Phagocytic activity of ethyl alcohol fraction and mitogenic activity of monoacetyldiglyceride isolated from deer antler for mouse peritoneal macrophages

Abstract

Macrophages perform critical functions in the immune system. They originate in the bone marrow and, through the blood stream, reach all the tissues in the organism. According to the specific needs, tissue macrophages either proliferate, further differentiate to more specialized macrophagic populations, or become activated. Macrophages are activated by a variety of stimuli in the course of an immune response. They are capable of ingesting and digesting a broad range of phathogens, including virus-infected cells, tumor cells, and intracellular bacteria. Chemiluminescence provides a simple method of assessing phagocyte function in vitro. The chemiluminescence response of macrophages obtained from the ethyl alcohol fraction of deer antler, Cervus Nippon (CN-E) administered mice was enhanced the phagocytic activity in murine peritoneal macrophages. We observed the phagocytic activity using fluorescein-conjugated E coli K-12 bio-particles. Engulfment of the particles in peritoneal macrophages obtained from CN-E administered mice was enhanced the phagocytic activity in murine peritoneal macrophages. Lucigenin chemiluminescence and the engulfment of fluorescein- conjugated E. coli particles were enhanced in peritoneal macrophages of mice, which received CN-E (100mg/kg). The mechanism of phagocytic activity of CN-E was investigated in vivo. Nitric oxide production was suppressed and the concentration in [Ca^2+]_i was enhanced by CN-E both in vitro and in vivo. The enhancement in [Ca^2+]_i was diminished by EGTA. These results indicate that CN-E enhances the phagocytic activity of peritoneal macrophage via suppression of nitric oxide production and an increase in [Ca^2+]_i. We have studied deer antler with interest in its biological function, and reported that 70% ethyl alcohol fraction of deer antler, Cervus Nippon, enhanced the phagocytosis on mouse peritoneal macrophages and suppressed hyphal growth in Candida albicans. As an extension of the study in finding new components with interest in immune response in antler, we newly isolated and characterized monoacetyldiglycerides (MADGs) from chloroform fraction of deer antler. The acetic acid as a natural triglyceride component was already found to exist in seed oils of Balsaminaceae, Celastraceae, and others. However, monoacetyldiglycerides were newly isolated from an animal tissue, bovine udder and deer antler. We synthesized one of monoacetyldiglycerides, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (rac-MADG) and its enantiomers (R-(+)-MADG, (S)-(-)-MADG) that can be new active immune stimulating agents and be synthesized in less cost effective way with no toxic effects. The present study was undertaken to elucidate the mitogenic activity and signaling pathway for peritoneal macrophages. A factor stimulating a mitogenic activity of peritoneal macrophages is purified from deer antler and bovine udder. It is identified as a triglyceride, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (rac-MADG). In this study, its enantiomers, R-(+)-and S-(-)-1-palmitoyl-2-linoleoyl-3-acetylglycerol (R-(+)-MADG, S-(-)-MADG) are synthesized. Among them, R-(+)-MADG enantiomer turns out to increase a mitogenic activity in mouse peritoneal macrophages. Also, (S)-(-)-MADG shows a low mitogenic activity. Treatment of a macrophage with R-(+)-MADG increases reactive oxygen species(ROS). Furthermore, treatment of macrophages with antioxidant, N-acetyl-L-cysteine (NAC), suppresses the R-(+)-MADG-dependent macrophage proliferation. Results show that the generation