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Immunomodulatory activity of natural polyamines in murine macrophages
New Biotechnology · Volume 31S · July 2014
PE-04 Immunomodulatory activity of natural polyamines in murine macrophages Sun Chul Kang ∗ , Anil Kumar Chauhan, Souren Paul, Rekha Jakhar Daegu University
Macrophages are the key players of innate immunity, defending the body from foreign invaders through phagocytosis. In the present study we investigated the potent role of natural polyamines in modulation of macrophage activity by analysing all the sequential steps involved during phagocytosis. Initially, we treated splenocytes with three natural polyamines (PUT, SPD and SPM) to test their ability in proliferation of cells and involvement in mitosis. Thereafter, we detected their role in promoting membrane fluidity on RAW 264.7 cells, which is essential during uptake of particles. Phagolysosome fusion activity of macrophages in the presence of polyamines was then evaluated by measuring acid phosphatase through p-nitro phenyl phosphate. Further, we determined the capacity of polyamines in generation of superoxide anion to create respiratory burst inside the macrophages. We find decrease in the proliferation of splenocytes in the presence of polyamines which proved their potent role as strong immunomodulator. Uptake capacity of macrophages was observed to be enhanced after treatment with polyamines and also increased lysosomal activity of macrophages was detected at a concentration dependent manner. Percentage of NBT reduction calculated for superoxide anion generation revealed that polyamines potentiated this property of macrophages too. We also determined the anti-complementory activity of polyamines by detecting classical pathway of complement which showed to be effective, suggesting its command over unwanted activation of complement. This study presents the potential role of polyamines as an immunostimulatory drug, which could be effective to treat various immunological disorders. http://dx.doi.org/10.1016/j.nbt.2014.05.1880
PE-05 Thymol protects gastric ulcer induced by alcohol through regulation of matrix metalloprotein 9 activity Anil Kumar Chauhan ∗ , Sun Chul Kang Daegu University
In gastrointestinal disorders, ulcer is a common disease with multiple etiologies and most of them are observed due to high consumption of alcohol. Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degradation of extracellular matrix and are key players in various inflammatory diseases and among them MMP-9 is found to play major role during gastric inflammation. In this study, we aimed to determine the roles of thymol in expression of MMP-9 during ethanol induced gastric ulcer model in vivo. Sprague-Dawley rats, pretreated with thymol (10 mg/kg) or normal saline as control were subjected to intragastric administration of 95% ethanol
BIOPHARMACEUTICALS
(5 ml/kg). Morphological examination includes ulcer index for hemorrhage and hematoxylin and eosin staining was performed to analyze the severity of ulceration. Gelatin zymography was done to determine the expression of MMP-9 and a number of biological and immunological tests were carried out to determine the antioxidant and cytokine levels. Rats challenged with alcohol, developed severe injury in gastric mucosa with increased level of pro-inflammatory mediators like TNF-␣, Prostaglandin E2 and nitric oxide. Expression of MMP-9 was up-regulated and less production of antioxidant enzymes (SOD and GSH) was documented after treatment with alcohol. In thymol pretreated rats, a significant decrease in ulcer index, level of pro-inflammatory cytokines and nitric oxide was observed. Expression of MMP-9 was downregulated and antioxidant enzymes production was increased in thymol pretreated group. In conclusion, study suggests that thymol protects gastric mucosa injury from ethanol consumption by down-regulation of MMP-9 and inflammatory cytokines. http://dx.doi.org/10.1016/j.nbt.2014.05.1881
PE-06 7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits metastasis in vitro and in vivo Hui Min Wang Kaohsiung Medical University
Melanoma is the deadliest cancer. We identified 7hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent against human melanoma A375.S2 cells. 7hydroxydehydronuciferine (7-HDNF) was known to induce autophagy and apoptosis response mechanisms, and antimigratory activity of melanoma in vitro and in vivo. Cell proliferation assay was used to test cell viability. Acridine orange (AO) staining and flow analysiswere applied to observe cell morphology. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained. Western blot was applied to examine protein expressions whereas wound healing assay was to examine cell activity. Strong anticancer effects of 7-HDNF were exhibited in a dose-dependent mannerand displayed minor cytotoxicities on normal human skin cells.7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). 7-HDNF increased the cellular arrest in cell cycle at G2/M phase. Cellular membrane asymmetry loss was confirmed. Protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. 7-HDNF presented the high-quality anti-migratory activity. 7-HDNF inhibited melanoma tumor growth in mice xenograft model, accompanied with a decrease of phosphorylation of AKT. We demonstrated the mechanism of this compound starting with the formation and accumulation of AVO leading to autophagy. 7-HDNF caused the cellular membrane asymmetry loss, triggering the G2/M cell cycle arrest in caspase-dependent apoptosis. 7-HDNF presented highwww.elsevier.com/locate/nbt S111
