ERK Inhibition Increases RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells by Stimulating AMPK Activation and RANK Expression and Inhibiting Anti-Osteoclastogenic Factor Expression

Bone absorption is necessary for the maintenance of bone homeostasis. An osteoclast (OC) is a monocyte–macrophage lineage cell that absorbs bone tissue. Extracellular signal-regulated kinases (ERKs) are known to play important roles in regulating OC growth and differentiation. In this study, we examined specific downstream signal pathways affected by ERK inhibition during OC differentiation. Our results showed that the ERK inhibitors PD98059 and U0126 increased receptor activator of NF-κB ligand (RANKL)-induced OC differentiation in RAW 264.7 cells, implying a negative role in OC differentiation. This is supported by the effect of ERK2-specific small interfering RNA on increasing OC differentiation. In contrast to our findings regarding the RAW 264.7 cells, the ERK inhibitors attenuated the differentiation of bone marrow-derived cells into OCs. The ERK inhibitors significantly increased the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) but not the activation of p38 MAPK, Lyn, and mTOR. In addition, while the ERK inhibition increased the expression of the RANKL receptor RANK, it decreased the expression of negative mediators of OC differentiation, such as interferon regulatory factor-8, B-cell lymphoma 6, and interferon-γ. These dichotomous effects of ERK inhibition suggest that while ERKs may play positive roles in bone marrow-derived cells, ERKs may also play negative regulatory roles in RAW 264.7 cells. These data provide important information for drug development utilizing ERK inhibitors in OC-related disease treatment.     

Benzoylpaeoniflorin Activates Anti-Inflammatory Mechanisms to Mitigate Sepsis in Cell-Culture and Mouse Sepsis Models

Xuebijing injection (XBJI) (comprising of five herbs) is a widely used traditional Chinese medicine for sepsis treatment. However, the bioactive components of XBJI and the mechanisms responsible for its sepsis-mitigating action have not been experimentally determined. One of the main bioactive compounds in XBJI—benzoylpaeoniflorin (BPF)—inhibits the expressions of key mediators of inflammation such as nuclear factor kappa B (NF-κB), cyclooxygenase-1 (COX-1), and COX-2. However, its effects on sepsis have not been determined yet. Therefore, here, we investigated the immunomodulatory effect of BPF on severely inflamed endothelial cells, THP-1 macrophages, peritoneal macrophages, and mice. Human umbilical vein endothelial cells (HUVECs) and THP-1-macrophages were activated using lipopolysaccharide (LPS) after pretreatment with BPF. Subsequently, changes in the expression profiles of pro-inflammatory molecules including inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were determined using quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis. Furthermore, we monitored the phosphorylation of NF-kB and mitogen-activated protein kinases (MAPKs) to determine their activation levels. Using the LPS-induced mouse model of sepsis, we studied the effects of BPF on inflammatory cytokine production, pulmonary histopathology, and survival rates. Finally, we evaluated whether BPF protects against cecal ligation and puncture (CLP)-induced sepsis, as it closely mimics human sepsis. BPF pretreatment inhibited LPS-induced increase in mRNA and protein levels of iNOS, TNF-α, and IL-6 in HUVECs and THP-1-macrophages. It also suppressed LPS-mediated phosphorylation of p65, p38, JNK, and ERK. Mice with LPS-induced-sepsis who were treated with BPF had lower serum levels of IL-6, TNF-α, IL-1β, CXCL1, and CXCL2 than the control mice treated with BPF. Histopathology revealed that BPF treatment alleviated LPS-induced lung damage. In addition, in mice given a lethal dose of LPS, BPF treatment showed a dose-dependent improvement in survival rates. BPF treatment dose-dependently inhibited the LPS-induced IL-6, TNF-α, and CXCL1 production in peritoneal macrophages. BPF treatment also dose-dependently improved the survival rates in mice with CLP-induced sepsis. These results show that BPF alleviates LPS-stimulated septic conditions and protects mice from CLP-induced sepsis. Our research marks BPF as a potential drug in the treatment of sepsis and various inflammatory diseases.     

The Neuroprotective Effects of Exosomes Derived from TSG101-Overexpressing Human Neural Stem Cells in a Stroke Model

