Mitigation of Hematopoietic Syndrome of Acute Radiation Syndrome by 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) is Associated with Regulation of Systemic Inflammation in a Murine Model of Total-Body Irradiation.

The growing risk of accidental radiation exposure due to increased usage of ionizing radiation, such as in nuclear power, industries and medicine, has increased the necessity for the development of radiation countermeasures. Previously, we demonstrated the therapeutic potential of the acetylated diacylglycerol, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), as a radiation countermeasure by mitigating radiation-associated mortality and hematopoietic acute radiation syndrome (H-ARS) in BALB/c mice after a lethal dose (LD70/30) of gamma-ray total-body irradiation (TBI). In this study, we show that PLAG mitigates symptoms of H-ARS, as characterized by mature blood cell recovery and restoration of bone marrow cellularity, by regulating systemic inflammation. Log-rank test demonstrated that high levels of WBCs, lymphocytes and neutrophils on day 10 post-TBI resulted in significantly improved survival rate. PLAG significantly enhanced the nadir values of all major blood cell types as well as bone marrow cellularity. A single TBI at LD70/30 induced an immediate increase in the blood levels of CXCL1 (12.5 fold), CXCL2 (1.5 fold), IL-6 (86.9 fold), C-reactive protein (CRP; 1.3 fold) and G-CSF (15.7 fold) at 6 h post-TBI, but the cytokine levels returned to baseline level afterward. When the irradiated mice started to die around 15 days post-TBI, they exhibited a second surge in blood levels of CXCL1 (49.3 fold), CXCL2 (87.1 fold), IL-6 (208 fold), CRP (3.6 fold) and G-CSF (265.7 fold). However, PLAG-treated groups showed a significant decrease in these same blood levels (P < 0.001). Considering the inverse correlation between inflammatory cytokine levels and hematological nadirs, PLAG exerts its therapeutic effects on H-ARS by regulating inflammatory cytokine production. These data suggest that PLAG has high potential as a radiation countermeasure to mitigate H-ARS after accidental exposure to radiation.

1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice.

Acute gouty arthritis is an auto-inflammatory disease caused by the deposition of monosodium urate (MSU) crystals in joints or tissues. Excessive neutrophil recruitment into gouty lesions is a general clinical sign and induces a pain phenotype. Attenuation of successive periods of neutrophil infiltration might be a beneficial approach to achieve therapeutic efficacy. In this study, the activity of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) in attenuation of excess neutrophil infiltration was assessed in gout-induced lesions of BALB/c mice. Neutrophil infiltration in MSU-induced gouty lesions was analyzed using immunohistochemical staining. ELISA and RT-PCR were used to measure attenuation of expression of the major neutrophil chemoattractant, CXC motif chemokine ligand 8 (CXCL8), in a PLAG-treated animal model and in cells in vitro. The animal model revealed massive increased neutrophil infiltration in the MSU-induced gouty lesions, but the PLAG-treated mice had significantly reduced neutrophil numbers in these lesions. The results also indicated that the MSU crystals stimulated a damage-associated molecular pattern that was recognized by the P2Y6 purinergic receptor. This MSU-stimulated P2Y6 receptor was destined to intracellular trafficking. During intracellular endosomal trafficking of the receptor, endosome-dependent signaling provided expression of CXCL8 chemokines for neutrophil recruitment. PLAG accelerated initiation of the intracellular trafficking of the P2Y6 receptor and returning the receptor to the membrane. This process shortened the intracellular retention time of the receptor anchoring endosome and subsequently attenuated endosome-dependent signaling for CXCL8 expression. These study results suggested that PLAG could be used for resolution of acute inflammation induced in gout lesions.

Mitigating Effect of 1-Palmitoyl-2-Linoleoyl-3-Acetyl-Rac-Glycerol (PLAG) on a Murine Model of 5-Fluorouracil-Induced Hematological Toxicity.

