The immune function of heme oxygenase-1 (HO-1) from Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Heme oxygenase-1 (HO-1), an inducible rate-limiting metabolic enzyme, exerts critical immunomodulatory functions by potential anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Although accumulative studies have focused on the immune functions of HO-1 in mammals, the roles in fish are poorly understood, and the reports on involvement in the defensive and immune response are very limited. In this study, On-HO-1 gene from Oreochromis niloticus was successfully cloned and identified, which contained an open reading frame (ORF) of 816 bp and coded for a protein of 271 amino acids. The On-HO-1 protein phylogenetically shared a high homology with HO-1 in other teleost fish (76.10%-98.89 %) and a lowly homology with HO-1 in mammals (38.98%-41.55 %). The expression levels of On-HO-1 were highest in the liver of healthy tilapias and sharply induced by Streptococcus agalactiae or Aeromonas hydrophila. Besides, On-HO-1 overexpression significantly increased non-specific immunological parameters in serum during bacterial infection, including LZM, SOD, CAT, ACP, and AKP. It also exerted anti-inflammatory and anti-apoptotic effects in response to the immune response of the infection with S. agalactiae or A. hydrophila by upregulating anti-inflammatory factors (IL-10, TGF-ß), autophagy factors (ATG6, ATG8) and immune-related pathway factors (P65, P38), and down-regulating pro-inflammatory factors (IL-1ß, IL-6, TNF-α), apoptotic factors (Caspase3, Caspase9), pyroptosis factor (Caspase1), and inflammasome (NLRP3). These results suggested that On-HO-1 involved in immunomodulatory functions and host defense in Nile tilapia.

Clostridium butyricum Alleviates Enterotoxigenic Escherichia coli K88-Induced Oxidative Damage Through Regulating the p62-Keap1-Nrf2 Signaling Pathway and Remodeling the Cecal Microbial Community.

Clostridium butyricum (CB) can enhance antioxidant capacity and alleviate oxidative damage, but the molecular mechanism by which this occurs remains unclear. This study used enterotoxigenic Escherichia coli (ETEC) K88 as a pathogenic model, and the p62-Keap1-Nrf2 signaling pathway and intestinal microbiota as the starting point to explore the mechanism through which CB alleviates oxidative damage. After pretreatment with CB for 15 d, mice were challenged with ETEC K88 for 24 h. The results suggest that CB pretreatment can dramatically reduce crypt depth (CD) and significantly increase villus height (VH) and VH/CD in the jejunum of ETEC K88-infected mice and relieve morphological lesions of the liver and jejunum. Additionally, compared with ETEC-infected group, pretreatment with 4.4×106 CFU/mL CB can significantly reduce malondialdehyde (MDA) level and dramatically increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in the serum. This pretreatment can also greatly increase the mRNA expression levels of tight junction proteins and genes related to the p62-Keap1-Nrf2 signaling pathway in the liver and jejunum in ETEC K88-infected mice. Meanwhile, 16S rDNA amplicon sequencing revealed that Clostridium disporicum was significantly enriched after ETEC K88 challenge relative to the control group, while Lactobacillus was significantly enriched after 4.4×106 CFU/mL CB treatment. Furthermore, 4.4×106 CFU/mL CB pretreatment increased the short-chain fatty acid (SCFA) contents in the cecum of ETEC K88-infected mice. Moreover, we found that Lachnoclostridium, Roseburia, Lactobacillus, Terrisporobacter, Akkermansia, and Bacteroides are closely related to SCFA contents and oxidative indicators. Taken together, 4.4×106 CFU/mL CB pretreatment can alleviate ETEC K88-induced oxidative damage through activating the p62-Keap1-Nrf2 signaling pathway and remodeling the cecal microbiota community in mice.

HO­1 knockdown upregulates the expression of VCAM­1 to induce neutrophil recruitment during renal ischemia­reperfusion injury.