PE-04 Immunomodulatory activity of natural polyamines in murine macrophages Sun Chul Kang ∗ , Anil Kumar Chauhan, Souren Paul, Rekha Jakhar Daegu University
Macrophages are the key players of innate immunity, defending the body from foreign invaders through phagocytosis. In the present study we investigated the potent role of natural polyamines in modulation of macrophage activity by analysing all the sequential steps involved during phagocytosis. Initially, we treated splenocytes with three natural polyamines (PUT, SPD and SPM) to test their ability in proliferation of cells and involvement in mitosis. Thereafter, we detected their role in promoting membrane fluidity on RAW 264.7 cells, which is essential during uptake of particles. Phagolysosome fusion activity of macrophages in the presence of polyamines was then evaluated by measuring acid phosphatase through p-nitro phenyl phosphate. Further, we determined the capacity of polyamines in generation of superoxide anion to create respiratory burst inside the macrophages. We find decrease in the proliferation of splenocytes in the presence of polyamines which proved their potent role as strong immunomodulator. Uptake capacity of macrophages was observed to be enhanced after treatment with polyamines and also increased lysosomal activity of macrophages was detected at a concentration dependent manner. Percentage of NBT reduction calculated for superoxide anion generation revealed that polyamines potentiated this property of macrophages too. We also determined the anti-complementory activity of polyamines by detecting classical pathway of complement which showed to be effective, suggesting its command over unwanted activation of complement. This study presents the potential role of polyamines as an immunostimulatory drug, which could be effective to treat various immunological disorders. http://dx.doi.org/10.1016/j.nbt.2014.05.1880
PE-05 Thymol protects gastric ulcer induced by alcohol through regulation of matrix metalloprotein 9 activity Anil Kumar Chauhan ∗ , Sun Chul Kang Daegu University
In gastrointestinal disorders, ulcer is a common disease with multiple etiologies and most of them are observed due to high consumption of alcohol. Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degradation of extracellular matrix and are key players in various inflammatory diseases and among them MMP-9 is found to play major role during gastric inflammation. In this study, we aimed to determine the roles of thymol in expression of MMP-9 during ethanol induced gastric ulcer model in vivo. Sprague-Dawley rats, pretreated with thymol (10 mg/kg) or normal saline as control were subjected to intragastric administration of 95% ethanol
BIOPHARMACEUTICALS
(5 ml/kg). Morphological examination includes ulcer index for hemorrhage and hematoxylin and eosin staining was performed to analyze the severity of ulceration. Gelatin zymography was done to determine the expression of MMP-9 and a number of biological and immunological tests were carried out to determine the antioxidant and cytokine levels. Rats challenged with alcohol, developed severe injury in gastric mucosa with increased level of pro-inflammatory mediators like TNF-␣, Prostaglandin E2 and nitric oxide. Expression of MMP-9 was up-regulated and less production of antioxidant enzymes (SOD and GSH) was documented after treatment with alcohol. In thymol pretreated rats, a significant decrease in ulcer index, level of pro-inflammatory cytokines and nitric oxide was observed. Expression of MMP-9 was downregulated and antioxidant enzymes production was increased in thymol pretreated group. In conclusion, study suggests that thymol protects gastric mucosa injury from ethanol consumption by down-regulation of MMP-9 and inflammatory cytokines. http://dx.doi.org/10.1016/j.nbt.2014.05.1881
PE-06 7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits metastasis in vitro and in vivo Hui Min Wang Kaohsiung Medical University
Melanoma is the deadliest cancer. We identified 7hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent against human melanoma A375.S2 cells. 7hydroxydehydronuciferine (7-HDNF) was known to induce autophagy and apoptosis response mechanisms, and antimigratory activity of melanoma in vitro and in vivo. Cell proliferation assay was used to test cell viability. Acridine orange (AO) staining and flow analysiswere applied to observe cell morphology. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained. Western blot was applied to examine protein expressions whereas wound healing assay was to examine cell activity. Strong anticancer effects of 7-HDNF were exhibited in a dose-dependent mannerand displayed minor cytotoxicities on normal human skin cells.7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). 7-HDNF increased the cellular arrest in cell cycle at G2/M phase. Cellular membrane asymmetry loss was confirmed. Protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. 7-HDNF presented the high-quality anti-migratory activity. 7-HDNF inhibited melanoma tumor growth in mice xenograft model, accompanied with a decrease of phosphorylation of AKT. We demonstrated the mechanism of this compound starting with the formation and accumulation of AVO leading to autophagy. 7-HDNF caused the cellular membrane asymmetry loss, triggering the G2/M cell cycle arrest in caspase-dependent apoptosis. 7-HDNF presented highwww.elsevier.com/locate/nbt S111