5-Fluorouracil (5-FU) is an antimetabolite chemotherapy widely used for the treatment of various cancers. However, many cancer patients experience hematological side effects following 5-FU treatment. Here, we investigated the protective effects of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) as a mitigator against 5-FU-induced hematologic toxicity, including neutropenia, monocytopenia, thrombocytopenia, and thrombocytosis, in Balb/c mice injected with 5-FU (100 mg/kg, i.p.). Administration of PLAG significantly and dose-dependently reduced the duration of neutropenia and improved the nadirs of absolute neutrophil counts (ANCs). Moreover, while the ANCs of all mice in the control fell to the severely neutropenic range, none of the mice in the PLAG 200 and 400 mg/kg-treated groups experienced severe neutropenia. Administration of PLAG significantly delayed the mean first day of monocytopenia and reduced the duration of monocytopenia. PLAG also effectively reduced extreme changes in platelet counts induced by 5-FU treatment, thus preventing 5-FU-induced thrombocytopenia and thrombocytosis. PLAG significantly decreased plasma levels of the chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, interleukin (IL)-6, and C-reactive protein (CRP), which were elevated consistently with the occurrence time of neutropenia, monocytopenia, and thrombocytopenia. When compared with olive oil and palmitic linoleic hydroxyl glycerol (PLH), only PLAG effectively mitigated 5-FU-induced hematological toxicity, indicating that it has a distinctive mechanism of action. In conclusion, PLAG may have therapeutic potential as a mitigator for 5-FU-induced neutropenia and other hematological disorders.

Mitigating Effects of 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on Hematopoietic Acute Radiation Syndrome after Total-Body Ionizing Irradiation in Mice.

Acute radiation syndrome (ARS) occurs as a result of partial- or whole-body, high-dose exposure to radiation in a very short period of time. Survival is dependent on the severity of the hematopoietic sub-syndrome of ARS. In this study, we investigated the mitigating effects of a lipid molecule, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), on the kinetics of hematopoietic cells, including absolute neutrophil count (ANC), red blood cells (RBCs) and platelet counts, in mice after gamma-ray total-body irradiation (TBI). Male and female BALB/c mice (11 weeks old) received a LD70/30 dose of TBI. PLAG significantly and dose-dependently attenuated radiation-induced mortality (P = 0.0041 for PLAG 50 mg/kg; P < 0.0001 for PLAG 250 mg/kg) and body weight loss (P < 0.0001 for PLAG 50 and 250 mg/kg) in mice. Single-fraction TBI sharply reduced ANC within 3 days postirradiation and maintained the neutropenic state (ANC < 500 cells/µl) by approximately 26.8 ± 0.8 days. However, administration of PLAG attenuated radiation-induced severe neutropenia (ANC < 100 cells/µl) by effectively delaying the mean day of its onset and decreasing its duration. PLAG also significantly mitigated radiation-induced thrombocytopenia (P < 0.0001 for PLAG 250 mg/kg) and anemia (P = 0.0023 for PLAG 250 mg/kg) by increasing mean platelet and RBC counts, as well as hemoglobin levels, in peripheral blood. Moreover, delayed administration of PLAG, even at 48 and 72 h after gamma-ray irradiation, significantly attenuated radiation-induced mortality in a time-dependent manner. When compared to olive oil and palmitic linoleic hydroxyl (PLH), only PLAG effectively attenuated radiation-induced mortality, indicating that it has a distinctive mechanism of action. Based on these preclinical observations, we concluded that PLAG has high potential as a radiation countermeasure for the improvement of survivability and the treatment of hematopoietic injury in gamma-ray-induced ARS.

1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation.