of ROS induces in R-(+)-MADG-dependent cell signaling. Treatment of a macrophage with R-(+)-MADG increases the activity of protein kinase C (PKC). Treatment of macrophages with calphostin C, PKC inhibitor, inhibits R-(+)-MADG-induced macrophage proliferation. Results suggest that R-(+)-MADG enhances the activity of protein kinase C (PKC) and stimulates the macrophage growth. In conclusions, R-(+)-MADG accelerates the production of ROS and increases the activity of PKC to eventually stimulate macrophage cell growth. The existence of rac-MADG in deer antler and bovine udder provides passive protection for the neonate and immunostimulatory capabilities. Thus, it seems responsible to expect that (R)-(+)-MADG may act as a growth inducer of mouse peritoneal macrophages and be important not only from an immunological perspective, but also in the wound healing process and clearance of inert particulates.;녹용(Cervus nippon)은 한방에서 강정, 강장의 목적으로 사용되어 왔던 약재로서 성장 촉진작용, 콜레스테롤 저하작용 및 조혈작용 등이 있음이 보고되었다. 특히 한방에서 보약의 대표적인 약재로 사용되고 있는 녹용의 주작용은 면역복합체 억제작용, NK 세포의 활성 증강작용, 세포성 및 체액성 면역 증강작용 및 macrophage의 활성화 작용 등 생체의 면역계를 증강시키는 작용임을 알 수 있다. 그러나 녹용에 함유된 면역증강 성분 및 작용기전에 대해서는 아직 명확히 규명되지 않고 있다. 최근 녹용을 hexane, chloroform 및 70% ethyl alchol로 계통 분획하여 실험한 결과 70% ethyl alcohol fraction에 조혈작용이 있음을 보고하였다. 녹용의 70% ethyl alcohol 추출물(CN-E)이 흉선, 비장세포의 세포생존율 증가와 비장세포 중 helper T 세포의 proliferation 및 Th1-derived cytokine인 interferone γ를 증가시켜 macrophage를 활성화시킴을 확인하였다. CN-E에 의해 활성화된 macrophage는 intracellular Ca^2+ 증가와 nitric oxide 생성의 감소로 phagocytic activity가 촉진되고, 또한 CN-E가 C. albicans에서 hyphal growth를 억제함을 보고하였다. 이는 녹용 에탄올 추출물이 상처치료와 외부물질의 침입에 대한 보호 등 면역조절작용에서 중요한 약물로 작용할 수 있을 것으로 기대된다. 녹용에서 조혈모세포 촉진인자를 개발하던 중 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (rac-MADG)를 녹용과 bovine udder에서 구조 분석하였으며 유기합성법으로 쉽게 합성하였다. Triacylglycerides의 구성 지방산 중 acetic acid로 존재하는 예가 식물조직에서는 보고되었으나, 동물조직에 존재하는 예는 녹용과 bovine udder에서 최초로 확인되었다. 녹용과 bovine udder에서 구조 분석된 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (rac-MADG)와 그 거울상이성질체; R-(+)-1-palmitoyl-2-linoleoyl-3-acetylglycerol (R-(+)-MADG)와 S-(-)-1-palmitoyl-2-linoleoyl-3-acetylglycerol (S-(-)-MADG)를 합성하여 mouse peritoneal macrophages의 증식능을 확인하고 그 기전을 규명하기 위해 macrophage가 증식할 때 분비되는 ROS와 protein kinase C 활성을 측정하였다. Macrophage 증식능은 GM-SCF에 의해 촉진되며, 그 downstream signaling pathway에는 tyrosine phosphorylation, Phosphatidyl Inositol 3-kinase(PI 3-kinase)와 PKC 활성이 증대됨이 보고되고 있다. GM-CSF는 또한 macrophages 증식에 reactive oxygen species(ROS)와 H_2O_2 생성이 촉진됨이 보고되고 있다. rac-MADG, R-(+)-MADG, S-(-)-MADG (MADGs) 중에서 R-(+)-MADG가 GM-CSF와 유사하게 mouse peritoneal macrophages의 증식능을 가장 촉진하였으나, S-(-)-MADG는 mouse peritoneal macrophages의 증식능을 촉진하지 않았다. mouse peritoneal macrophages에 MADGs를 처리한 결과R-(+)-MADG에 의해 reactive oxygen species (ROS) 생성이 순간적으로 상승하였으며, antioxidant인 N-acetyl-L-cysteine에 의해 R-(+)-MADG로 촉진된 peritoneal macrophages의 증식능이 억제되었다. mouse peritoneal macrophages에MADGs를 처리한 결과 R-(+)-MADG가 가장 protein kinase C (PKC) 을 촉진하였으며, PKC inhibitor인 calphostin C 처리결과 R-(+)-MADG로 촉진된 peritoneal macrophages의 증식능이 억제되었다. 이 결과는 R-(+)-MADG가 mouse peritoneal macrophages에서reactive oxygen species (ROS) 생성과 protein kinase C (PKC) 활성을 촉진하여 결국 peritoneal macrophages의 증식능을 증대하였다. 이는 녹용, bovine udder 및 milk에 존재하는 racemates인 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (rac-MADG)와 그 합성한 거울상이성질체중 R-(+)-1-palmitoyl-2-linoleoyl-3-acetylglycerol이 macrophages proliferation과 phagocytosos를 촉진하여 세균 또는 손상되거나 노쇠한 세포, 암세포 등을 제거하고 항균작용을 촉진하여 방어와 감시역할 외에도 특이적 면역반응의 유도에 중요하게 기여할 것으로 기대된다. 녹용, bovine udder 및 milk에 존재하는 racemates인 rac-MADG와 그 합성한 거울상이성질체중 R-(+)-MADG가 mouse peritoneal macrophage에 직접적으로 작용하여 macrophage proliferation을 촉진하는지 또는 GM-CSF를 유도하여 GM-CSF가 macrophage proliferation을 촉진하는지 여부를 검토할 예정이다.