Low-dose Lipopolysaccharide Research Articles

Lipopolysaccharide aggravates canine influenza a (H3N2) virus infection and lung damage via mTOR/autophagy in vivo and in vitro.

Influenza A (H3N2) accounts for the majority of influenza worldwide and continues to challenge human health. Disturbance in the gut microbiota caused by many diseases leads to increased production of lipopolysaccharide (LPS), and LPS induces sepsis and conditions associated with local or systemic inflammation. However, to date, little attention has been paid to the potential impact of LPS on influenza A (H3N2) infection and the potential mechanism. Hence, in this study we used canine influenza A (H3N2) virus (CIV) as a model of influenza A virus to investigate the effect of low-dose of LPS on CIV replication and lung damage and explore the underlying mechanism in mice and A549 and HPAEpiC cells. The results showed that LPS (25μg/kg) increased CIV infection and lung damage in mice, as indicated by pulmonary virus titer, viral NP levels, lung index, and pulmonary histopathology. LPS (1μg/ml) also increased CIV replication in A549cells as indicated by the above same parameters. Furthermore, low doses of LPS reduced CIV-induced p-mTOR protein expression and enhanced CIV-induced autophagy-related mRNA/protein expressions in vivo and in vitro. In addition, the use of the mTOR activator, MHY1485, reversed CIV-induced autophagy and CIV replication in A549 and HPAEpiC cells, respectively. siATG5 alleviated CIV replication exacerbated by LPS in the two lines. In conclusion, LPS aggravates CIV infection and lung damage via mTOR/autophagy.

Chronic exposure to low-level lipopolysaccharide dampens influenza-mediated inflammatory response via A20 and PPAR network.

Influenza A virus (IAV) infection leads to severe inflammation, and while epithelial-driven inflammatory responses occur via activation of NF-κB, the factors that modulate inflammation, particularly the negative regulators are less well-defined. In this study we show that A20 is a crucial molecular switch that dampens IAV-induced inflammatory responses. Chronic exposure to low-dose LPS environment can restrict this excessive inflammation. The mechanisms that this environment provides to suppress inflammation remain elusive. Here, our evidences show that chronic exposure to low-dose LPS suppressed IAV infection or LPS stimulation-induced inflammation in vitro and in vivo. Chronic low-dose LPS environment increases A20 expression, which in turn positively regulates PPAR-α and -γ, thus dampens the NF-κB signaling pathway and NLRP3 inflammasome activation. Knockout of A20 abolished the inhibitory effect on inflammation. Thus, A20 and its induced PPAR-α and -γ play a key role in suppressing excessive inflammatory responses in the chronic low-dose LPS environment.

Liposaccharide-induced sustained mild inflammation fragments social behavior and alters basolateral amygdala activity.

Conditions with sustained low-grade inflammation have high comorbidity with depression and anxiety and are associated with social withdrawal. The basolateral amygdala (BLA) is critical for affective and social behaviors and is sensitive to inflammatory challenges. Large systemic doses of lipopolysaccharide (LPS) initiate peripheral inflammation, increase BLA neuronal activity, and disrupt social and affective measures in rodents. However, LPS doses commonly used in behavioral studies are high enough to evoke sickness syndrome, which can confound interpretation of amygdala-associated behaviors. The objectives of this study were to find a LPS dose that triggers mild peripheral inflammation but not observable sickness syndrome in adult male rats, to test the effects of sustained mild inflammation on BLA and social behaviors. To accomplish this, we administered single doses of LPS (0-100μg/kg, intraperitoneally) and measured open field behavior, or repeated LPS (5μg/kg, 3 consecutive days), and measured BLA neuronal firing, social interaction, and elevated plus maze behavior. Repeated low-dose LPS decreased BLA neuron firing rate but increased the total number of active BLA neurons. Repeated low-dose LPS also caused early disengagement during social bouts and less anogenital investigation and an overall pattern of heightened social caution associated with reduced gain of social familiarity over the course of a social session. These results provide evidence for parallel shifts in social interaction and amygdala activity caused by prolonged mild inflammation. This effect of inflammation may contribute to social symptoms associated with comorbid depression and chronic inflammatory conditions.

