Berberine Protects against Neuronal Damage via Suppression of Glia-Mediated Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) triggers a series of neuroinflammatory processes that contribute to evolution of neuronal injury. The present study investigated the neuroprotective effects and anti-inflammatory actions of berberine, an isoquinoline alkaloid, in both in vitro and in vivo TBI models. Mice subjected to controlled cortical impact injury were injected with berberine (10 mg·kg⁻¹) or vehicle 10 min after injury. In addition to behavioral studies and histology analysis, blood-brain barrier (BBB) permeability and brain water content were determined. Expression of PI3K/Akt and Erk signaling and inflammatory mediators were also analyzed. The protective effect of berberine was also investigated in cultured neurons either subjected to stretch injury or exposed to conditioned media with activated microglia. Berberine significantly attenuated functional deficits and brain damage associated with TBI up to day 28 post-injury. Berberine also reduced neuronal death, apoptosis, BBB permeability, and brain edema at day 1 post-injury. These changes coincided with a marked reduction in leukocyte infiltration, microglial activation, matrix metalloproteinase-9 activity, and expression of inflammatory mediators. Berberine had no effect on Akt or Erk 1/2 phosphorylation. In mixed glial cultures, berberine reduced TLR4/MyD88/NF-κB signaling. Berberine also attenuated neuronal death induced by microglial conditioned media; however, it did not directly protect cultured neurons subjected to stretch injury. Moreover, administration of berberine at 3 h post-injury also reduced TBI-induced neuronal damage, apoptosis and inflammation in vivo. Berberine reduces TBI-induced brain damage by limiting the production of inflammatory mediators by glial cells, rather than by a direct neuroprotective effect.     

An agent-based model of inflammation and fibrosis following particulate exposure in the lung

Inflammation and airway remodeling occur in a variety of airway diseases. Modeling aspects of the inflammatory and fibrotic processes following repeated exposure to particulate matter may provide insights into a spectrum of airway diseases, as well as prevention/treatment strategies. An agent-based model (ABM) was created to examine the response of an abstracted population of inflammatory cells (nominally macrophages, but possibly including other inflammatory cells such as lymphocytes) and cells involved in remodeling (nominally fibroblasts) to particulate exposure. The model focused on a limited number of relevant interactions, specifically those among macrophages, fibroblasts, a pro-inflammatory cytokine (TNF-α), an anti-inflammatory cytokine (TGF-β1), collagen deposition, and tissue damage. The model yielded three distinct states that were equated with (1) self-resolving inflammation and a return to baseline, (2) a pro-inflammatory process of localized tissue damage and fibrosis, and (3) elevated pro- and anti-inflammatory cytokines, persistent tissue damage, and fibrosis outcomes. Experimental results consistent with these predicted states were observed in histology sections of lung tissue from mice exposed to particulate matter. Systematic in silico studies suggested that the development of each state depended primarily upon the degree and duration of exposure. Thus, a relatively simple ABM resulted in several, biologically feasible, emergent states, suggesting that the model captures certain salient features of inflammation following exposure of the lung to particulate matter. This ABM may hold future utility in the setting of airway disease resulting from inflammation and fibrosis following particulate exposure.     

Quantification of cytokines and inflammatory mediators in a three-dimensional model of inflammatory arthritis

Human recombinant IL-1beta and TNFalpha have been previously used to induce a cytokine response in canine chondrocytes. In order to establish this functional relation in a homologous system in vitro, we have developed both 2D and 3D models of inflammatory arthritis using canine recombinant cytokines in canine articular chondrocytes. IL-1beta and TNFalpha were cloned and subsequently expressed in Escherichia coli. The purified recombinant canine cytokines were used to simulate inflammation in vitro and the expression of typical inflammation markers such as proinflammatory cytokines (IL-1beta, IL-6, IL-8, GM-CSF and TNFalpha), enzyme mediators (MMP-3 MMP-13, iNOS, COX-2) and their catabolites (NO, PGE(2)) was measured. High expression of proinflammatory cytokines, enzyme mediators and their catabolites was only observed in IL-1beta/TNFalpha stimulated cells. We conclude that the canine IL-1beta and TNFalpha generated in this study are biologically active and equally effective in the canine cell culture systems. Inducing an inflammatory pathway by canine exogenous cytokines in canine chondrocytes provides a useful tool for the study of canine inflammatory arthritis.     

