Garlic-derived compounds: Epigenetic modulators and their antitumor effects.

Cancer is a highly heterogeneous disease that poses a serious threat to human health worldwide. Despite significant advances in the diagnosis and treatment of cancer, the prognosis and survival rate of cancer remain poor due to late diagnosis, drug resistance, and adverse reactions. Therefore, it is very necessary to study the development mechanism of cancer and formulate effective therapeutic interventions. As widely available bioactive substances, natural products have shown obvious anticancer potential, especially by targeting abnormal epigenetic changes. The main active part of garlic is organic sulfur compounds, of which diallyl trisulfide (DATS) content is the highest, accounting for more than 40% of the total composition. The garlic-derived compounds have been recognized as an antioxidant for cancer prevention and treatment. However, the molecular mechanism of the antitumor effect of garlic-derived compounds remains unclear. Recent studies have identified garlic-derived compound DATS that plays critical roles in enhancing CpG demethylation or promoting histone acetylation as an epigenetic inhibitor. Here, we review the therapeutic progress of garlic-derived compounds against cancer through epigenetic pathways.

Single injection by LC-ESI-MS/MS for simultaneous determination of organophosphate tri- and di-esters in plant tissue based on ultrasonic-assisted sequential extraction and single-step purification.

A novel methodology based on ultrasonic-assisted sequential extraction, dispersive-SPE purification, and single-injection on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is proposed, for the first time, to simultaneously measure 14 tri-OPEs and 9 di-OPEs in plant tissues. The samples were successively ultrasonicated with a mixture of hexane:dichloromethane (1:1, v/v) and 8% acetic acid in acetonitrile for extracting tri- and di-OPEs purified with graphitized carbon black and quantitated on LC-MS/MS at the same time. The recoveries of targeted tri- and di-OPEs in the matrix spike ranged from 66% to 120% and 71% to 110% respectively. The proposed method was validated by processing eight types of common vegetables including spinach (Spinacia oleracea L.), lettuce (Lactuca sativa), carrot (Daucus carota var. sativa Hoffm.), sweet potato (Solanum tuberosum L.), cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), green beans (Phaseolus vulgaris), and cowpeas (Vigna unguiculata), with the recoveries of surrogates ranging from 84% to 98%.

Quantitative analysis and hepatoprotective mechanism of Cistanche deserticola Y. C. Ma against alcohol-induced liver injury in mice.

Cistanche deserticola Y. C. Ma (CD), known as "desert ginseng", has been found to have hepatoprotective effect. This research aimed to investigate the quality control and its alleviating effect on alcoholic liver injury in mice. In this study, for the first time, a sensitive and efficient ultra-high-performance liquid chromatography with quadrupole ion-trap mass spectrometry (UPLC-Q-TRAP/MS) method was developed to rapidly characterize nine representative phenylethanoid glycosides (PhGs) in the CD extract within 14 min, offering a reference for the quality control standard of this plant. In addition, we found that the CD extract significantly inhibited the weight loss, decreased the liver index, and attenuated excessive lipid deposition, inflammatory and oxidative stress in the mice liver. With the help of the high-throughput lipidomics technique, we discovered that CD markedly reversed 17 lipid metabolites and their involved linoleic acid, arachidonic acid and glycerophospholipid metabolic pathways. As these metabolites are mainly associated with lipid metabolism and liver damage, we further used molecular biological tests to found that CD could regulate the upstream genes and proteins of the lipid metabolism pathway, including adenosine 5'-monophosphate-activated protein kinase (AMPK), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and peroxidase proliferators activate receptors α (PPARα). In conclusion, this study elucidates the modulatory effects of CD on lipid metabolism disorders in alcoholic fatty liver from holistic system and provides a reference for further research and development of CD as a therapeutic agent.

Impacts of Fructose on Intestinal Barrier Function, Inflammation and Microbiota in a Piglet Model.