The enhancement effect of low-dose dietary lipopolysaccharide on the growth and immunity of Litopenaeus vannamei, and transcriptome analysis

In this study, the effects of dietary lipopolysaccharide (LPS) on Litopenaeus vannamei were investigated to determine whether LPS could play a role as a potential immunostimulant in shrimp. L. vannamei with an initial body weight of 0.30 ± 0.02 g were fed a diet containing LPS at doses of 0, 0.2, 1, 5, 25 or 125 mg kg−1 for eight weeks (groups LPS0, LPS0.2, LPS1, LPS5, LPS25 and LPS125, respectively). After eight weeks of feeding, the growth performance, immunity and transcriptome response of L. vannamei were analysed. Only dietary LPS at 0.2 and 1 mg kg−1 resulted in a significant increase in the growth of L. vannamei (P < 0.05). According to the weight gain rate (WGR) and specific growth rate (SGR), the optimum dietary LPS level was 2.462 and 2.455 mg kg−1, respectively. When compared with the control group, the survival rate (SR) of L. vannamei in the LPS0.2 group was significantly increased after white spot syndrome virus (WSSV) infection and the SR of L. vannamei in the LPS1 group was significantly increased after Vibrio parahaemolyticus infection (both P < 0.05). Compared with the LPS0 group, immune enzyme activity in the serum of L. vannamei could be significantly increased and the content of maleic dialdehyde (MDA) significantly decreased by dietary LPS. Transcriptome analysis of the haemocytes of L. vannamei identified 399 up-regulated differentially expressed genes (DEGs) and 5000 down-regulated DEGs in the LPS0.2 compared to the control group. Most of the DEGs were significantly enriched in the following pathways: phosphatidylinositol signalling, Wnt signalling, Jak-STAT signalling and inositol phosphate metabolism. In conclusion, this study revealed that diets supplemented with low-dose LPS had positive effects on the growth and immunity of L. vannamei.

Neuroprotection by Preconditioning in Mice is Dependent on MyD88-Mediated CXCL10 Expression in Endothelial Cells.

The central nervous system (CNS) can be preconditioned to resist damage by peripheral pretreatment with low-dose gram-negative bacterial endotoxin lipopolysaccharide (LPS). Underlying mechanisms associated with transient protection of the cerebral cortex against traumatic brain injury include increased neuronal production of antiapoptotic and neurotrophic molecules, microglial-mediated displacement of inhibitory presynaptic terminals innervating the soma of cortical projection neurons, and synchronized firing of cortical projection neurons. However, the cell types and signaling responsible for these neuronal and microglial changes are unknown. A fundamental question is whether LPS penetrates the CNS or acts on the luminal surface of brain endothelial cells, thereby triggering an indirect parenchymal neuroprotective response. The present study shows that a low-dose intraperitoneal LPS treatment increases brain endothelial cell activation markers CD54, but does not open the blood-brain barrier or alter brain endothelial cell tight junctions as assessed by electron microscopy. NanoString nCounter transcript analyses of CD31-positive brain endothelial cells further revealed significant upregulation of Cxcl10, C3, Ccl2, Il1β, Cxcl2, and Cxcl1, consistent with identification of myeloid differentiation primary response 88 (MyD88) as a regulator of these transcripts by pathway analysis. Conditional genetic endothelial cell gene ablation approaches demonstrated that both MyD88-dependent Toll-like receptor 4 (TLR4) signaling and Cxcl10 expression are essential for LPS-induced neuroprotection and microglial activation. These results suggest that C-X-C motif chemokine ligand 10 (CXCL10) production by endothelial cells in response to circulating TLR ligands may directly or indirectly signal to CXCR3 on neurons and/or microglia. Targeted activation of brain endothelial receptors may thus provide an attractive approach for inducing transient neuroprotection.

Effects of Continuous LPS Induction on Oxidative Stress and Liver Injury in Weaned Piglets