诱导型一氧化氮合酶抑制剂研发现状

一氧化氮(NO)对炎症性疾病的治疗作用近来引起了广泛的重视。诱导型一氧化合成酶(iNOS)被发现广泛地参与趋炎因子表达和反应性氧化产物(ROS)/反应性氮化产物(RNS)的产生,从而进一步证明了一氧化氮在炎症病理发生发展中的关键作用。由于传统的抗炎药物环氧合酶-2(COX-2)抑制剂被报导有较多副作用,新型抑制炎症药物的研究开发势在必行。本文分别介绍了化学来源、生物来源、植物来原性iNOS抑制剂阻的开发、研究现状,阐述了其在断炎症信息传递通道中的作用。表明了iNOS抑制剂防止炎症损害的相关机理,提出iNOs不仅能在初始阶段影响炎症的发生,也对抑制和终结炎症有作用。最后进一步介绍了用中草药研发iNOs抑制剂的可能性,展望了于中药在该领域内的巨大前景。     

Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

Background

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response.

Methods

Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation.

Results

MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury.

Conclusions

Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.     

Different inflammatory mediators induce inflammation and pain after application of liquid nitrogen to the skin

The application of liquid nitrogen to the skin induces inflammation and pain. However, there is little data on the role of inflammatory mediators in the production of these symptoms. We have developed an experimental model to study some aspects of the inflammatory response and its mediators following the application of cold. We have applied liquid nitrogen jets to subcutaneous air pouches in the dorsal skin of rats to study the kinetics of the migration of inflammatory cells; also to the ear for histopathological analysis and on the paws for edema and pain. Inflammatory mediators were identified by pharmacological means. The results showed that the cellular inflammatory response was characterized by persistent cell migration, mainly of granulocytes. Histopathology of the ears confirmed these findings. Histamine and sympathomimetic mediators were mainly responsible for the resultant swelling. However, the hypernociception that resulted involved other mediators including IL-1 and eicosanoids. These data suggest that interference with the release of inflammatory mediators might reduce the side effects of cryosurgery and prevent hyperalgesia and inflammation at the site of application of cold.     

Different inflammatory mediators induce inflammation and pain after application of liquid nitrogen to the skin

The application of liquid nitrogen to the skin induces inflammation and pain. However, there is little data on the role of inflammatory mediators in the production of these symptoms. We have developed an experimental model to study some aspects of the inflammatory response and its mediators following the application of cold. We have applied liquid nitrogen jets to subcutaneous air pouches in the dorsal skin of rats to study the kinetics of the migration of inflammatory cells; also to the ear for histopathological analysis and on the paws for edema and pain. Inflammatory mediators were identified by pharmacological means. The results showed that the cellular inflammatory response was characterized by persistent cell migration, mainly of granulocytes. Histopathology of the ears confirmed these findings. Histamine and sympathomimetic mediators were mainly responsible for the resultant swelling. However, the hypernociception that resulted involved other mediators including IL-1 and eicosanoids. These data suggest that interference with the release of inflammatory mediators might reduce the side effects of cryosurgery and prevent hyperalgesia and inflammation at the site of application of cold.     