Heme oxygenase­1 (HO­1) has been reported to be upregulated following renal ischemia­reperfusion injury (IRI) and plays a key cytoprotective role; however, the underlying molecular mechanisms of its protective effects remain poorly understood. In the present study, in order to further elucidate the molecular mechanisms underlying the cytoprotective role of HO­1 in renal IRI, HO­1+/+ and HO­1+/­ mice were subjected to renal ischemia and subsequent reperfusion followed by the analysis of blood urea nitrogen (BUN) and serum creatinine (SCr) levels, the severity of histological changes, HO­1 and vascular cell adhesion molecule­1 (VCAM­1) protein expression, the mRNA expression of inflammatory factors and the effects of VCAM­1 blockade. The results of the present study demonstrated that the upregulated expression levels of VCAM­1 in HO­1+/­ mice during IRI increased the extent of renal tissue damage and activated the inflammatory response. These effects were subsequently reversed following infusion with an anti­VCAM­1 antibody. In addition, the upregulated expression of VCAM­1 in mouse glomerulus vascular endothelial cells isolated from HO­1+/­ mice increased the adhesion and migration of neutrophils, effects which were also reversed upon incubation with an anti­VCAM­1 antibody. These results indicated that HO­1 knockdown may upregulate the expression of VCAM­1 during renal IRI, resulting in increased neutrophil recruitment and the activation of the inflammatory response, thereby exacerbating renal IRI. The present study thus highlights the regulatory mechanisms of HO­1 in renal IRI and provides a potential target for the clinical treatment of IRI following renal transplantation.

Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions.

Nrf2 is a redox-sensitive transcription factor that is thought to be important in protection against intracellular pathogens. To determine the protective role of Nrf2 in the host defense against Mycobacterium avium complex (MAC), both wild-type and Nrf2-deficient mice were intranasally infected with MAC bacteria. Nrf2-deficient mice were highly susceptible to MAC bacteria compared with wild-type mice. There were no significant changes in the levels of oxidative stress and Th1 cytokine production between genotypes. Comprehensive transcriptome analysis showed that the expressions of Nramp1 and HO-1 were much lower in the infected lungs, and the expression of Nramp1 was especially lower in alveolar macrophages of Nrf2-deficient mice than of wild-type mice. Electron microscopy showed that many infected alveolar macrophages from Nrf2-deficient mice contained a large number of intracellular MAC bacteria with little formation of phagolysosomes, compared with those from wild-type mice. Treatment with sulforaphane, an activator of Nrf2, increased resistance to MAC with increased lung expression of Nramp1 and HO-1 in wild-type mice. These results indicate that Nramp1 and HO-1, regulated by Nrf2, are essential in defending against MAC infection due to the promotion of phagolysosome fusion and granuloma formation, respectively. Thus, Nrf2 is thought to be a critical determinant of host resistance to MAC infection.IMPORTANCE Nontuberculous mycobacteria (NTM) are an important cause of morbidity and mortality in pulmonary infections. Among them, Mycobacterium avium complex (MAC) is the most common cause of pulmonary NTM disease worldwide. It is thought that both environmental exposure and host susceptibility are required for the establishment of pulmonary MAC disease, because pulmonary MAC diseases are most commonly observed in slender, postmenopausal women without a clearly recognized immunodeficiency. However, host factors that regulate MAC susceptibility have not been elucidated until now. This study shows that Nrf2 is a critical regulator of host susceptibility to pulmonary MAC disease by promoting phagolysosome fusion and granuloma formation via activating Nramp1 and HO-1 genes, respectively. The Nrf2 system is activated in alveolar macrophages, the most important cells during MAC infection, as both the main reservoir of infection and bacillus-killing cells. Thus, augmentation of Nrf2 might be a useful therapeutic approach for protection against pulmonary MAC disease.

Regulation of inflammation by the antioxidant haem oxygenase 1.

Haem oxygenase 1 (HO-1), an inducible enzyme responsible for the breakdown of haem, is primarily considered an antioxidant, and has long been overlooked by immunologists. However, research over the past two decades in particular has demonstrated that HO-1 also exhibits numerous anti-inflammatory properties. These emerging immunomodulatory functions have made HO-1 an appealing target for treatment of diseases characterized by high levels of chronic inflammation. In this Review, we present an introduction to HO-1 for immunologists, including an overview of its roles in iron metabolism and antioxidant defence, and the factors which regulate its expression. We discuss the impact of HO-1 induction in specific immune cell populations and provide new insights into the immunomodulation that accompanies haem catabolism, including its relationship to immunometabolism. Furthermore, we highlight the therapeutic potential of HO-1 induction to treat chronic inflammatory and autoimmune diseases, and the issues faced when trying to translate such therapies to the clinic. Finally, we examine a number of alternative, safer strategies that are under investigation to harness the therapeutic potential of HO-1, including the use of phytochemicals, novel HO-1 inducers and carbon monoxide-based therapies.