Due to imperfections in their immune and digestive systems, weaned piglets are susceptible to invasions of the external environment and diseases, especially bacterial infections, which lead to slow growth, tissue damage, and even the death of piglets. Here, a model of weaned piglets induced by Escherichia coli lipopolysaccharide (LPS) was established to explore the effects of continuous low-dose LPS induction on the mechanism of liver injury. A total of forty-eight healthy 28-day-old weaned piglets (weight = 6.65 ± 1.19 kg) were randomly divided into two groups: the CON group and LPS group. During the experimental period of thirteen days, the LPS group was injected intraperitoneally with LPS (100 μg/kg) once per day, and the CON group was treated with the same volume of 0.9% NaCl solution. On the 1st, 5th, 9th, and 13th days, the serum and liver of the piglets were collected for the determination of serum biochemical indexes, an antioxidant capacity evaluation, and histopathological examinations. In addition, the mRNA expression levels of the TLR4 pathway and inflammatory cytokines were detected. The results showed that the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the serum increased after LPS induction. The activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) in the serum and liver homogenate of the LPS group were lower than those of the CON group, while the malondialdehyde (MDA) content in the serum and the activities of catalase (CAT) and superoxide dismutase (SOD) in the liver of the LPS group were higher than those in the CON group. At the same time, morphological impairment of the livers occurred, including hepatocyte caryolysis, hepatocyte vacuolization, karyopycnosis, and inflammatory cell infiltration, and the mRNA expression levels of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-10 were upregulated in the livers after LPS induction. The above results were more obvious on the 1st and 5th days of LPS induction, while the trend during the later period was not significant. It was concluded that the oxidative stress and liver injury occurred at the early stage of LPS induction, while the liver damage weakened at the later stage. The weaned piglets probably gradually developed tolerance to the endotoxin after the continuous low-dose induction of LPS.

Unique Features of the Immune Response in BTBR Mice.

Inflammation plays a considerable role in the pathogenesis of many diseases, including neurodegenerative and psychiatric ones. Elucidation of the specific features of an immune response in various model organisms, and studying the relation of these features with the behavioral phenotype, can improve the understanding of the molecular mechanisms of many psychopathologies. In this work, we focused on BTBR mice, which have a pronounced autism-like behavioral phenotype, elevated levels of oxidative-stress markers, an abnormal immune response, several structural aberrations in the brain, and other unique traits. Although some studies have already shown an abnormal immune response in BTBR mice, the existing literature data are still fragmentary. Here, we used inflammation induced by low-dose lipopolysaccharide, polyinosinic:polycytidylic acid, or their combinations, in mice of strains BTBR T+Itpr3tf/J (BTBR) and C57BL6/J. Peripheral inflammation was assessed by means of a complete blood count, lymphocyte immunophenotyping, and expression levels of cytokines in the spleen. Neuroinflammation was evaluated in the hypothalamus and prefrontal cortex by analysis of mRNA levels of proinflammatory cytokines (tumor necrosis factor, Tnf), (interleukin-1 beta, Il-1β), and (interleukin-6, Il-6) and of markers of microglia activation (allograft inflammatory factor 1, Aif1) and astroglia activation (glial fibrillary acidic protein, Gfap). We found that in both strains of mice, the most severe inflammatory response was caused by the administration of polyinosinic:polycytidylic acid, whereas the combined administration of the two toll-like receptor (TLR) agonists did not enhance this response. Nonetheless, BTBR mice showed a more pronounced response to low-dose lipopolysaccharide, an altered lymphocytosis ratio due to an increase in the number of CD4+ lymphocytes, and high expression of markers of activated microglia (Aif1) and astroglia (Gfap) in various brain regions as compared to C57BL6/J mice. Thus, in addition to research into mechanisms of autism-like behavior, BTBR mice can be used as a model of TLR3/TLR4-induced neuroinflammation and a unique model for finding and evaluating the effectiveness of various TLR antagonists aimed at reducing neuroinflammation.

Effect of multifactorial therapeutic approach on axonal regeneration and cell viability in an in-vitro model of spinal-derived neural injury.

The highly debilitated nature of spinal cord injuries (SCI) creates an inhibitory repair environment that limits the recovery rate and therefore single interventional treatment has been resulted in incomplete recovery. A multifactorial approach that combines several therapeutic approaches may address diverse aspects of SCI pathology and enhance the recovery rate over single therapy. Accordingly, in this study, we aimed to investigate the effect of combined olfactory ensheathing cells (OECs) (to transport trophic factor, mediate immunomodulation, provide a suitable environment for cell survival), G-CSF (to establish a favorable environment for cell survival) and lipopolysaccharide (LPS) (to boost the protective activity of OEC) therapy on the cell viability after a scratch injury caused by a cataract knife on cells in an in-vitro model of spinal-derived neural injury. In this study, we used mixed neuronal-glial cultures, which are widely used for an in vitro study of neuronal damage. Scratch insult was made on cells using a cataract knife. The cells were divided into 8 groups (two control groups with and without olfactory ensheathing cells (OECs) treatment, injury group, three injury groups with single therapy by using super low dose of LPS (SLD-LPS) (100pg/ml), OEC group, and G-CSF (100ng/ml) group, and two injury groups with combined therapy (OEC with SLD-LPS and with all three treatments)). We found a significant decrease in the survival rate of injured cells (p < 0.001) 24h after scratching insult. Our results indicated morphological alterations in cells in the acute phase (1, 2 and 6h) after injury, with significant increased gap size at 6h after induction of injury. Our combined therapy, significantly prevented cell death and decreased the size of the gap over time. We found that combined therapy promoted cell survival following spinal injury by providing a neuroprotective environment for cells. Therefore, our findings provide new insight into the combined therapy, which can be considered for promising preclinical therapeutic strategy for SCI toward clinical trials.