Down-regulation of proinflammatory capacity during apoptosis in human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) are essential to innate immunity in humans and contribute significantly to inflammation. Although progress has been made, the molecular basis for termination of inflammation in humans is incompletely characterized. We used human oligonucleotide microarrays to identify genes encoding inflammatory mediators that were differentially regulated during the induction of apoptosis. One hundred thirty-three of 212 differentially expressed genes encoding proinflammatory factors, signal transduction mediators, adhesion molecules, and other proteins that facilitate the inflammatory response were down-regulated during the induction of apoptosis following PMN phagocytosis. Among these, 42 genes encoded proteins critical to the inflammatory response, including receptors for IL-8 beta, IL-10 alpha, IL-13 alpha 1, IL-15 alpha, IL-17, IL-18, C1q, low-density lipoprotein, IgG Fc (CD32), and formyl peptide, Toll-like receptor 6, platelet/endothelial cell adhesion molecule-1 (CD31), P-selectin (CD62), IL-1 alpha, IL-16, and granulocyte chemoattractant protein-2 were down-regulated. Many of these genes were similarly down-regulated during Fas-mediated or camptothecin-induced apoptosis. We used flow cytometry to confirm that IL-8R beta (CXCR2) and IL-1 alpha were significantly down-regulated during PMN apoptosis. We also discovered that 23 genes encoding phosphoinositide and calcium-mediated signal transduction components, which comprise complex pathways essential to the inflammatory response of host cells, were differentially regulated during PMN apoptosis. Importantly, our data demonstrate that PMNs down-regulate proinflammatory capacity at the level of gene expression during induction of apoptosis. These findings provide new insight into the molecular events that resolve inflammation following PMN activation in humans.     

Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise

There has been no investigation to determine if the widely used over-the-counter, water-soluble antioxidants vitamin C and N-acetyl-cysteine (NAC) could act as pro-oxidants in humans during inflammatory conditions. We induced an acute-phase inflammatory response by an eccentric arm muscle injury. The inflammation was characterized by edema, swelling, pain, and increases in plasma inflammatory indicators, myeloperoxidase and interleukin-6. Immediately following the injury, subjects consumed a placebo or vitamin C (12.5 mg/kg body weight) and NAC (10 mg/kg body weight) for 7 d. The resulting muscle injury caused increased levels of serum bleomycin-detectable iron and the amount of iron was higher in the vitamin C and NAC group. The concentrations of lactate dehydrogenase (LDH), creatine kinase (CK), and myoglobin were significantly elevated 2, 3, and 4 d postinjury and returned to baseline levels by day 7. In addition, LDH and CK activities were elevated to a greater extent in the vitamin C and NAC group. Levels of markers for oxidative stress (lipid hydroperoxides and 8-iso prostaglandin F2alpha; 8-Iso-PGF2alpha) and antioxidant enzyme activities were also elevated post-injury. The subjects receiving vitamin C and NAC had higher levels of lipid hydroperoxides and 8-Iso-PGF2alpha 2 d after the exercise. This acute human inflammatory model strongly suggests that vitamin C and NAC supplementation immediately post-injury, transiently increases tissue damage and oxidative stress.     

Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells attenuates Escherichia coli-induced acute lung injury in mice

Background

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response.

Methods

Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation.

Results

MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury.

Conclusions

Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.     

Computational Identification of Mechanistic Factors That Determine the Timing and Intensity of the Inflammatory Response

Timely resolution of inflammation is critical for the restoration of homeostasis in injured or infected tissue. Chronic inflammation is often characterized by a persistent increase in the concentrations of inflammatory cells and molecular mediators, whose distinct amount and timing characteristics offer an opportunity to identify effective therapeutic regulatory targets. Here, we used our recently developed computational model of local inflammation to identify potential targets for molecular interventions and to investigate the effects of individual and combined inhibition of such targets. This was accomplished via the development and application of computational strategies involving the simulation and analysis of thousands of inflammatory scenarios. We found that modulation of macrophage influx and efflux is an effective potential strategy to regulate the amount of inflammatory cells and molecular mediators in both normal and chronic inflammatory scenarios. We identified three molecular mediators − tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and the chemokine CXCL8 − as potential molecular targets whose individual or combined inhibition may robustly regulate both the amount and timing properties of the kinetic trajectories for neutrophils and macrophages in chronic inflammation. Modulation of macrophage flux, as well as of the abundance of TNF-α, TGF-β, and CXCL8, may improve the resolution of chronic inflammation.     