The immune function of heme oxygenase-1 from grass carp (Ctenopharyngodon idellus) in response to bacterial infection.

Heme oxygenase (HO)-1, a rate-limiting enzyme in heme catabolism, results in the formation of equivalent amounts of biliverdin (BV), carbon monoxide (CO) and ferrous iron (Fe2+). Previous studies have revealed that HO-1 plays an important role in immune responses. However, the mechanism underlying the immune responses against bacterial infection of teleost HO-1 remains enigmatic. To decipher the mechanisms, we have cloned and characterized the HO-1 gene of grass carp (designated as GcHO-1) in this research. The results showed that the open reading frame (ORF) of GcHO-1 was 819 bp, which encoded a putative protein of 272 amino acids. The deduced amino acid sequence phylogenetically shared the highest identity with other teleosts, and contained two domains of heme-oxygenase and a single-pass transmembrane domain. The mRNA expressions of GcHO-1 in healthy grass carp have widely existed in examined tissues in the following order of spleen > head-kidney > middle head-kidney > intestines > liver > gills > heart > muscle > brain. Besides, the mRNA and protein transcription of GcHO-1 were both significantly up-regulated in the liver and head-kidney tissues after Staphylococcus aureus and Aeromonas hydrophila infection. In addition, overexpression of GcHO-1 in kidney cell line (CIK) cells of grass carp could reduce the expression of inflammatory cytokines (IL-1ß, IL-8, TNFα, CCL1 and IL-6). Herein, we demonstrate that GcHO-1 plays an anti-inflammatory role in innate immunity. Our results shed new light on the mechanisms underlying the antibacterial immunity of teleost.

ß-Hydroxyisovalerylshikonin inhibits IL-1ß-induced chondrocyte inflammation via Nrf2 and retards osteoarthritis in mice.

Osteoarthritis is a chronic degenerative disease characterized by cartilage destruction. It is the fourth most disabling disease worldwide and is currently incurable. Inflammation and extracellular matrix (ECM) degradation are considered to be substantial reasons for accelerating the progression of OA. ß-Hydroxyisoamylshikonin (ß-HIVS) is a natural naphthoquinone compound with anti-inflammatory and antioxidant activity. However, the effect of ß-HIVS on OA is still unclear. In this study, we found that ß-HIVS can down-regulate the expression of NO, PEG2, IL-6, TNF-α, COX-2, and iNOS, suggesting its anti-inflammatory effects in chondrocytes; we also found that ß-HIVS may down-regulate the expression of ADAMTS5 and MMP13 and up-regulate the expression of aggrecan and collagen II to inhibit the degradation of ECM. Mechanistically, ß-HIVS inhibited the NFκB pathway by activating the Nrf2/HO-1 axis, thereby exerting its anti-inflammatory and inhibitory effects on ECM degradation. In vivo experiments also proved the therapeutic effects of ß-HIVS on OA in mice, and Nrf2 is the target of ß-HIVS. These findings indicate that ß-HIVS may become a new drug for the treatment of OA.

Heme oxygenase-1 orchestrates the immunosuppressive program of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) contribute to the maintenance of a strong immunosuppressive environment, supporting tumor progression and resistance to treatment. To date, the mechanisms that drive acquisition of these immunosuppressive features are still poorly defined. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme that catabolizes free heme. It displays important cytoprotective, antiinflammatory, and antioxidant properties. A growing body of evidence suggests that HO-1 may also promote tumor development. Herein, we show that HO-1 is highly expressed in monocytic cells in the tumor microenvironment (TME) once they differentiate into TAMs. Deletion of HO-1 in the myeloid compartment enhances the beneficial effects of a therapeutic antitumor vaccine by restoring CD8+ T cell proliferation and cytotoxicity. We further show that induction of HO-1 plays a major role in monocyte education by tumor cells by modulating their transcriptional and epigenetic programs. These results identify HO-1 as a valuable therapeutic target to reprogram the TME and synergize with current cancer therapies to facilitate antitumor response.