The NLRC4 Inflammasome Drives Myelodysplastic Syndrome By Linking Epigenetic Reprogramming and Innate Immune Signaling

Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignancies characterized by dysplastic and ineffective hematopoiesis. Despite emerging evidence that chronic inflammation contributes to the development of MDS, the mechanisms underlying the dysregulation of innate immune and inflammatory signaling pathways in hematopoietic stem and progenitor cells (HSPCs) remain poorly characterized. We have used multiple MDS mouse models to identify a tumor suppressive role for the EP300 epigenetic histone acetyltransferase enzyme in the progression of MDS to AML. Based on the gene expression changes we observed, we have used one specific mouse model, that lacks Tet2 expression, to define the role that chronic inflammation plays in promoting MDS development and the mechanisms involved. Methods We have conducted a series of in vitro, in vivo, and in silico experiments using Tet2-deficient mice, and chronic, low-dose LPS treatment to model chronic inflammation. Cytokine array immunoassays were performed to evaluate the inflammatory response of WT or Tet2-deficient mice to LPS treatment. Chimeric mice transplanted with bone marrow (BM) cells isolated from LPS or vehicle (PBS) treated WT or Tet2-deficient mice were monitored for the development of MDS and survival. Serial competitive bone marrow transplantation assays and flow cytometry profiling were used to determine the competitive advantage of LPS or PBS-treated BM cells, and their differentiation. Bioinformatic analyses of RNA sequencing (RNA-seq) results from our mouse model were performed to characterize the transcriptional profiling and identify novel innate immune pathways. Colony-forming unit (CFU) and qPCR assays were used to evaluate HSPC proliferative capacity, self-renewal, and gene expression changes. Results We find that mice lacking Tet2 develop a hyper-inflammatory status, marked by the expression of a number of pro-inflammatory cytokines, such as Il-1b and Il-6, which are further fueled by LPS-induced chronic inflammation. Chimeric mice transplanted with BM cells isolated from LPS-treated Tet2-deficient mice exhibit a significantly shorter life span and early onset of MDS phenotypes, including dysplastic hematopoiesis and thrombocytopenia, compared to age-matched chimeric mice transplanted with BM cells isolated from PBS-treated Tet2-deficient mice or LPS-treated WT mice. Chronic inflammation promotes the self-renewal and myeloid differentiation of Tet2-deficient HSPCs in the short term; however, it accelerates HSC exhaustion in the long run. Transcription profiling and subsequent bioinformatic analyses identify functional clusters in LPS-treated Tet2-deficient HSPCs, including genes involved in the regulation of epigenetics, immunity, metabolism, and cell cycle. Detailed inspection of the most highly differentially expressed genes reveals that many genes in the Nlrc4 inflammasome pathway are upregulated in LPS-treated Tet2-deficient HSPCs. We validate changes in the expression of these genes by qPCR, and then show that deleting Nlrc4 suppresses the colony formation of Tet2-deficient HSPCs in vitro. Ongoing work focuses on evaluating the effects of deleting Nlrc4 or blocking Nlrc4 function using a small molecule inhibitor on MDS development in vivo. Conclusions The combinatory role of intrinsic and extrinsic factors drives the development of MDS in this Tet2-deficient MDS mouse model. It is likely that different genetic mutations generate unique sensitivity to certain signaling pathways, and we demonstrate the epigenetic rewiring and activation of the Nlrc4 inflammasome pathway as a mechanistic basis for the inflammatory component of MDS [or clonal hematopoiesis of indeterminate potential (CHIP)] pathogenesis in Tet2-deficient cells. Our work has the potential to provide novel targeting opportunities in MDS management and confirm the importance of different components of inflammasome signaling in MDS.

