•ICV-LPS treated rats exhibited memory deficits and anxiety-like behavior in rats.•The neuroprotective effect of plumbagin mediated through mitigation of oxidative stress and inflammation.•In-silico ...study revealed binding affinity of plumbagin on acetylcholinesterase site.•Plumbagin could be considered as promising therapeutic agent in the neuroprotection against behavioral disorders.
Plumbagin (5‑hydroxy-2-methyl-1,4-naphthoquinone), a phytoconstituent has been shown to exhibit potent antioxidant and anti-inflammatory effects. In the present investigation, we explored the neuroprotective effects of plumbagin against intracerebroventricular (ICV) lipopolysaccharide (LPS)-induced behavioral deficits. We found that ICV administration of LPS (5 μg/5μl in each ICV injection) bilaterally in the hippocampus caused significant reduction in learning and memory abilities of rats. Moreover, ICV-LPS treated rats displayed anxiety-like behavior evident by open field test and elevated plus maze test. Biochemical analysis in the hippocampus revealed oxidative-nitrosative stress, mitochondrial complexes (I, II, and III) impairment and cholinergic dysfunction in ICV-LPS rats. However, pre- and post-treatment with plumbagin (10 and 20 mg/kg) dose-dependently attenuated ICV-LPS induced oxidative stress (reduction of MDA and nitrite with GSH elevation), mitochondrial impairment, and cholinergic dysfunction (reduction of AChE) in the hippocampus. The pro-inflammatory cytokine (IL-1β) was also found to be increased in the ICV-LPS treated group which was markedly reduced by the treatment of Plumbagin-20 mg/kg. Further, in-silico analysis revealed that the calculated dock score (-5.116 kcal/mol) and predicted binding energy (-45.800 kcal/mol) supports the binding of plumbagin in the binding site of AChE. All these findings support the neuroprotective effect of plumbagin against ICV-LPS induced behavioral and neurochemical deficits in rats. Thus, plumbagin may be considered an intriguing pharmacological intervention for the management of behavioral dysfunction.
Lipopolysaccharide (LPS) triggers an inflammatory response, causing impairment of cardiomyocyte Ca2+ and Na + regulation. This study aimed to determine whether piscidin-1 (PCD-1), an antimicrobial ...peptide, improves intracellular Ca2+ and Na + regulation in LPS-challenged atrial cardiomyocytes. Rabbit atrial cardiomyocytes were enzymatically isolated from the left atria. Patch-clamp ionic current recording, intracellular Ca2+ monitoring using Fluo-3, and detection of cytosolic reactive oxygen species production were conducted in control, LPS-challenged, and LPS + PCD-1-treated atrial cardiomyocytes. LPS-challenged cardiomyocytes showed shortened durations of action potential at their 50% and 90% repolarizations, which was reversed by PCD-1 treatment. LPS-challenged cardiomyocytes showed decreased L-type Ca2+ channel currents and larger Na+/Ca2+ exchange currents compared to controls. While LPS did not affect the sodium current, an enhanced late sodium current with increased cytosolic Na+ levels was observed in LPS-challenged cardiomyocytes. These LPS-induced alterations in the ionic current were ameliorated by PCD-1 treatment. LPS-challenged cardiomyocytes displayed lowered Ca2+ transient amplitudes and decreased Ca2+ stores and greater Ca2+ leakage in the sarcoplasmic reticulum compared to the control. Exposure to PCD-1 attenuated LPS-induced alterations in Ca2+ regulation. The elevated reactive oxygen species levels observed in LPS-challenged myocytes were suppressed after PCD-1 treatment. The protein levels of NF-κB and IL-6 increased following LPS treatment. Decreased sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a protein levels were observed in LPS-challenged cardiomyocytes. PCD-1 modulates LPS-induced alterations in inflammatory and Ca2+ regulatory protein levels. Our results suggest that PCD-1 modulates LPS-induced alterations in intracellular Ca2+ and Na + homeostasis, reactive oxygen species production, and the NF-κB inflammatory pathway in atrial cardiomyocytes.
Neuroinflammation has been well recognized as a key pathological event in acute glaucoma. The medical therapy of acute glaucoma mainly focuses on lowering intraocular pressure (IOP), while there are ...still scarce anti-inflammatory agents in the clinical treatment of acute glaucoma. Here we reported that β,3α,5α-trihydroxy-androst-6-one (sterone), a novel synthetic polyhydric steroid, blocked neuroinflammation mediated by microglia/macrophages and alleviated the loss of retinal ganglion cells (RGCs) caused by acute intraocular hypertension (AIH). The results showed that sterone significantly inhibited the morphological changes, the up-regulation of inflammatory biomarker ionized calcium-binding adapter molecule 1 (Iba-1), and the mRNA increase of proinflammatory tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in BV2 microglia and RAW264.7 macrophages. Moreover, immunofluorescence and western blotting analysis revealed that sterone markedly abrogated the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB) p65 subunit. Furthermore, sterone significantly suppressed the inflammatory microglial activation and RGCs' reduction caused by retinal ischemia/reperfusion (I/R) injury in a rat AIH model. These results suggest sterone may be a potential candidate in the treatment of acute glaucoma caused by microglial activation-mediated neuroinflammatory injury.