Activation of PPAR-γ induces macrophage polarization and reduces neutrophil migration mediated by heme oxygenase 1.

Natural or synthetic ligands for peroxisome proliferator-activated receptor gamma (PPAR-γ) represent an interesting tool for pharmacological interventions to treat inflammatory conditions. In particular, PPAR-γ activation prevents pain and inflammation in the temporomandibular joint (TMJ) by decreasing cytokine release and stimulating the synthesis of endogenous opioids. The goal of this study was to clarify whether PPAR-γ activation induces macrophage polarization, inhibiting inflammatory cytokine release and leukocyte recruitment. In addition, we investigated the involvement of heme oxygenase 1 (HO-1) in downstream events after PPAR-γ activation. Our results demonstrate that PPAR-γ activation ablates cytokine release by Bone Marrow-Derived Macrophages (BMDM) in vitro. 15d-PGJ2 induces the PPAR-γ heterodimer activation from rat macrophages, with macrophage polarization from M1-like cells toward M2-like cells. This response is mediated through HO-1. PPAR-γ activation diminished neutrophil migration induced by carrageenan, which was also HO-1 dependent. Ca2+/calmodulin expression did not change after PPAR-γ activation indicating that is not required for the activation of the intracellular L-arginine/NO/cGMP/K+ATP channel pathway. In summary, the anti-inflammatory actions induced by PPAR-γ activation involve macrophage polarization. HO-1 expression is increased and HO-1 activity is required for the suppression of neutrophil migration.

Heme oxygenase-1 alleviates eosinophilic inflammation by inhibiting STAT3-SOCS3 signaling.

Airway inflammation of eosinophilic asthma (EA) attributes to Th2 response, leaving the role of Th17 response unknown. Signal transducer and activator of transcription 3 (STAT3) induce both suppressors of cytokine signaling 3 (SOCS3) and retinoic acid receptor-related orphan nuclear receptor γ (RORγt) to initiate Th17 cell differentiation which is inhibited by SOCS3, a negative feedback regulator of STAT3. Heme oxygenase-1 (HO-1) is a stress-responsive, cytoprotective, and immunoregulatory molecular. Two other isoforms of the enzyme includes HO-2 and HO-3. Because HO-2 does not exhibit stress-related upregulation and distributes mainly in nervous system and HO-3 shows a low enzymatic activity, we tested a hypothesized anti-inflammatory role for HO-1 in EA by inhibiting STAT3-SOCS3 signaling. Animal model was established with Ovalbumin in wild type Balb/C mice. Hemin or SNPP was intraperitoneally (IP) injected ahead of the animal model to induce or inhibit HO-1 expression. Airway inflammation was evaluated by bronchoalveolar lavage, hematoxyline and eosin staining, enzyme-linked immunosorbent assay, and Western blot analysis. In vivo results showed that HO-1 induction inhibited phosphorylation of STAT3 and expression of SOCS3 and RORγt, decreased Th2 and Th17 immune responses, and alleviated airway inflammation. In vitro results revealed that HO-1 inhibited phosphorylation of STAT3 and expression of SOCS3 in naive CD4+ T cells. These findings identify HO-1 induction as a potential therapeutic strategy for EA treatment by reducing STAT3 phosphorylation, STAT3-SOCS3-mediated Th2/Th17 immune responses, and ultimate allergic airway inflammation.

The Good and the Bad Side of Heme-Oxygenase-1 in the Gut.