LPS-Induced Inhibition of miR-143 Expression in Brown Adipocytes Promotes Thermogenesis and Fever.

Fever is an important part of inflammatory response to infection. Although brown adipose tissue (BAT) thermogenesis is known to be potently influenced by systemic inflammation, the role of BAT during infection-induced fever remains largely unknown. Here, we injected mice with a low dose of LPS and found that low-dose LPS can directly induce thermogenesis of brown adipocytes. It is known that miR-143 is highly expressed in the BAT, and miR-143 knockout mice exhibited stronger thermogenesis under cold exposure. Interestingly, miR-143 was negatively correlated with an LPS-induced increase of TNFα and IL-6 mRNA levels, and the IL-6 pathway may mediate the inhibition of miR-143 expression. Moreover, miR-143 is down-regulated by LPS, and overexpression of miR-143 in brown adipocytes by lentivirus could rescue the enhancement of UCP1 protein expression caused by LPS, hinting miR-143 may be an important regulator of the thermogenesis in brown adipocytes. More importantly, the knockout of miR-143 further enhanced the LPS-induced increase of body temperature and BAT thermogenesis, and this result was further confirmed by in vitro experiments by using primary brown adipocytes. Mechanistically, adenylate cyclase 9 (AC9) is a new target gene of miR-143 and LPS increases BAT thermogenesis by a way of inhibiting miR-143 expression, a negative regulator for AC9. Our study considerably improves our collective understanding of the important function of miR-143 in inflammatory BAT thermogenesis.

Hydrogen-Rich Water Mitigates LPS-Induced Chronic Intestinal Inflammatory Response in Rats via Nrf-2 and NF-κB Signaling Pathways.

Long-term exposure to low-dose lipopolysaccharide can impair intestinal barriers, causing intestinal inflammation and leading to systemic inflammation. Hydrogen-rich water possesses antioxidant and anti-inflammatory functions and exerts inhibitory effects on various inflammatory diseases. In this study, we investigated whether oral hydrogen-rich water could prevent lipopolysaccharide-induced chronic intestinal inflammation. An experimental model was established by feeding hydrogen-rich water, followed by the injection of lipopolysaccharide (200 μg/kg) in the tail vein of rats after seven months. ELISA, Western blot, immunohistochemistry, and other methods were used to detect related cytokines, proteins related to the NF-κB and Nrf-2 signaling pathways, and tight-junction proteins to study the anti-inflammatory and antioxidant effects of hydrogen-rich water. The obtained results show that hydrogen-rich water significantly increased the levels of superoxide dismutase and structural proteins; activated the Nrf-2 signaling pathway; downregulated the expression of inflammatory factors cyclooxygenase-2, myeloperoxidase, and ROS; and decreased the activation of the NF-κB signaling pathway. These results suggest that hydrogen-rich water could protect against chronic intestinal inflammation in rats caused by lipopolysaccharide-induced activation of the NF-κB signaling pathway by regulating the Nrf-2 signaling pathway.

Influences of calorie restriction and lipopolysaccharide therapy on inflammation, cytokine response, and cell proliferation in pancreatic adenocarcinoma mouse model.

The study aimed to investigate the effects of lipopolysaccharide (LPS) alone and in combination with calorie restriction (CR) on the pancreatic tissues in C57BL/6 mice modeled with pancreatic ductal adenocarcinoma (PDAC). Forty male C57BL/6 mice (10-13 weeks old) were divided into five groups; LPS,LPS + CR, PDAC, PDAC + LPS, and PDAC + LPS + CR. Nuclear factor kappa B (NF-κβ), interleukin-6 (IL-6), and c-Jun N-terminal kinases (JNK) mRNA expression levels were measured in pancreatic tissues. NF-κβ, IL-6, JNK, and proliferating cell nuclear antigen(PCNA) peptide levels were determined by immunohistochemistry. Oxidative stress markers and antioxidant enzyme activities were determined spectrophotometrically. TH1/TH2 cytokine measurements were determined by a flow cytometer. It was detected that the number of PCNA immune + cells in the PDAC + LPS + CR group was significantly lower than in the PDAC and PDAC + LPS groups (p < 0.01, p < 0.05 respectively). PDAC + LPS + CR group's plasma interferon-gamma(IFN-γ), IL-6, IL-2, tumor necrosis factor-alpha, IL-3, and IL-4 levels were found to be significantly lower than the PDAC group (p < 0.01, p < 0.001, p < 0.01, p < 0.05, p < 0.01, and p < 0.05 respectively). According to our findings, the combination of low-dose LPS and 40% CR was found to be more effective in PDAC model mice.