Eicosanoid modulation by butyrate has been reported in various cells and conditions. Recently, comprehensive analyses of lipid mediators using liquid chromatography/tandem mass spectrometry has been ...reported. We hypothesized that tributyrin, a prodrug of butyrate, may attenuate LPS-induced liver injury in rats by suppressing the production of pro-inflammatory lipid mediators and/or by inducing anti-inflammatory specialized proresolving mediators. To test this, groups of Wistar rats were orally administered tributyrin (1 g/kg body weight) or vehicle 1 h before intraperitoneal injection of LPS. The livers were collected at 0, 1.5, 6, and 24 h later and analyzed: lipid mediators were profiled by liquid chromatography/tandem mass spectrometry; expression of cyclooxygenase-2, 5-lipoxygenase (LOX), 12/15-LOX, and leukotriene (LT) A
hydrolase, and nuclear translocation of 5-LOX were evaluated by western blot analysis; and induction of liver injury was assessed by immunostaining for 8-hydroxy-2'-deoxyguanosine, an indicator of oxidative DNA damage. We found that tributyrin treatment attenuated LPS-induced production of pro-inflammatory LTB
(p < 0.05) and decreased oxidative stress levels in the liver. Tributyrin also attenuated the nuclear translocation of 5-LOX in response to LPS, suggesting a possible mechanism for the LTB
reduction. LPS-induced changes in other lipid mediators were not significantly affected by tributyrin treatment up to 24 h after LPS injection. Our results suggest that oral tributyrin administration protects against endotoxemia-associated liver damage by reducing production of the pro-inflammatory eicosanoid LTB
.
In inflamed tissues or during ischemia-reperfusion episodes, activated macrophages produce large amounts of reactive species and are, thus, exposed to the damaging effects of reactive species. Here, ...our goal was to investigate the mechanism whereby activated macrophages protect themselves from oxidant stress-induced cell death. Hydrogen peroxide-treated mouse bone marrow-derived macrophages (BMDM) and THP-1 human monocyte-derived cells were chosen as models. We found a gradual development of resistance: first in monocyte-to-macrophage differentiation, and subsequently after lipopolysaccharide (LPS) exposure. Investigating the mechanism of the latter, we found that exposure to intense hydrogen peroxide stress causes poly(ADP-ribose) polymerase-1 (PARP-1) dependent programmed necrotic cell death, also known as parthanatos, as indicated by the protected status of PARP-1 knockout BMDMs and the protective effect of the PARP inhibitor PJ-34. In hydrogen peroxide-treated macrophages, however, apoptosis inducing factor (AIF) proved dispensable for parthanatos; nuclear translocation of AIF was not observed. A key event in LPS-mediated protection against the hydrogen peroxide-induced AIF independent parthanatos was downregulation of PARP1 mRNA and protein. The importance of this event was confirmed by overexpression of PARP1 in THP1 cells using a viral promoter, which lead to stable PARP1 levels even after LPS treatment and unresponsiveness to LPS-induced cytoprotection. In BMDMs, LPS-induced PARP1 suppression lead to prevention of NAD+ depletion. Moreover, LPS also induced expression of antioxidant proteins (superoxide dismutase-2, thioredoxin reductase 1 and peroxiredoxin) and triggered a metabolic shift to aerobic glycolysis, also known as the Warburg effect. In summary, we provide evidence that in macrophages intense hydrogen peroxide stress causes AIF-independent parthanatos from which LPS provides protection. The mechanism of LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD+ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.
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•High concentrations of hydrogen peroxide causes PARP-1 mediated necrotic cell death (parthanatos) in macrophages.•Apoptosis inducing factor (AIF) is not required for hydrogen peroxide-induced parthanatos in macrophages.•LPS-stimulated macrophages are protected from hydrogen peroxide-induced cell death.•LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD+ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.
Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most Gram-negative bacteria. LPS transport from the inner membrane (IM) to the OM is achieved by seven ...lipopolysaccharide transport proteins (LptA-G). LptB2FG, an type VI ATP-binding cassette (ABC) transporter, forms a stable complex with LptC, extracts LPS from the IM and powers LPS transport to the OM. Here we report the cryo-EM structures of LptB2FG and LptB2FGC from Klebsiella pneumoniae in complex with LPS. The KpLptB2FG-LPS structure provides detailed interactions between LPS and the transporter, while the KpLptB2FGC-LPS structure may represent an intermediate state that the transmembrane helix of LptC has not been fully inserted into the transmembrane domains of LptB2FG.