The metabolic disorder caused by excessive fructose intake was reported extensively and often accompanied by intestinal barrier dysfunction. And the rising dietary fructose was consumed at an early age of human. However, related researches were almost conducted in rodent models, while in the anatomy and physiology of gastrointestinal tract, pig is more similar to human beings than rodents. Hence, weaned piglets were chosen as the model animals in our study to investigate the fructose's impacts on intestinal tight junction, inflammation response and microbiota structure of piglets. Herein, growth performance, inflammatory response, oxidation resistance and ileal and colonic microbiota of piglet were detected after 35-day fructose supplementation. Our results showed decreased tight junction gene expressions in piglets after fructose addition, with no obvious changes in the growth performance, antioxidant resistance and inflammatory response. Moreover, fructose supplementation differently modified the microbiota structures in ileum and colon. In ileum, the proportions of Streptococcus and Faecalibacterium were higher in Fru group (fructose supplementation). In colon, the proportions of Blautia and Clostridium sensu stricto 1 were higher in Fru group. All the results suggested that tight junction dysfunction might be an earlier fructose-induced event than inflammatory response and oxidant stress and that altered microbes in ileum and colon might be the potential candidates to alleviate fructose-induced intestinal permeability alteration.

Endogenous Regulation and Pharmacological Modulation of Sepsis-Induced HMGB1 Release and Action: An Updated Review.

Sepsis remains a common cause of death in intensive care units, accounting for approximately 20% of total deaths worldwide. Its pathogenesis is partly attributable to dysregulated inflammatory responses to bacterial endotoxins (such as lipopolysaccharide, LPS), which stimulate innate immune cells to sequentially release early cytokines (such as tumor necrosis factor (TNF) and interferons (IFNs)) and late mediators (such as high-mobility group box 1, HMGB1). Despite difficulties in translating mechanistic insights into effective therapies, an improved understanding of the complex mechanisms underlying the pathogenesis of sepsis is still urgently needed. Here, we review recent progress in elucidating the intricate mechanisms underlying the regulation of HMGB1 release and action, and propose a few potential therapeutic candidates for future clinical investigations.

Dietary Betaine Addition Promotes Hepatic Cholesterol Synthesis, Bile Acid Conversion, and Export in Rats.

It is widely reported how betaine addition regulates lipid metabolism but how betaine affects cholesterol metabolism is still unknown. This study aimed to investigate the role of betaine in hepatic cholesterol metabolism of Sprague-Dawley rats. Rats were randomly allocated to four groups and fed with a basal diet or a high-fat diet with or without 1% betaine. The experiment lasted 28 days. The results showed that dietary betaine supplementation reduced the feed intake of rats with final weight unchanged. Serum low-density-lipoprotein cholesterol was increased with the high-fat diet. The high-fat diet promoted cholesterol synthesis and excretion by enhancing the HMG-CoA reductase and ABCG5/G8, respectively, which lead to a balance of hepatic cholesterol. Rats in betaine groups showed a higher level of hepatic total cholesterol. Dietary betaine addition enhanced cholesterol synthesis as well as conversion of bile acid from cholesterol by increasing the levels of HMGCR and CYP7A1. The high-fat diet decreased the level of bile salt export pump, while dietary betaine addition inhibited this decrease and promoted bile acid efflux and increased total bile acid levels in the intestine. In summary, dietary betaine addition promoted hepatic cholesterol metabolism, including cholesterol synthesis, conversion of bile acids, and bile acid export.

Melatonin protects against maternal obesity-associated oxidative stress and meiotic defects in oocytes via the SIRT3-SOD2-dependent pathway.

Maternal obesity in humans is associated with poor outcomes across the reproductive spectrum. Emerging evidence indicates that these defects are likely attributed to factors within the oocyte. Although various molecules and pathways may contribute to impaired oocyte quality, prevention of fertility issues associated with maternal obesity is a challenge. Using mice fed a high-fat diet (HFD) as an obesity model, we document spindle disorganization, chromosome misalignment, and elevated reactive oxygen species (ROS) levels in oocytes from obese mice. Oral administration of melatonin to HFD mice not only reduces ROS generation, but also prevents spindle/chromosome anomalies in oocytes, consequently promoting the developmental potential of early embryos. Consistent with this finding, we find that melatonin supplement during in vitro maturation also markedly attenuates oxidative stress and meiotic defects in HFD oocytes. Finally, by performing morpholino knockdown and acetylation-mimetic mutant overexpression assays, we reveal that melatonin ameliorates maternal obesity-induced defective phenotypes in oocytes through the SIRT3-SOD2-dependent mechanism. In sum, our data uncover the marked beneficial effects of melatonin on oocyte quality from obese females; this opens a new area for optimizing culture system as well as fertility management.