Glial functions in the blood-brain communication at the circumventricular organs.

The circumventricular organs (CVOs) are located around the brain ventricles, lack a blood-brain barrier (BBB) and sense blood-derived molecules. This review discusses recent advances in the importance of CVO functions, especially glial cells transferring periphery inflammation signals to the brain. The CVOs show size-limited vascular permeability, allowing the passage of molecules with molecular weight <10,000. This indicates that the lack of an endothelial cell barrier does not mean the free movement of blood-derived molecules into the CVO parenchyma. Astrocytes and tanycytes constitute a dense barrier at the distal CVO subdivision, preventing the free diffusion of blood-derived molecules into neighboring brain regions. Tanycytes in the CVOs mediate communication between cerebrospinal fluid and brain parenchyma via transcytosis. Microglia and macrophages of the CVOs are essential for transmitting peripheral information to other brain regions via toll-like receptor 2 (TLR2). Inhibition of TLR2 signaling or depletion of microglia and macrophages in the brain eliminates TLR2-dependent inflammatory responses. In contrast to TLR2, astrocytes and tanycytes in the CVOs of the brain are crucial for initiating lipopolysaccharide (LPS)-induced inflammatory responses via TLR4. Depletion of microglia and macrophages augments LPS-induced fever and chronic sickness responses. Microglia and macrophages in the CVOs are continuously activated, even under normal physiological conditions, as they exhibit activated morphology and express the M1/M2 marker proteins. Moreover, the microglial proliferation occurs in various regions, such as the hypothalamus, medulla oblongata, and telencephalon, with a marked increase in the CVOs, due to low-dose LPS administration, and after high-dose LPS administration, proliferation is seen in most brain regions, except for the cerebral cortex and hippocampus. A transient increase in the microglial population is beneficial during LPS-induced inflammation for attenuating sickness response. Transient receptor potential receptor vanilloid 1 expressed in astrocytes and tanycytes of the CVOs is responsible for thermoregulation upon exposure to a warm environment less than 37°C. Alternatively, Nax expressed in astrocytes and tanycytes of the CVOs is crucial for maintaining body fluid homeostasis. Thus, recent findings indicate that glial cells in the brain CVOs are essential for initiating neuroinflammatory responses and maintaining body fluid and thermal homeostasis.

Inflammation aggravated the hepatotoxicity of triptolide by oxidative stress, lipid metabolism disorder, autophagy, and apoptosis in zebrafish.

Triptolide is a major compound isolated from the Tripterygium wilfordii Hook that is mainly used for the treatment of autoimmune disorders and inflammatory diseases. Though triptolide-induced hepatotoxicity has been widely reported, the hepatic effects when the patients are in an inflammatory state are not clear. In this study, we used low-dose Lipopolysaccharides (LPS) to disrupt the inflammation homeostasis in the liver of zebrafish and explored the hepatotoxicity of triptolide under an inflammatory state. Compared with the Triptolide group, LPS-Triptolide cotreatment exacerbate the liver injury with a remarkable decrease of liver size and liver-specific fluorescence intensity, accompanied by significant elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Liver cell damages were further demonstrated by histological staining and scanning electron microscopy observation. Lipid metabolism was severely impaired as indicated by delayed yolk sac absorption, accumulated triglycerides in the liver, and dysregulation of the related genes, such as ppar-α, cpt-1, mgst, srebf1/2, and fasn. Oxidative stress could be involved in the molecular mechanism as the Nrf2/keap1 antioxidant pathways were down-regulated when the zebrafish in an inflammatory state. Moreover, the expression of autophagy-related genes such as beclin, atg5, map1lc3b, and atg3 was also dysregulated. Finally, apoptosis was significantly induced in responses to LPS-Triptolide co-treatment. We speculate that triptolide could exacerbate the immune response and impair lipid metabolism, resulting in enhanced sensitivity of the zebrafish liver to triptolide-induced toxic effects through disruption of the antioxidant system and induction of apoptosis.