•Cryo-EM structure of KpLptB2FG-LPS at 3.85 Å•Cryo-EM structure of KpLptB2FGC-LPS at 4.3 Å•KpLptB2FGC-LPS structure may present in an immediate state during LPS transport
Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated ...the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling.
•LPS inhibites osteogenic differentiation in HBMSCs via suppression of p38 MAPK signaling pathway.•HAMSCs promote LPS-induced HBMSCs osteogenic differentiation through p38 MAPK signaling pathway.•HAMSCs reverse LPS-induced oxidative stress in LPS-induced HBMSCs through p38 MAPK signaling pathway.
Context: The flavonoid and phenolic compounds found in Artocarpus altilis have made their popularity. This plant has been used as a traditional medicine in Indonesia for various diseases. Aims: To ...evaluate the toxic concentration of the A. altilis extract and to investigate the capacity of the extract to inhibit inflammation against the macrophage cell line RAW 264.7, which has been induced with lipopolysaccharide (LPS). Methods: The MTT assay was used to determine the level of toxicity shown by the extract. The qRT-PCR method was used to examine the anti-inflammatory activity of the sample by comparing the relative expression levels of IL-1β, IL-6, and IL-10 mRNA in LPS-stimulated RAW 264.7 cells to those of dexamethasone, which served as the control. Results: The results of the test for toxicity indicated that the extract is safe. In RAW 264.7 cells that had been stimulated with LPS, we observed that the addition of A. altilis leaf extract drastically reduced the expression of IL-1β and IL-6 mRNA while simultaneously increasing the production of IL-10. Conclusions: The ethanol extract of A. altilis leaf was nontoxic and exhibited anti-inflammatory activity on LPS-induced RAW 264.7 cells.
Toll-like receptors (TLRs) are now recognized as the major receptors for microbial pathogens on cells of the innate immune system. Recently, TLRs were also identified in many organs including the ...kidney. However, the cellular distribution and role of these renal TLRs remain largely unknown. In this paper, we investigated the expression of TLR4 in a cecal ligation and puncture (CLP) model of sepsis in Sprague-Dawley rats utilizing fluorescence microscopy. In sham animals, TLR4 was expressed predominantly in Tamm-Horsfall protein (THP)-positive tubules. In CLP animals, TLR4 expression increased markedly in all tubules (proximal and distal), glomeruli, and the renal vasculature. The staining showed a strong apical distribution in all tubules. A moderately less intense cellular signal colocalized partially with the Golgi apparatus. In addition, kidneys from septic rats showed increased expression of CD14 and THP. They each colocalized strongly with TLR4, albeit in different tubular segments. We also imaged the kidneys of live septic animals with two-photon microscopy after fluorescent lipopolysaccharide (LPS) injection. Within 10 min, LPS was seen at the brush border of some proximal tubules. Within 60 min, LPS was fully cytoplasmic in proximal tubules. Conversely, distal tubules showed no LPS uptake. We conclude that TLR4, CD14, and THP have specific renal cellular and tubular expression patterns that are markedly affected by sepsis. Systemic endotoxin can freely access the tubular and cellular sites where these proteins are present. Therefore, locally expressed TLRs and other interacting proteins could potentially modulate the renal response to systemic sepsis.
Abstract
The intestinal barrier plays a crucial role in maintaining gut health, and an increased permeability has been linked to several intestinal and extra-intestinal disorders. There is an ...increasing demand for interventions aimed at strengthening this barrier and for in vivo challenge models to assess their efficiency. This study investigated the effect of sauna-induced dehydration on intestinal barrier function (clinicaltrials.gov: NCT03620825). Twenty healthy subjects underwent three conditions in random order: (1) Sauna dehydration (loss of 3% body weight), (2) non-steroidal anti-inflammatory drug (NSAID) intake, (3) negative control. Intestinal permeability was assessed by a multi-sugar urinary recovery test, while intestinal damage, bacterial translocation and cytokines were assessed by plasma markers. The sauna dehydration protocol resulted in an increase in gastroduodenal and small intestinal permeability. Presumably, this increase occurred without substantial damage to the enterocytes as plasma intestinal fatty acid-binding protein (I-FABP) and liver fatty acid-binding protein (L-FABP) were not affected. In addition, we observed significant increases in levels of lipopolysaccharide-binding protein (LBP), IL-6 and IL-8, while sCD14, IL-10, IFN-ɣ and TNF-α were not affected. These results suggest that sauna dehydration increased intestinal permeability and could be applied as a new physiological in vivo challenge model for intestinal barrier function.
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