Significance: Mucosal immunity in the gut has the important task of protecting an organism against potential danger, but at the same time of staying silent in response to harmless antigens present in the intestinal lumen. The delicate balance between immune activation and tolerance is referred to as gut homeostasis. Recent Advances: It has become clear that different types of immune cells and several factors participate in the maintenance of gut homeostasis, having as a final goal the prevention of non-necessary inflammation. Immune cells of the myeloid lineage, such as macrophages located in the lamina propria, represent the most abundant leukocyte population in the intestine and play a critical role in keeping the immune system silent, via the production of the anti-inflammatory cytokine interleukin-10. Critical Issues: Gut macrophages are an important source of the oxidative enzyme heme-oxygenase-1 (HO-1), which has crucial immune-modulatory properties. The protective role of HO-1 in the control of the intestinal inflammation, and its connection with the enteric flora have been demonstrated in experimental settings as well as in human biological samples. Future Directions: Loss of the gut homeostasis gives rise to conditions of acute inflammation that may degenerate into chronic disease, eventually leading to carcinogenesis. Understanding the mechanisms that regulate this enzyme will disclose novel therapeutic approaches that are designed to control chronic inflammation in the intestine.

The protective effect of Sargassum horneri against particulate matter-induced inflammation in lung tissues of an in vivo mouse asthma model.

Sargassum horneri is an edible brown seaweed with potential anti-inflammatory properties. The present study was designed to evaluate the anti-inflammatory properties of S. horneri using an in vivo mouse asthma model following exposure to particulate matter (PM). 7-8 week old BALB/c mice (20-25 g) were randomly divided into seven groups (n = 4) as follows: 1: no treatment, 2: OVA (ovalbumin) + PM, 3: OVA + PM + SHE (S. horneri ethanol extract) 200 mg kg-1, 4: OVA + PM + SHE 400 mg kg-1, 5: OVA + PM + prednisone 5 mg kg-1, 6: OVA only, and 7: PM only. All mice (except healthy controls) were sensitized on the first day by intraperitoneal injection of 10 µg OVA and 2 mg Al(OH)3 in 200 µL of saline. Starting from day 15, mice (except groups 1 and 6) were exposed to sonicated PM (5 mg m-3, 30 min day-1) through a nebulizer daily for 7 consecutive days. Mice exposed to PM and OVA showed up-regulated expression of MAPKs and pro-inflammatory cytokine production in the lungs. Furthermore, PM-exposed lungs had significantly reduced expression of Nrf2 and HO-1 genes. However, oral administration of the SHE reduced the phosphorylation levels of MAPKs, iNOS and COX2 expression levels, and mRNA expression levels of pro-inflammatory cytokines. In addition, SHE treated group mice had up-regulated anti-oxidant gene expression levels in the lungs compared to group 2. These findings demonstrate that oral administration of the SHE re-establishes PM-induced inflammation and oxidative stress in the lungs. Taken together, the SHE has therapeutic potential in preventing PM-induced inflammation and oxidative stress.

Trametes orientalis polysaccharide alleviates PM2.5-induced lung injury in mice through its antioxidant and anti-inflammatory activities.

This paper examined the molecular conformation of Trametes orientalis polysaccharide (TOP-2) and evaluated the ameliorative effects of TOP-2 on PM2.5-induced lung injury in mice. The Congo red test and transmission electron microscopy (TEM) showed that TOP-2 had a triple-helical structure. PM2.5-induced pulmonary edema was ameliorated by TOP-2 intervention. PM2.5 notably increased the number of inflammatory cells and percentages of neutrophils in bronchoalveolar lavage fluid (BALF), and notably reduced the percentages of macrophages in BALF, while TOP-2 abolished these effects. The increased levels of total protein, albumin, C-reactive protein (CRP), myeloperoxidase (MPO), lactate dehydrogenase (LDH), alkaline phosphatase (AKP), acid sphingomyelinase (ASM), TNF-α, IL-1ß and IL-6 in BALF after PM2.5 exposure were inhibited by TOP-2. In addition, TOP-2 could not only remarkably promote the activities of antioxidant enzymes, but also reduce the levels of malondialdehyde (MDA), protein carbonyl group (PCG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Furthermore, TOP-2 up-regulated the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and inhibited the activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome in the lung tissue. These results hint that TOP-2 could alleviate PM2.5-induced lung injury in mice via its antioxidant and anti-inflammatory activities, and the underlying mechanisms, at least partly, depended on activation of the Nrf2/HO-1 pathway and inhibition of NLRP3 inflammasome.

MicroRNA-155 and microRNA-181a, via HO-1, participate in regulating the immunotoxicity of cadmium in the kidneys of exposed Cyprinus carpio.