Akebia saponin D protects hippocampal neurogenesis from microglia-mediated inflammation and ameliorates depressive-like behaviors and cognitive impairment in mice through the PI3K-Akt pathway.

Given the ability of akebia saponin D (ASD) to protect various types of stem cells, in the present study, we hypothesized that ASD could promote the proliferation, differentiation, and survival of neural stem/precursor cells (NSPCs), even in a microglia-mediated inflammatory environment, thereby mitigating inflammation-related neuropsychopathology. We established a mouse model of chronic neuroinflammation by exposing animals to low-dose lipopolysaccharide (LPS, 0.25 mg/kg/d) for 14 days. The results showed that chronic exposure to LPS strikingly reduced hippocampal levels of PI3K and pAkt and neurogenesis in mice. In the presen of a microglia-mediated inflammatory niche, the PI3K-Akt signaling in cultured NSPCs was inhibited, promoting their apoptosis and differentiation into astrocytes, while decreasing neurogenesis. Conversely, ASD strongly increased the levels of PI3K and pAkt and stimulated NSPC proliferation, survival and neuronal differentiation in the microglia-mediated inflammatory niche in vitro and in vivo. ASD also restored the synaptic function of hippocampal neurons and ameliorated depressive- and anxiety-like behaviors and cognitive impairment in mice chronically exposed to LPS. The results from network pharmacology analysis showed that the PI3K-AKT pathway is one of the targets of ASD to against major depressive disorder (MDD), anxiety and Alzheimer’s disease (AD). And the results from molecular docking based on computer modeling showed that ASD is bound to the interaction interface of the PI3K and AKT. The PI3K-Akt inhibitor LY294002 blocked the therapeutic effects of ASD in vitro and in vivo. These results suggested that ASD protects NSPCs from the microglia-mediated inflammatory niche, promoting their proliferation, survival and neuronal differentiation, as well as ameliorating depressive- and anxiety-like behaviors and cognitive impairment by activating the PI3K-AKT pathway. Our work suggests the potential of ASD for treating Alzheimer’s disease, depression and other cognitive disorders involving impaired neurogenesis by microglia-mediated inflammation.

A pulmonary endothelial amplification loop aggravates ex-vivo transfusion-related acute lung injury via increased toll-like receptor 4 and intra-cellular adhesion molecule-1 expression.

Transfusion-Related Acute Lung Injury (TRALI) is a life-threatening complication of blood transfusions characterized by pulmonary endothelial cell damage and edema, with a high incidence in critically ill patients. The pathophysiology of TRALI is unresolved, but can generally be hypothesized to follow a 2-hit model in which the first hit is elicited by the underlying clinical condition of the patient (e.g., inflammation, which can be reflected by LPS in experimental models), and the second hit is delivered by the blood transfusion product (e.g., HLA class I antibodies). Here, we report a synergistic role for LPS and HLA class I antibody binding to pulmonary endothelium resulting in enhanced inflammatory responses. Pulmonary endothelial cells were treated with PBS or low-dose LPS, exclusively or in combination with anti-HLA class I. Endothelial surface expression of HLA class I, TLR4, and inflammatory marker ICAM-1 were measured, and trans-endothelial migration (TEM) of neutrophils was investigated. LPS treatment of pulmonary endothelium enhanced HLA class I antibody binding, and combined LPS and HLA class I antibody binding enhanced TLR4 (LPS receptor) and ICAM-1 expression on the endothelial cell surface. Low-dose LPS and HLA antibody together also increased neutrophil TEM under physiological flow by on average 5-fold. We conclude that LPS and anti-HLA class I antibody have the ability to activate the pulmonary endothelium into a spiral of increasing inflammation, opening the opportunity to potentially block TLR4 to prevent or reduce the severity of TRALI in vivo.

Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a–/–) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146–/– podocytes from these animals also showed a basally upregulated TGFβ-Smad3 signaling in vitro. Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFβ, and linked downstream signaling.