Involvement of PKCα and ERK1/2 signaling pathways in EGCG's protection against stress-induced neural injuries in Wistar rats.

Stress-induced neural injuries are closely linked to the pathogenesis of various neuropsychiatric disorders and psychosomatic diseases. We and others have previously demonstrated certain protective effects of epigallocatechin-3-gallate (EGCG) in stress-induced cerebral impairments, but the underlying protective mechanisms still remain poorly elucidated. Here we provide evidence to support the possible involvement of PKCα and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways in EGCG-mediated protection against restraint stress-induced neural injuries in rats. In both open-field and step-through behavioral tests, the restraint stress-induced neuronal impairments were significantly ameliorated by administration of EGCG or green tea polyphenols (GTPs), which was associated with a partial restoration of normal plasma glucocorticoid, dopamine and serotonin levels. Furthermore, the stress-induced decrease of PKCα and ERK1/2 expression and phosphorylation was significantly attenuated by EGCG and to a less extent by GTP administration. Additionally, EGCG supplementation restored the production of adenosine triphosphate (ATP) and the expression of a key regulator of cellular energy metabolism, the peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α), in stressed animals. In conclusion, PKCα and ERK1/2 signaling pathways as well as PGC-1α-mediated ATP production might be involved in EGCG-mediated protection against stress-induced neural injuries.

A novel recombinant 6Aß15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-ß (Aß) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aß15-THc-C immunogen was formulated with alum adjuvant as a novel Aß B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aß-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aß or oligomeric forms of Aß, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aß levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aß-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine.

Ascorbic Acid Attenuates Hyperoxia-Compromised Host Defense against Pulmonary Bacterial Infection.

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial infections, causing ventilator-associated pneumonia. The phagocytic activity of macrophages is impaired by hyperoxia-induced increases in the levels of reactive oxygen species (ROS) and extracellular high-mobility group box protein B1 (HMGB1). Ascorbic acid (AA), an essential nutrient and antioxidant, has been shown to be beneficial in various animal models of ROS-mediated diseases. The aim of this study was to determine whether AA could attenuate hyperoxia-compromised host defense and improve macrophage functions against bacterial infections. C57BL/6 male mice were exposed to hyperoxia (≥98% O2, 48 h), followed by intratracheal inoculation with Pseudomonas aeruginosa, and simultaneous intraperitoneal administration of AA. AA (50 mg/kg) significantly improved bacterial clearance in the lungs and airways, and significantly reduced HMGB1 accumulation in the airways. The incubation of RAW 264.7 cells (a macrophage-like cell line) with AA (0-1,000 µM) before hyperoxic exposure (95% O2) stabilized the phagocytic activity of macrophages in a concentration-dependent manner. The AA-enhanced macrophage function was associated with significantly decreased production of intracellular ROS and accumulation of extracellular HMGB1. These data suggest that AA supplementation can prevent or attenuate the development of ventilator-associated pneumonia in patients receiving oxygen support.

Plumbagin Protects Mice from Lethal Sepsis by Modulating Immunometabolism Upstream of PKM2.