Cadmium (Cd) is a nonessential metal that is a contaminant in aquatic ecosystems. Cd can accumulate in aquatic animals, leading to detrimental effects in tissues, and Cd exposure can induce immunotoxicity in fish. MicroRNAs (miRNAs) play critical roles in immune responses, yet the participation of miRNAs in Cd-induced immunotoxicity remains poorly understood. The present study evaluated the effects of Cd exposure on the immune responses and the mRNAs and miRNAs expressions of immune-related genes in Cyprinus carpio (C. carpio). Then, microRNA-155 (miR-155) was overexpressed and microRNA-181a (miR-181a) was knocked down to determine which miRNA plays a key role in the immune response to Cd. The results showed that 0.5 mg/L Cd2+ significantly decreased the activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the kidneys of C. carpio. Cd exposure upregulated the mRNA expressions of interleukin (IL)-1ß, IL-8, nuclear factor-kappa B (NF-κB), tumour necrosis factor-α (TNF-α), and Toll-like receptor 4(TLR-4) and downregulated those of IL-10 and heme oxygenase-1 (HO-1) in C. carpio kidneys. Cd exposure also led to upregulation of miR-155 and miR-181a expressions. Furthermore, AKP and ACP activity in the kidneys was markedly changed after intraperitoneal injection of C. carpio with miR-155 agomir and miR-181a antagomir. All detected mRNA expressions were significantly decreased after injection of miR-155 agomir, and IL-10, NF-κB, TNF-α, and HO-1 mRNA expressions were markedly increased after injection of miR-181a antagomir. The results of this study demonstrate that Cd exposure can immunocompromise C. carpio by targeting HO-1 through miR-155 and miR-181a. This is the first study to reveal that Cd exposure induces immunotoxicity through miR-155 and miR-181a in the kidneys of C. carpio.

Non-extractable polyphenols from cranberries: potential anti-inflammation and anti-colon-cancer agents.

Cranberries (Vaccinium macrocarpon) are full of polyphenols, which display various health benefits. Most studies have focused on extractable polyphenols (EPs) rather than non-extractable polyphenols (NEPs) but NEPs may possess important biological functions. The objective of this work was to characterize EP and NEP fractions from whole cranberries and determine their potential as anti-inflammation and anti-colon-cancer agents. Our results showed that of the identified polyphenols, anthocyanins were the major ones in the cranberry EP fraction, while phenolic acids were most abundant in the NEP fraction. The oxygen radical absorbance capacity (ORAC) of the NEPs was significantly higher than that of the EPs. Both the EPs and NEPs showed anti-inflammatory effects in inhibiting LPS-induced production of nitric oxide in macrophages. At the concentrations tested, the NEPs showed significantly higher inhibition of the production of nitric oxide in macrophages than the EPs, which was accompanied by decreased expression of inducible nitric oxide synthase (iNOS) and increased expression of HO-1. EP and NEP samples showed anti-cancer capacities in HCT116 cells. And the NEPs showed stronger inhibitory effects on the viability and colony formation capacity of human colon cancer HCT116 cells than the EPs. In a flow cytometry analysis, the NEPs caused cell cycle arrest at the G0/G1 phase and induced significant cellular apoptosis in colon cancer cells. Overall, our results suggested that both the EP and NEP fractions from cranberries were bioactive, and importantly, the NEP fraction showed promising anti-inflammation and anti-colon-cancer potential.

Heracleum moellendorffii roots inhibit the production of pro-inflammatory mediators through the inhibition of NF-κB and MAPK signaling, and activation of ROS/Nrf2/HO-1 signaling in LPS-stimulated RAW264.7 cells.

