF-α, IL-6, and IL-1β. In contrast, Poly I:C treatment resulted in a less-pronounced inflammatory response. Interestingly, the female placenta showed higher sensitivity to LPS than male placenta. Anti-inflammatory agents, curcumin, 1α,25- dihydroxyvitamin D3, and progesterone attenuated the LPS-induced proinflammatory cytokine response at both mRNA and protein levels. We conclude that rat placental slices represent a novel alternative model to study the role of sexual dimorphism in the acute inflammatory response and immune activation in pregnancy.

RCsHscB Derived from Clonorchis sinensis: A Carcinogenic Liver Fluke Ameliorates LPS-Induced Acute Hepatic Injury by Repression of Inflammation.

Sepsis-associated acute liver injury caused by spillovers of bacteria and endotoxins (lipopolysaccharide, LPS) into the liver remains a public health issue due to the lack of specific therapeutic approaches. Previous studies showed that the recombinant protein HscB (rCsHscB) of Clonorchis sinensis, a carcinogenic liver fluke, had an anti-inflammatory effect and could alleviate inflammatory diseases such as enteritis; however, whether it can prevent sepsis-associated acute liver injury induced by LPS is still unknown. In our current study, the therapeutic effects and the potential mechanisms of rCsHscB on LPS-induced acute liver injury were investigated both in vivo and in vitro. The data showed that rCsHscB prevented LPS-induced liver damage, as demonstrated by histopathological observation and hepatic damage markers (the activities of serum ALT and AST) in a murine model of sepsis-associated acute liver injury. rCsHscB also significantly reversed the high levels of serum IL-6 and MCP-1 induced by LPS. In addition, rCsHscB attenuated the production of LPS-induced proinflammatory cytokines, including IL-6 and TNF-α, in a macrophage cell line-RAW264.7, through possible mediation by the MAPK signaling pathway in vitro. In conclusion, the present study demonstrates that rCsHscB derived from a fluke C. sinensis protects against sepsis-associated acute liver injury induced by LPS, which may be attributed to the inhibition of the MAPK signaling pathway. Our present study provides a potential therapeutic strategy for sepsis-associated acute liver injury.

490. Brain-derived neurotrophic factor (BDNF) is a prognostic biomarker and therapeutic target through AMP-activated protein kinase (AMPK) signal pathway for sepsis

Abstract Background Brain-derived neurotrophic factor (BDNF) is a pleiotropic cytokine that not only acts on the central nervous system, but is also implicated in metabolic regulation, development of lung diseases, and support of hematopoiesis. Recently, a study report that the plasma levels of BDNF were independently associated with mortality in critically ill patients. We aimed to evaluate the role of BDNF in sepsis patients and animal sepsis model. Methods Prospectively enrolled adult sepsis patients (n=168) who visited the emergency room (ER) of Korea University Ansan Hospital from January 2017 to December 2018., and healthy volunteers (n=48) were included in the present study. Serum BDNF level was determined and analyzed in septic patients and healthy volunteers. The effects of BDNF on pro-inflammatory cytokines, AMP-activated protein kinase (AMPK) and NF-κB signaling in septic condition of RAW264.7 cells and mouse peritoneal macrophage, and mouse sepsis model by cecal ligation and puncture (CLP). Results Serum BDNF levels (mean) were significantly decreased in sepsis patients compared to healthy controls (11.78ng/ml vs 18.91ng/ml, p< 0.001) and significantly decreased in non-survivors compared to survivors at discharge (3.16ng/ml vs 13.86ng/ml, p< 0.05). BDNF treatment suppressed LPS-induced pro-inflammatory cytokine (TNF-α, iL-6, and iL-1β) in RAW264.7 cells. Although LPS stimulation diminished AMPK phosphorylation, BDNF was able to decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on down stream signaling events, particularly LPS-induced IκBα degradation in RAW cells and peritoneal macrophage. In CLP model, intra-muscular injection of BDNF after CLP significantly decreased serum levels of TNF-α which was increased compared to sham, and significantly improved survival of mouse (p< 0.01). Conclusion Sepsis results in a decreased production of BDNF, which might be a potential prognostic biomarker and therapeutic target through AMPK signal pathway for sepsis. Disclosures All Authors: No reported disclosures.

Selective CB2 Receptor Agonist, HU-308, Reduces Systemic Inflammation in Endotoxin Model of Pneumonia-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) and sepsis are risk factors contributing to mortality in patients with pneumonia. In ARDS, also termed acute lung injury (ALI), pulmonary immune responses lead to excessive pro-inflammatory cytokine release and aberrant alveolar neutrophil infiltration. Systemic spread of cytokines is associated with systemic complications including sepsis, multi-organ failure, and death. Thus, dampening pro-inflammatory cytokine release is a viable strategy to improve outcome. Activation of cannabinoid type II receptor (CB2) has been shown to reduce cytokine release in various in vivo and in vitro studies. Herein, we investigated the effect of HU-308, a specific CB2 agonist, on systemic and pulmonary inflammation in a model of pneumonia-induced ALI. C57Bl/6 mice received intranasal endotoxin or saline, followed by intravenous HU-308, dexamethasone, or vehicle. ALI was scored by histology and plasma levels of select inflammatory mediators were assessed by Luminex assay. Intravital microscopy (IVM) was performed to assess leukocyte adhesion and capillary perfusion in intestinal and pulmonary microcirculation. HU-308 and dexamethasone attenuated LPS-induced cytokine release and intestinal microcirculatory impairment. HU-308 modestly reduced ALI score, while dexamethasone abolished it. These results suggest administration of HU-308 can reduce systemic inflammation without suppressing pulmonary immune response in pneumonia-induced ALI and systemic inflammation.

Neuroprotective and Anti-Neuroinflammatory Properties of Vignae Radiatae Semen in Neuronal HT22 and Microglial BV2 Cell Lines.

The important factors in the pathogenesis of neurodegenerative disorders include oxidative stress and neuron-glia system inflammation. Vignae Radiatae Semen (VRS) exhibits antihypertensive, anticancer, anti-melanogenesis, hepatoprotective, and immunomodulatory properties. However, the neuroprotective effects and anti-neuroinflammatory activities of VRS ethanol extract (VRSE) remained unknown. Thus, this study aimed to investigate the neuroprotective and anti-inflammatory activities of VRSE against hydrogen peroxide (H2O2)-induced neuronal cell death in mouse hippocampal HT22 cells and lipopolysaccharide (LPS)-stimulated BV2 microglial activation, respectively. This study revealed that VRSE pretreatment had significantly prevented H2O2-induced neuronal cell death and attenuated reactive oxygen species generations in HT22 cells. Additionally, VRSE attenuated the apoptosis protein expression while increasing the anti-apoptotic protein expression. Further, VRSE showed significant inhibitory effects on LPS-induced pro-inflammatory cytokines in BV2 microglia. Moreover, VRSE pretreatment significantly activated the tropomyosin-related kinase receptor B/cAMP response element-binding protein, brain-derived neurotrophic factor and nuclear factor erythroid 2-related factor 2, and heme oxygenase-1 signaling pathways in HT22 cells exposed to H2O2 and inhibited the activation of the mitogen-activated protein kinase and nuclear factor-κB mechanism in BV2 cells stimulated with LPS. Therefore, VRSE exerts therapeutic potential against neurodegenerative diseases related to oxidative stress and pathological inflammatory responses.