Sepsis is characterized by dysregulated systemic inflammation with release of early (for example, interleukin (IL)-1ß) and late (for example, HMGB1) proinflammatory mediators from macrophages. Plumbagin, a medicinal plant-derived naphthoquinone, has been reported to exhibit antiinflammatory activity, but the underling mechanisms remain unclear. Here, we have demonstrated that plumbagin inhibits the inflammatory response through interfering with the immunometabolism pathway in activated macrophages. Remarkably, plumbagin inhibited lipopolysaccharide (LPS)-induced aerobic glycolysis by downregulating the expression of pyruvate kinase M2 (PKM2), a protein kinase responsible for the final and rate-limiting reaction step of the glycolytic pathway. Moreover, the NADPH oxidase 4 (NOX4)-mediated oxidative stress was required for LPS-induced PKM2 expression, because pharmacologic or genetic inhibition of NOX4 by plumbagin or RNA interference limited LPS-induced PKM2 expression, lactate production and subsequent proinflammatory cytokine (IL-1ß and HMGB1) release in macrophages. Finally, plumbagin protected mice from lethal endotoxemia and polymicrobial sepsis induced by cecal ligation and puncture. These findings identify a new approach for inhibiting the NOX4/PKM2-dependent immunometabolism pathway in the treatment of sepsis and inflammatory diseases.

EGCG induces G-CSF expression and neutrophilia in experimental sepsis.

A major green tea component, epigallocatechin-3-gallate (EGCG), has been proven protective against lethal sepsis in experimental setting, but its protective mechanisms remain incompletely understood. Here, we provide evidence to support EGCG's capacities in stimulating G-CSF production and neutrophilia in vivo. In an animal model of sepsis, EGCG significantly elevated peritoneal levels of G-CSF and several chemokines (e.g., MCP-1/CCL2 and MIP-1γ/CCL9), and consequently increased peritoneal neutrophil numbers (neutrophilia) at a late stage. In vitro, EGCG divergently affected HMGB1-mediated production of several chemokines: reducing CXCL15 and RANTES/CCL5, but elevating G-CSF and MIP-1α/CCL3 production by peritoneal macrophages. Similarly, it significantly induced the expression and secretion of G-CSF and MIP-1α/CCL3 in human peripheral blood mononuclear cells. Based on our preliminary data, it may be important to search for anti-inflammatory and G-CSF-stimulating agents for the clinical management of inflammatory diseases.

An ongoing search for potential targets and therapies for lethal sepsis.

Sepsis, which refers to a systemic inflammatory response syndrome resulting from a microbial infection, represents the leading cause of death in intensive care units. The pathogenesis of sepsis remains poorly understood although it is attributable to dysregulated immune responses orchestrated by innate immune cells that are sequentially released early (e.g., tumor necrosis factor(TNF), interleukin-1(IL-1), and interferon-γ(IFN-γ)) and late (e.g., high mobility group box 1(HMGB1)) pro-inflammatory mediators. As a ubiquitous nuclear protein, HMGB1 can be passively released from pathologically damaged cells, thereby converging infection and injury on commonly dysregulated inflammatory responses. We review evidence that supports extracellular HMGB1 as a late mediator of inflammatory diseases and discuss the potential of several Chinese herbal components as HMGB1-targeting therapies. We propose that it is important to develop strategies for specifically attenuating injury-elicited inflammatory responses without compromising the infection-mediated innate immunity for the clinical management of sepsis and other inflammatory diseases.

Novel Mechanisms of Herbal Therapies for Inhibiting HMGB1 Secretion or Action.

High mobility group box 1 (HMGB1) is an evolutionarily conserved protein and is constitutively expressed in virtually all types of cells. In response to microbial infections, HMGB1 is secreted from activated immune cells to orchestrate rigorous inflammatory responses. Here we review the distinct mechanisms by which several herbal components inhibit HMGB1 action or secretion, such as by modulating inflammasome activation, autophagic degradation, or endocytic uptake. In light of the reciprocal interactions between these cellular processes, it is possible to develop more effective combinational herbal therapies for the clinical management of inflammatory diseases.

Targeting HMGB1 in the treatment of sepsis.