Although tissue-type plasminogen activator was approved by the FDA for early reperfusion of occluded vessels, there is a need for an effective neuroprotective drug for stroke patients. In this study, we established tumor susceptibility gene (TSG)101-overexpressing human neural stem cells (F3.TSG) and investigated whether they showed enhanced secretion of exosomes and whether treatment with exosomes during reperfusion alleviated ischemia-reperfusion-mediated brain damage. F3.TSG cells secreted higher amounts of exosomes than the parental F3 cells. In N2A cells subjected to oxygen–glucose deprivation (OGD), treatment with exosomes or coculture with F3.TSG cells significantly attenuated lactate dehydrogenase release, the mRNA expression of proinflammatory factors, and the protein expression of DNA-damage-related proteins. In a middle cerebral artery occlusion (MCAO) rat model, treatment with exosomes, F3 cells, or F3.TSG cells after 2 h of occlusion followed by reperfusion reduced the infarction volume and suppressed inflammatory cytokines, DNA-damage-related proteins, and glial fibrillary acidic protein, and upregulated several neurotrophic factors. Thus, TSG101-overexpressing neural stem cells showed enhanced exosome secretion; exosome treatment protected against MCAO-induced brain damage via anti-inflammatory activities, DNA damage pathway inhibition, and growth/trophic factor induction. Therefore, exosomes and F3.TSG cells can affect neuroprotection and functional recovery in acute stroke patients.     

Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.     

Factors and Pathways Modulating Endothelial Cell Senescence in Vascular Aging

Aging causes a progressive decline in the structure and function of organs. With advancing age, an accumulation of senescent endothelial cells (ECs) contributes to the risk of developing vascular dysfunction and cardiovascular diseases, including hypertension, diabetes, atherosclerosis, and neurodegeneration. Senescent ECs undergo phenotypic changes that alter the pattern of expressed proteins, as well as their morphologies and functions, and have been linked to vascular impairments, such as aortic stiffness, enhanced inflammation, and dysregulated vascular tone. Numerous molecules and pathways, including sirtuins, Klotho, RAAS, IGFBP, NRF2, and mTOR, have been implicated in promoting EC senescence. This review summarizes the molecular players and signaling pathways driving EC senescence and identifies targets with possible therapeutic value in age-related vascular diseases.     

Trifuhalol A Suppresses Allergic Inflammation through Dual Inhibition of TAK1 and MK2 Mediated by IgE and IL-33

The activation and degranulation of immune cells play a pivotal role in allergic inflammation, a pathological condition that includes anaphylaxis, pruritus, and allergic march-related diseases. In this study, trifuhalol A, a phlorotannin isolated from Agarum cribrosum, inhibited the degranulation of immune cells and the biosynthesis of IL-33 and IgE in differentiated B cells and keratinocytes, respectively. Additionally, trifuhalol A suppressed the IL-33 and IgE-mediated activation of RBL-2H3 cells through the regulation of the TAK1 and MK2 pathways. Hence, the effect of trifuhalol A on allergic inflammation was evaluated using a Compound 48/80-induced systemic anaphylaxis mouse model and a house dust mite (HDM)-induced atopic dermatitis (AD) mouse model. Trifuhalol A alleviated anaphylactic death and pruritus, which appeared as an early-phase reaction to allergic inflammation in the Compound 48/80-induced systemic anaphylaxis model. In addition, trifuhalol A improved symptoms such as itching, edema, erythema, and hyperkeratinization in HDM-induced AD mice as a late-phase reaction. Moreover, the expression of IL-33 and thymic stromal lymphopoietin, inflammatory cytokines secreted from activated keratinocytes, was significantly reduced by trifuhalol A administration, resulting in the reduced infiltration of immune cells into the skin and a reduction in the blood levels of IgE and IL-4. In summarizing the above results, these results confirm that trifuhalol A is a potential therapeutic candidate for the regulation of allergic inflammation.     

Chronic Alcohol Exposure Promotes Cancer Stemness and Glycolysis in Oral/Oropharyngeal Squamous Cell Carcinoma Cell Lines by Activating NFAT Signaling

Alcohol consumption is associated with an increased risk of several cancers, including oral/oropharyngeal squamous cell carcinoma (OSCC). Alcohol also enhances the progression and aggressiveness of existing cancers; however, its underlying molecular mechanism remains elusive. Especially, the local carcinogenic effects of alcohol on OSCC in closest contact with ingestion of alcohol are poorly understood. We demonstrated that chronic ethanol exposure to OSCC increased cancer stem cell (CSC) populations and their stemness features, including self-renewal capacity, expression of stem cell markers, ALDH activity, and migration ability. The ethanol exposure also led to a significant increase in aerobic glycolysis. Moreover, increased aerobic glycolytic activity was required to support the stemness phenotype of ethanol-exposed OSCC, suggesting a molecular coupling between cancer stemness and metabolic reprogramming. We further demonstrated that chronic ethanol exposure activated NFAT (nuclear factor of activated T cells) signaling in OSCC. Functional studies revealed that pharmacological and genetic inhibition of NFAT suppressed CSC phenotype and aerobic glycolysis in ethanol-exposed OSCC. Collectively, chronic ethanol exposure promotes cancer stemness and aerobic glycolysis via activation of NFAT signaling. Our study provides a novel insight into the roles of cancer stemness and metabolic reprogramming in the molecular mechanism of alcohol-mediated carcinogenesis.