Combining experiments with multi-cell agent-based modeling to study biological tissue patterning

Agent-based modeling (ABM), also termed ‘Individual-basedmodeling (IBM)’, is a computational approach that simulatesthe interactions of autonomous entities (agents, or individualcells) with each other and their local environment to predicthigher level emergent patterns. A literature-derived rule setgoverns the actions of each individual agent. While this techniquehas been widely used in the ecological and social sciences,it has only recently been applied in biomedical research. Thepurpose of this review is to provide an introduction to ABMas it has been used to study complex multi-cell biological phenomena,underscore the importance of coupling models with experimentalwork, and outline future challenges for the ABM field and itsapplication to biomedical research. We highlight a number ofpublished examples of ABM, focusing on work that has combinedexperimental with ABM analyses and how this pairing producesnew understanding. We conclude with suggestions for moving forwardwith this parallel approach.      

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H₂S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H₂S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund''s adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO₂⁻), PGE₂, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.     

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo

Background

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo.

Methods

Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured.

Results

In WT mice, CpG-ODN induced a strong activation of pulmonary NFκB as well as a significant increase in pulmonary TNF-α and IL-1β mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice.

Conclusion

This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.     

Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament

Denervation degrades normal ligament properties and impairs ligament healing. This suggests that secreted neuromediators, such as neuropeptides, could be modulating cell metabolism in ligament and scar tissue. To test this hypothesis we investigated the effect of exogenous substance P (SP), neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP) on the mRNA levels for proteins associated with inflammation, angiogenesis, and matrix production in tissue-cultured specimens of normal and injured medial collateral ligament. SP and NPY induced increased mRNA levels for several inflammatory mediators in the 2-week post-injury specimens. All three neuropeptides induced decreases in mRNA levels for healing-associated growth factors and matrix molecules, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and collagen types I and III. The results indicate that neuropeptides strongly influence the metabolic activity of cells in healing ligament, particularly at early time points after injury.     

Central Role for MCP-1/CCL2 in Injury-Induced Inflammation Revealed by In Vitro,In Silico,and Clinical Studies

The translation of in vitro findings to clinical outcomes is often elusive. Trauma/hemorrhagic shock (T/HS) results in hepatic hypoxia that drives inflammation. We hypothesize that in silico methods would help bridge in vitro hepatocyte data and clinical T/HS, in which the liver is a primary site of inflammation. Primary mouse hepatocytes were cultured under hypoxia (1% O₂) or normoxia (21% O₂) for 1–72 h, and both the cell supernatants and protein lysates were assayed for 18 inflammatory mediators by Luminex™ technology. Statistical analysis and data-driven modeling were employed to characterize the main components of the cellular response. Statistical analyses, hierarchical and k-means clustering, Principal Component Analysis, and Dynamic Network Analysis suggested MCP-1/CCL2 and IL-1α as central coordinators of hepatocyte-mediated inflammation in C57BL/6 mouse hepatocytes. Hepatocytes from MCP-1-null mice had altered dynamic inflammatory networks. Circulating MCP-1 levels segregated human T/HS survivors from non-survivors. Furthermore, T/HS survivors with elevated early levels of plasma MCP-1 post-injury had longer total lengths of stay, longer intensive care unit lengths of stay, and prolonged requirement for mechanical ventilation vs. those with low plasma MCP-1. This study identifies MCP-1 as a main driver of the response of hepatocytes in vitro and as a biomarker for clinical outcomes in T/HS, and suggests an experimental and computational framework for discovery of novel clinical biomarkers in inflammatory diseases.     

Sphingosine kinase: Role in regulation of bioactive sphingolipid mediators in inflammation

Sphingolipids and their synthetic enzymes are emerging as important mediators in inflammatory responses and as regulators of immune cell functions. In particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P) have been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. SK1 has been shown to be activated by cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin1-β (IL1-β). The activation of SK1 in this pathway has been shown to be, at least in part, required for mediating TNF-α and IL1-β inflammatory responses in cells, including induction of cyclo-oxygenase 2 (COX2). In addition to their role in inflammatory signaling, SK and S1P have also been implicated in various immune cell functions including, mast cell degranulation, migration of neutrophils, and migration and maturation of lymphocytes. The involvement of sphingolipids and sphingolipid metabolizing enzymes in inflammatory signaling and immune cell functions has implicated these mediators in numerous inflammatory disease states as well. The contribution of these mediators, specifically SK1 and S1P, to inflammation and disease are discussed in this review.