Cancer patients treated with chemotherapy often experience a rapid decline of blood neutrophils, a dose-limiting side effect called chemotherapy-induced neutropenia. This complication brings about dose reductions or cessation of chemotherapy during treatment of cancer patients because a rapid decline of neutrophil counts increases susceptibility to infection. Here, we found that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation via the inhibition of neutrophil-attracting chemokine production in macrophages using in vivo and in vitro approaches. A single intraperitoneal administration of gemcitabine induced the migration of circulating neutrophils into the peritoneal cavity in normal mice, and PLAG effectively decreased neutrophil migration by inhibiting the expression of adhesion molecules, L-selectin and LFA-1. Inhibition of CXCR2 by its antagonist, reparixin, abrogated gemcitabine-induced neutrophil migration, indicating that chemokines produced by gemcitabine mainly support neutrophil activation. In vitro experiments demonstrated that PLAG inhibited NADPH oxidase 2 (NOX2)-mediated reactive oxygen species production induced by gemcitabine, which is the upstream of MIP-2 and/or CXCL8. Importantly, PLAG down-regulated gemcitabine-induced membrane translocation of the cytosolic NOX subunit, Rac1, and phosphorylation of p47phox. The activation of upstream signaling molecules of p47phox phosphorylation, phospholipase C ß3 and protein kinase C, were effectively regulated by PLAG. We also demonstrated that 1-palmitoyl-2-linoleic-3-hydroxyl-rac-glycerol (PLH), the natural form of diacylglycerol, has no effects on gemcitabine-induced CXCL8 production and dHL-60 migration, suggesting that an acetyl group at the third position of the glycerol backbone may have a key role in the regulation of neutrophil activation. Altogether, this study suggests the potential of PLAG as a therapeutic strategy to modulate chemotherapy-induced neutrophil activation for cancer patients undergoing chemotherapeutic treatment.

PLAG alleviates chemotherapy-induced thrombocytopenia via promotion of megakaryocyte/erythrocyte progenitor differentiation in mice.

Previously, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride) was reported to have an effect on the proliferation of hematopoietic stem cells (HSCs) or to contribute to the prevention of chemotherapy-induced neutropenia. In this study, we examined the role of PLAG in the differentiation of bone marrow cells from HSCs into progenitor cells in mice. After 15days, the lineage-negative cells, especially megakaryocyte/erythrocyte progenitors (MEP), were significantly increased in mice that received daily PLAG administration compared to those in the untreated mice. Furthermore, we explored the possibility that the PLAG-induced increase in MEP will contribute to reduction of chemotherapy-induced thrombocytopenia (CIT) in a thrombocytopenia mouse model. Mice were administrated 5-fluorouracil (5-FU) and PLAG. After 7days, bone marrow cells were analyzed. Treatment with 5-FU powerfully decreased myeloid precursor populations and treatment with 5-FU/PLAG resulted in reduction of decreased myeloid progenitor cell numbers. In addition, numbers of circulating platelets were also increased by PLAG treatment. Taken together, PLAG plays a role in differentiating HSCs toward MEP and alleviating chemotherapy-induced bone marrow cell reduction. Thus PLAG shows its potential to augment the therapeutic effect of anti-cancer drugs-induced thrombocytopenia.

PLAG (1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol) Modulates Eosinophil Chemotaxis by Regulating CCL26 Expression from Epithelial Cells.

Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.

Ingenane-type diterpene compounds from Euphorbia kansui modulate IFN-γ production through NF-κB activation.

BACKGROUND: Euphorbia kansui, a traditional medical herb, has been shown to have anti-tumour and anti-viral activities. Previously, we have reported that E. kansui increases interferon-gamma (IFN-γ) production in natural killer (NK) cells. However, it is not clear how E. kansui regulates IFN-γ secretion by NK cells. RESULTS: In this study, E. kansui was separated into six individual compounds from the same chloroform fraction so that the activity of each compound could be compared. E. kansui compounds induced IFN-γ secretion through the phosphorylation of protein kinase D and IκB kinase pathways. Furthermore, E. kansui compounds activated the translocation of p65, a sub-unit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to the nucleus and induced NF-κB at the transcriptional level. CONCLUSION: These findings suggest that E. kansui enhances IFN-γ secretion through the NF-κB pathway in NK cells. © 2015 Society of Chemical Industry.