Baicalein inhibits IL-1β- and TNF-α-induced inflammatory cytokine production from human mast cells via regulation of the NF-κB pathway

<p>Abstract</p> <p>Background</p> <p>Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Baicalein (BAI), isolated from the traditional Chinese herbal medicine Huangqin (<it>Scutellaria baicalensis Georgi</it>), has been shown to have anti-inflammatory effects. We examined its effects and mechanisms on the expression of inflammatory cytokines in an IL-1β- and TNF-α-activated human mast cell line, HMC-1.</p> <p>Methods</p> <p>HMC-1 cells were stimulated either with IL-1β (10 ng/ml) or TNF-α (100 U/ml) in the presence or absence of BAI. We assessed the expression of IL-6, IL-8, and MCP-1 by ELISA and RT-PCR, NF-κB activation by electrophoretic mobility shift assay (EMSA), and IκBα activation by Western blot.</p> <p>Results</p> <p>BAI (1.8 to 30 μM) significantly inhibited production of IL-6, IL-8, and MCP-1 in a dose-dependent manner in IL-1β-activated HMC-1. BAI (30 μM) also significantly inhibited production of IL-6, IL-8, and MCP-1 in TNF-α-activated HMC-1. Inhibitory effects appear to involve the NF-κB pathway. BAI inhibited NF-κB activation in IL-1β- and TNF-α-activated HMC-1. Furthermore, BAI increased cytoplasmic IκBα proteins in IL-1β- and TNF-α-activated HMC-1.</p> <p>Conclusion</p> <p>Our results showed that BAI inhibited the production of inflammatory cytokines through inhibition of NF-κB activation and IκBα phosphorylation and degradation in human mast cells. This inhibitory effect of BAI on the expression of inflammatory cytokines suggests its usefulness in the development of novel anti-inflammatory therapies.</p

Baicalein Inhibits IL-1β- and TNF-α-Induced Inflammatory Cytokine Production From Human Mast Cells via Regulation of the NF-κB Pathway

Background: Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Baicalein (BAI), isolated from the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi), has been shown to have anti-inflammatory effects. We examined its effects and mechanisms on the expression of inflammatory cytokines in an IL-1β- and TNF-α-activated human mast cell line, HMC-1. Methods: HMC-1 cells were stimulated either with IL-1β (10 ng/ml) or TNF-α (100 U/ml) in the presence or absence of BAI. We assessed the expression of IL-6, IL-8, and MCP-1 by ELISA and RT-PCR, NF-κB activation by electrophoretic mobility shift assay (EMSA), and IκBα activation by Western blot. Results: BAI (1.8 to 30 μM) significantly inhibited production of IL-6, IL-8, and MCP-1 in a dose-dependent manner in IL-1β-activated HMC-1. BAI (30 μM) also significantly inhibited production of IL-6, IL-8, and MCP-1 in TNF-α-activated HMC-1. Inhibitory effects appear to involve the NF-κB pathway. BAI inhibited NF-κB activation in IL-1β- and TNF-α-activated HMC-1. Furthermore, BAI increased cytoplasmic IκBα proteins in IL-1β- and TNF-α-activated HMC-1. Conclusion: Our results showed that BAI inhibited the production of inflammatory cytokines through inhibition of NF-κB activation and IκBα phosphorylation and degradation in human mast cells. This inhibitory effect of BAI on the expression of inflammatory cytokines suggests its usefulness in the development of novel anti-inflammatory therapies

Lactate and Immunosuppression in Sepsis

Serum lactate levels are traditionally interpreted as a marker of tissue hypoxia and often used clinically as an indicator of severity and outcome of sepsis/septic shock. Interestingly, recent studies involving the effects of tumor-derived lactate suggest that lactate itself may have an immunosuppressive effect in its local environment. This finding adds to the recent advances in immunometabolism that shed light on the importance of metabolism and metabolic intermediates in the regulation of innate immune and inflammatory responses in sepsis. In this article, we summarize recent studies, showing that the activation of immune cells requires aerobic glycolytic metabolism and that lactate produced by aerobic glycolysis may play an immunosuppressive role in sepsis

Lactate Suppresses Macrophage Pro-Inflammatory Response to LPS Stimulation by Inhibition of YAP and NF-κB Activation via GPR81-Mediated Signaling

Recent evidence from cancer research indicates that lactate exerts a suppressive effect on innate immune responses in cancer. This study investigated the mechanisms by which lactate suppresses macrophage pro-inflammatory responses. Macrophages [Raw 264.7 and bone marrow derived macrophages (BMDMs)] were treated with LPS in the presence or absence of lactate. Pro-inflammatory cytokines, NF-κB and YAP activation and nuclear translocation were examined. Our results show that lactate significantly attenuates LPS stimulated macrophage TNF-α and IL-6 production. Lactate also suppresses LPS stimulated macrophage NF-κB and YAP activation and nuclear translocation in macrophages. Interestingly, YAP activation and nuclear translocation are required for LPS stimulated macrophage NF-κB activation and TNFα production. Importantly, lactate suppressed YAP activation and nuclear translocation is mediated by GPR81 dependent AMKP and LATS activation which phosphorylates YAP, resulting in YAP inactivation. Finally, we demonstrated that LPS stimulation induces an interaction between YAP and NF-κB subunit p65, while lactate decreases the interaction of YAP and NF-κB, thus suppressing LPS induced pro-inflammatory cytokine production. Our study demonstrates that lactate exerts a previously unknown role in the suppression of macrophage pro-inflammatory cytokine production via GPR81 mediated YAP inactivation, resulting in disruption of YAP and NF-κB interaction and nuclear translocation in macrophages