Empirical Study of Anti-Inflammatory Effects of Kecombrang (Etlingera elatior) in Mus musculus Sepsis Model

Mice used as experimental animals in the laboratory belong to the genus Mus, subfamily Murinae, family Muridae, superfamily Muroidea, order Rodentia, and class M. musculus , these mice are also referred to as home mice. Mice have been used as standard animals in toxicology, teratology, and carcinogenesis tests, even today, mice have also been used for behavioral, neurologic, nutritional, genetic, immunological, infectious, metabolic, and degenerative disease studies. Animal models of sepsis with intraperitoneal or intravenous injection of lipopolysaccharide (LPS) have been widely used for sepsis research. LPS induces systemic inflammation that mimics the early phase of sepsis. LPS injection causes kidney injury, including a decrease in glomerular filtration rate, an increase in blood urea nitrogen, and an increase in neutrophil infiltration in the kidney. The injectable dose of LPS can be titrated to mimic early sepsis without hemodynamic compromise, which has been useful for studying the systemic and renal responses. The response during the early phase of sepsis is that doses of LPS are usually used to induce systemic hypotension and decrease glomerular perfusion, whereas low doses of LPS do not cause systemic hypotension but still decrease glomerular perfusion. There are several advantages of LPS compared to others, namely, the method used is simple and the model is very controlled and standardized. The dose of endotoxin that causes 50% mortality in mice is 1–25 mg/kg. In this study, mice were given intraperitoneal injection of LPS at a dose of 0.3 mg/kg BW. LPS injection was given to the positive control group and treatment group 1, treatment group 2, and treatment group 3 at the start of the study.

Suppression of Inflammatory Cytokine Genes Expression in Vascular Endothelial Cells by Super-low Dose Lipopolysaccharide-activated Macrophages.

Vascular endothelial cells play an important role in regulating immune responses and in keeping the balance between blood coagulation and fibrinolysis. Inflammatory cytokines produced by activated macrophages and vascular endothelial cells excessively activate vascular endothelial cells, leading to an imbalance in the expression of blood coagulation- and fibrinolysis-related factors. The dysfunction of vascular endothelial cells can lead to development of various diseases. In a previous study the increased expression of inflammatory cytokines in adipocytes was shown to be suppressed by the culture medium of macrophages activated by low-dose lipopolysaccharide (LPS). Suppressing inflammatory cytokine gene expression of low-dose LPS-activated macrophages may allow for the regulation of the dysfunction in vascular endothelial cells. Human monocytes THP-1 cells were differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and were activated with LPS. The culture medium of the LPS-activated THP-1 was added to human aortic endothelial cells (HAoEC). After five days, the expression of inflammatory cytokine genes interleukin (IL)1B, IL6, IL8, and tumor necrosis factor (TNF)A, blood coagulation-related genes SERPINE1, tissue factor (TF), and thrombopoietin (TM), and fibrinolysis-related gene tissue-type plasminogen activator (t-PA) was analyzed using quantitative real-time PCR. IL1B, IL8, SERPINE1, TF, and TM expression in HAoEC was significantly reduced in the culture medium of super-low dose (0.1 ng/ml) LPS-activated macrophages. Super-low dose LPS-activated macrophages can suppress vascular endothelial cell inflammation and may be useful in preventing various diseases caused by the dysfunction of activated vascular endothelial cells.

IL-25 directly modulates adipocyte function and inflammation through the regulation of adiponectin.

This study aimed to investigate the direct role of IL-25 in modulating adipocyte function during homeostasis and low-grade inflammation induced by lipopolysaccharide (LPS). The 3T3-L1 preadipocyte cell lines and primary cultures of adipose-derived stromal vascular precursor cells of wild-type and IL-17RB-deficient mice were used to determine the direct function of IL-25. The expression of IL-17RB in differentiating adipocyte was determined using real-time PCR and flow cytometry analysis. The effect of IL-25 on lipid accumulation, triglyceride content, lipolysis, glucose uptake, and adipokine expression in the mature adipocytes was evaluated. IL-25 modulating the expression of inflammatory cytokines in adipocytes induced by low dose LPS was determined using real-time PCR and ELISA. The receptor for IL-25 was up-regulated during adipocyte differentiation and IL-25 directly modulated adipocyte function by reducing lipid accumulation and triglyceride concentration and enhancing lipolysis without affecting an insulin-stimulated glucose uptake. Interestingly, IL-25 induced adiponectin secretion through the PI3K/AKT signaling pathway. In 3T3-L1 adipocytes under low-grade inflammation, IL-25 attenuated the expression of IL-6 and CCL5 through the induction of adiponectin. Our studies suggest that IL-25 directly regulates adipocyte function by maintaining the adiponectin level during homeostasis and by alleviating inflammatory response through the regulation of adiponectin during low-grade inflammation in adipocytes.