INTRODUCTION: Sepsis refers to the host's deleterious and non-resolving systemic inflammatory response to microbial infections and represents the leading cause of death in the intensive care unit. The pathogenesis of sepsis is complex, but partly mediated by a newly identified alarmin molecule, the high mobility group box 1 (HMGB1). AREAS COVERED: Here we review the evidence that support extracellular HMGB1 as a late mediator of experimental sepsis with a wider therapeutic window and discuss the therapeutic potential of HMGB1-neutralizing antibodies and small molecule inhibitors (herbal components) in experimental sepsis. EXPERT OPINION: It will be important to evaluate the efficacy of HMGB1-targeting strategies for the clinical management of human sepsis in the future.

It Is Not Just Folklore: The Aqueous Extract of Mung Bean Coat Is Protective against Sepsis.

Mung bean (Vigna Radiata) has been traditionally used in China both as nutritional food and herbal medicine against a number of inflammatory conditions since the 1050s. A nucleosomal protein, HMGB1, has recently been established as a late mediator of lethal systemic inflammation with a relatively wider therapeutic window for pharmacological interventions. Here we explored the HMGB1-inhibiting capacity and therapeutic potential of mung bean coat (MBC) extract in vitro and in vivo. We found that MBC extract dose-dependently attenuated LPS-induced release of HMGB1 and several chemokines in macrophage cultures. Oral administration of MBC extract significantly increased animal survival rates from 29.4% (in saline group, N = 17 mice) to 70% (in experimental MBC extract group, N = 17 mice, P < 0.05). In vitro, MBC extract stimulated HMGB1 protein aggregation and facilitated both the formation of microtubule-associatedprotein-1-light-chain-3-(LC3-)containing cytoplasmic vesicles, and the production of LC3-II in macrophage cultures. Consequently, MBC extract treatment led to reduction of cellular HMGB1 levels in macrophage cultures, which was impaired by coaddition of two autophagy inhibitors (bafilomycin A1 and 3-methyladenine). Conclusion. MBC extract is protective against lethal sepsis possibly by stimulating autophagic HMGB1 degradation.

Novel role of PKR in inflammasome activation and HMGB1 release.

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1ß, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1ß, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.

Use of animal model of sepsis to evaluate novel herbal therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP). CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis. Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-γ) and late proinflammatory mediators (e.g., HMGB1). Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

High mobility group box 1 protein as a potential drug target for infection- and injury-elicited inflammation.

In response to infection or injury, a ubiquitous nucleosomal protein, HMGB1 is secreted actively by innate immune cells, and / or released passively by injured/damaged cells. Subsequently, extracellular HMGB1 alerts, recruits, and activates various innate immune cells to sustain a rigorous inflammatory response. A growing number of HMGB1 inhibitors ranging from neutralizing antibodies, endogenous hormones, to medicinal herb-derived small molecule HMGB1 inhibitors (such as nicotine, glycyrrhizin, tanshinones, and EGCG) are proven protective against lethal infection and ischemic injury. Here we review emerging evidence that support extracellular HMGB1 as a proinflammatory alarmin(g) danger signal, and discuss a wide array of HMGB1 inhibitors as potential therapeutic agents for sepsis and ischemic injury.

Caging a Beast in the Inflammation Arena: Use of Chinese Medicinal Herbs to Inhibit a Late Mediator of Lethal Sepsis, HMGB1.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, which kills > 225,000 people annually in the U.S. alone. The high mortality of sepsis is partly mediated by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF) and late (e.g., HMGB1) pro-inflammatory cytokines. Although early proinflammatory cytokines may be protective against infection, excessive accumulation of late-acting proinflammatory mediators (such as HMGB1) may sustain a potentially injurious inflammatory response. Agents capable of inhibiting HMGB1 activities (e.g., neutralizing antibodies) or release [e.g., Chinese herbs, Danggui (Angelica sinensis), Danshen (Salvia miltiorrhiza) and Green tea (Camellia sinensis)] rescue mice from lethal sepsis even when given 24 hours after onset of the disease. Here we review emerging evidence that support a critical role for extracellular HMGB1 as a late mediator of lethal sepsis, and several commonly used Chinese herbs (Danggui, Danshen and Green tea) as potential HMGB1- targeting therapeutic agents in experimental sepsis.