TLR3 Mediates Repair and Regeneration of Damaged Neonatal Heart through Glycolysis Dependent YAP1 Regulated miR-152 Expression

The present study investigated whether TLR3 is required for neonatal heart repair and regeneration following myocardial infarction (MI). TLR3 deficient neonatal mice exhibited impaired cardiac functional recovery and a larger infarct size, while wild type neonatal mice showed cardiac functional recovery and small infarct size after MI. The data suggest that TLR3 is essential for the regeneration and repair of damaged neonatal myocardium. In vitro treatment of neonatal cardiomyocytes with a TLR3 ligand, Poly (I:C), significantly enhances glycolytic metabolism, YAP1 activation and proliferation of cardiomyocytes which were prevented by a glycolysis inhibitor, 2-deoxyglucose (2-DG). Administration of 2-DG to neonatal mice abolished cardiac functional recovery and YAP activation after MI, suggesting that TLR3-mediated regeneration and repair of the damaged neonatal myocardium is through glycolytic-dependent YAP1 activation. Inhibition of YAP1 activation abolished Poly (I:C) induced proliferation of neonatal cardiomyocytes. Interestingly, activation of YAP1 increases the expression of miR-152 which represses the expression of cell cycle inhibitory proteins, P27kip1 and DNMT1, leading to cardiomyocyte proliferation. We conclude that TLR3 is required for neonatal heart regeneration and repair after MI. The mechanisms involve glycolytic-dependent YAP1 activation, resulting in miR-152 expression which targets DNMT1/p27kip1

Endothelial HSPA12B Exerts Protection Against Sepsis-Induced Severe Cardiomyopathy via Suppression of Adhesion Molecule Expression by miR-126

Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells (ECs) and has been reported to protect against cardiac dysfunction from endotoxemia or myocardial infarction. This study investigated the mechanisms by which endothelial HSPA12B protects polymicrobial sepsis–induced cardiomyopathy. Wild-type (WT) and endothelial HSPA12B knockout (HSPA12B–/–) mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP). Cecal ligation and puncture sepsis accelerated mortality and caused severe cardiac dysfunction in HSPA12B–/– mice compared with WT septic mice. The levels of adhesion molecules and the infiltrated immune cells in the myocardium of HSPA12B–/– septic mice were markedly greater than in WT septic mice. The levels of microRNA-126 (miR-126), which targets adhesion molecules, in serum exosomes from HSPA12B–/– septic mice were significantly lower than in WT septic mice. Transfection of ECs with adenovirus expressing HSPA12B significantly increased miR-126 levels. Increased miR-126 levels in ECs prevented LPS-stimulated expression of adhesion molecules. In vivo delivery of miR-126 carried by exosomes into the myocardium of HSPA12B–/– mice significantly attenuated CLP sepsis increased levels of adhesion molecules, and improved CLP sepsis–induced cardiac dysfunction. The data suggest that HSPA12B protects against sepsis-induced severe cardiomyopathy via regulating miR-126 expression which targets adhesion molecules, thus decreasing the accumulation of immune cells in the myocardium

MicroRNA-214 Protects Against Hypoxia/Reoxygenation Induced Cell Damage and Myocardial Ischemia/Reperfusion Injury via Suppression of PTEN and Bim1 Expression

Background: Myocardial apoptosis plays an important role in myocardial ischemia/reperfusion (I/R) injury. Activation of PI3K/Akt signaling protects the myocardium from I/R injury. This study investigated the role of miR-214 in hypoxia/ reoxygenation (H/R)-induced cell damage in vitro and myocardial I/R injury in vivo. Methods and Results: H9C2 cardiomyoblasts were transfected with lentivirus expressing miR-214 (LmiR-214) or lentivirus expressing scrambled miR-control (LmiR-control) respectively, to establish cell lines of LmiR-214 and LmiR-control. The cells were subjected to hypoxia for 4 h followed by reoxygenation for 24 h. Transfection of LmiR-214 suppresses PTEN expression, significantly increases the levels of Akt phosphorylation, markedly attenuates LDH release, and enhances the viability of the cells subjected to H/R. In vivo transfection of mouse hearts with LmiR-214 significantly attenuates I/R induced cardiac dysfunction and reduces I/Rinduced myocardial infarct size. LmiR-214 transfection significantly attenuates I/Rinduced myocardial apoptosis and caspase-3/7 and caspase-8 activity. Increased expression of miR-214 by transfection of LmiR-214 suppresses PTEN expression, increases the levels of phosphorylated Akt, represses Bim1 expression and induces Bad phosphorylation in the myocardium. In addition, in vitro data shows transfection of miR-214 mimics to H9C2 cells suppresses the expression and translocation of Bim1 from cytosol to mitochondria and induces Bad phosphorylation. Conclusions: Our in vitro and in vivo data suggests that miR-214 protects cells from H/R induced damage and attenuates I/R induced myocardial injury. The mechanisms involve activation of PI3K/Akt signaling by targeting PTEN expression, induction of Bad phosphorylation, and suppression of Bim1 expression, resulting in decreases in I/R-induced myocardial apoptosis