BACKGROUND: Heracleum moellendorffii roots (HM-R) have been long treated for inflammatory diseases such as arthritis, backache and fever. However, an anti-inflammatory effect and the specific mechanism of HM-R were not yet clear. In this study, we for the first time explored the anti-inflammatory of HM-R. METHODS: The cytotoxicity of HM-R against RAW264.7 cells was evaluated using MTT assay. The inhibition of NO and PGE2 production by HM-R was evaluated using Griess reagent and Prostaglandin E2 ELISA Kit, respectively. The changes in mRNA or protein level following HM-R treatment were assessed by RT-PCR and Western blot analysis, respectively. RESULTS: HM-R dose-dependently blocked LPS-induced NO and PGE2 production. In addition, HM-R inhibited LPS-induced overexpression of iNOS, COX-2, IL-1ß and IL-6 in RAW264.7 cells. HM-R inhibited LPS-induced NF-κB signaling activation through blocking IκB-α degradation and p65 nuclear accumulation. Furthermore, HM-R inhibited MAPK signaling activation by attenuating the phosphorylation of ERK1/2, p38 and JNK. HM-R increased nuclear accumulation of Nrf2 and HO-1 expression. However, NAC reduced the increased nuclear accumulation of Nrf2 and HO-1 expression by HM-R. In HPLC analysis, falcarinol was detected from HM-R as an anti-inflammatory compound. CONCLUSIONS: These results indicate that HM-R may exert anti-inflammatory activity by inhibiting NF-κB and MAPK signaling, and activating ROS/Nrf2/HO-1 signaling. These findings suggest that HM-R has a potential as a natural material for the development of anti-inflammatory drugs.

Antcamphin M Inhibits TLR4-Mediated Inflammatory Responses by Upregulating the Nrf2/HO-1 Pathway and Suppressing the NLRP3 Inflammasome Pathway in Macrophages.

The medicinal mushroom Antrodia cinnamomea has been demonstrated to have anti-inflammatory properties. However, the bioactive compounds in A. cinnamomea need further investigation. The present study aimed to understand the mechanism of action of antcamphin M, an ergostanoid isolated from A. cinnamomea mycelium and to clarify its underlying mechanisms of action. RAW264.7 cells were pretreated with the indicated concentrations of antcamphin M, prior to stimulation with lipopolysaccharide (LPS). Cell viability, production of nitric oxide (NO), prostaglandin E2 (PGE2), cytokines, and chemokines, as well as the inflammation-related signaling pathways were investigated. The study revealed that antcamphin M significantly decreased the LPS-induced production of NO, PGE2, pro-inflammatory cytokines, and keratinocyte chemoattractant CXCL1 (KC), along with the levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins without significant cytotoxicity, indicating it had a better anti-inflammatory activity than that of gisenoside Rb1 and Rg1. Additionally, antcamphin M significantly inhibited the activation of MAPKs (p38, ERK, and JNK), NFκB, and components of the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) signaling pathways and also increased the levels of nuclear factor erythroid-2-related factor (Nrf2) and heme oxygenase-1 (HO-1). These findings suggest that antcamphin M possesses potent anti-inflammatory activities and could be a potential candidate for the development of anti-inflammatory drugs.

p-Coumaric-Acid-Containing Adenostemma lavenia Ameliorates Acute Lung Injury by Activating AMPK/Nrf2/HO-1 Signaling and Improving the Anti-oxidant Response.

Adenostemma lavenia is a perennial herb belonging to the Compositae family and is widely distributed in the tropical parts of Asia. It has been widely used as medicine in Taiwan with the whole plant used to treat pulmonary congestion, pneumonia, bacterial infections of the respiratory tract, edema, and inflammation. This study sought to investigate the anti-inflammatory effects of A. lavenia in vitro and in animal models. The anti-inflammatory effects of ethyl acetate fractions of A. lavenia (EAAL) were stimulated with lipopolysaccharide (LPS) murine macrophages (RAW 264.7) and lung injury in mice. EAAL reduced proinflammatory cytokine responses. Preoral EAAL alleviated LPS-induced histological alterations in lung tissue and inhibited the infiltration of inflammatory cells and protein concentrations in bronchoalveolar lavage fluid (BALF). EAAL prevented protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2); phosphorylation of IκB-α, MAPKs, and AMP-activated protein kinase (AMPK); and activated anti-oxidant enzymes (catalase, SOD, and GPx), heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) in LPS-stimulated cells and lung tissues. Fingerprinting of EAAL was performed with HPLC to control its quality, and p-coumaric acid was found to be a major constituent. This study suggests that EAAL is a potential therapeutic agent to treat inflammatory disorders.

Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages.

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHinT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1ß induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.