Lactate Promotes Macrophage HMGB1 Lactylation, Acetylation, and Exosomal Release in Polymicrobial Sepsis

High circulating levels of lactate and high mobility group box-1 (HMGB1) are associated with the severity and mortality of sepsis. However, it is unclear whether lactate could promote HMGB1 release during sepsis. The present study demonstrated a novel role of lactate in HMGB1 lactylation and acetylation in macrophages during polymicrobial sepsis. We found that macrophages can uptake extracellular lactate via monocarboxylate transporters (MCTs) to promote HMGB1 lactylation via a p300/CBP-dependent mechanism. We also observed that lactate stimulates HMGB1 acetylation by Hippo/YAP-mediated suppression of deacetylase SIRT1 and β-arrestin2-mediated recruitment of acetylases p300/CBP to the nucleus via G protein-coupled receptor 81 (GPR81). The lactylated/acetylated HMGB1 is released from macrophages via exosome secretion which increases endothelium permeability. In vivo reduction of lactate production and/or inhibition of GPR81-mediated signaling decreases circulating exosomal HMGB1 levels and improves survival outcome in polymicrobial sepsis. Our results provide the basis for targeting lactate/lactate-associated signaling to combat sepsis

TIR/BB-Loop Mimetic AS-1 Attenuates Cardiac Ischemia/Reperfusion Injury via a Caveolae and Caveolin-3-Dependent Mechanism

AS-1, the TIR/BB loop mimetic, plays a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. The muscle specific caveolin3 (Cav-3) and the caveolae have been found to be critical for cardioprotection. This study aimed to evaluate our hypothesis that caveolae and Cav-3 are essential for AS-1-induced cardioprotection against myocardial I/R injury. To address these issues, we analyzed the involvement of Cav-3 in AS-1 mediated cardioprotection both in vivo and in vitro. We demonstrate that AS-1 administration significantly decreased infarct size, improved cardiac function after myocardial I/R and modulated membrane caveolae and Cav-3 expression in the myocardium. For in vitro studies, AS-1 treatment prevented Cav-3 re-distribution induced by H/R injury. In contrast, disruption of caveolae by MCD treatment or Cav-3 knockdown abolished the protection against H/R-induced myocytes injury by AS-1. Our findings reveal that AS-1 attenuates myocardial I/R injury through caveolae and Cav-3 dependent mechanism

The TLR9 ligand, CpG-ODN, Induces Protection Against Cerebral Ischemia/Reperfusion Injury via Activation of pi3k/Akt Signaling.

Toll-like receptors (TLRs) have been shown to be involved in cerebral ischemia/reperfusion (I/R) injury. TLR9 is located in intracellular compartments and recognizes CpG-DNA. This study examined the effect of CpG-ODN on cerebral I/R injury. C57BL/6 mice were treated with CpG-ODN by i.p. injection 1 hour before the mice were subjected to cerebral ischemia (60 minutes) followed by reperfusion (24 hours). Scrambled-ODN served as control-ODN. Untreated mice, subjected to cerebral I/R, served as I/R control. The effect of inhibitory CpG-ODN (iCpG-ODN) on cerebral I/R injury was also examined. In addition, we examined the therapeutic effect of CpG-ODN on cerebral I/R injury by administration of CpG-ODN 15 minutes after cerebral ischemia. CpG-ODN administration significantly decreased cerebral I/R-induced infarct volume by 69.7% (6.4±1.80% vs 21.0±2.85%, P\u3c0.05), improved neurological scores, and increased survival rate, when compared with the untreated I/R group. Therapeutic administration of CpG-ODN also significantly reduced infarct volume by 44.7% (12.6±2.03% vs 22.8±2.54%, P\u3c0.05) compared with untreated I/R mice. Neither control-ODN, nor iCpG-ODN altered I/R-induced cerebral injury or neurological deficits. Nissl staining showed that CpG-ODN treatment preserved neuronal morphology in the ischemic hippocampus. Immunoblot showed that CpG-ODN administration increased Bcl-2 levels by 41% and attenuated I/R-increased levels of Bax and caspase-3 activity in ischemic brain tissues. Importantly, CpG-ODN treatment induced Akt and GSK-3β phosphorylation in brain tissue and cultured microglial cells. PI3K inhibition with LY294002 abolished CpG-ODN-induced protection. CpG-ODN significantly reduces cerebral I/R injury via a PI3K/Akt-dependent mechanism. Our data also indicate that CpG-ODN may be useful in the therapy of cerebral I/R injury