Zhixue Wang1, Zijing Liu2, Lijng Zhou1, Tingting Long1, Xing Zhou1, Yixi Bao3. 1. Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China. 2. Department of Clinical Medicine, Xinjiang Medical University, Urumqi 830054, China. 3. Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China. Electronic address: yixibao@163.com.
Abstract
OBJECTIVE: This study is to investigate the role of second messengers and TLR4/NF-κB signaling pathway in the immunomodulatory activities of Astragalus polysaccharide (APS) and Polysaccharopeptide (PSP) in macrophages. METHODS: RAW 264.7 macrophage cells were treated with APS, PSP, lipopolysaccharide (LPS), or NiCl2. Power-spectral method was used to detect protein kinase C (PKC) and Griess reaction to detect nitric oxide (NO). ELISA was conducted to detect cyclic adenosine monophosphate (cAMP), diglycerides (DAG), inositol 1, 4, 5-triphosphate (IP3), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Confocal laser scanning microscopy was performed to detect calcium level. qRT-PCR and Western blot was used to detect mRNA and protein expression of NF-κB. RESULTS: APS and PSP significantly increased the concentrations of intracellular second messengers (NO, cAMP, DAG, IP3, Ca2+) and the activity of PKC in macrophages (p<0.05).The intracellular NF-κB mRNA and protein levels were significantly increased in macrophages treated by APS and PSP (p<0.05), whereas those were significantly decreased after NiCl2 incubation (p<0.05). Similarly, the secretion of TNF-α and IL-6 were significantly decreased by the treatment of NiCl2. CONCLUSION: Our findings strongly suggest that Ca2+-cAMP and TLR4/NF-κB signaling pathways are, at least partly, involved in APS and PSP mediated immunomodulatory activities in macrophages. Copyright Â
OBJECTIVE: This study is to investigate the role of second messengers and TLR4/NF-κB signaling pathway in the immunomodulatory activities of Astragalus polysaccharide (APS) and Polysaccharopeptide (PSP) in macrophages. METHODS: RAW 264.7 macrophage cells were treated with APS, PSP, lipopolysaccharide (LPS), or NiCl2. Power-spectral method was used to detect protein kinase C (PKC) and Griess reaction to detect nitric oxide (NO). ELISA was conducted to detect cyclic adenosine monophosphate (cAMP), diglycerides (DAG), inositol 1, 4, 5-triphosphate (IP3), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Confocal laser scanning microscopy was performed to detect calcium level. qRT-PCR and Western blot was used to detect mRNA and protein expression of NF-κB. RESULTS:APS and PSP significantly increased the concentrations of intracellular second messengers (NO, cAMP, DAG, IP3, Ca2+) and the activity of PKC in macrophages (p<0.05).The intracellular NF-κB mRNA and protein levels were significantly increased in macrophages treated by APS and PSP (p<0.05), whereas those were significantly decreased after NiCl2 incubation (p<0.05). Similarly, the secretion of TNF-α and IL-6 were significantly decreased by the treatment of NiCl2. CONCLUSION: Our findings strongly suggest that Ca2+-cAMP and TLR4/NF-κB signaling pathways are, at least partly, involved in APS and PSP mediated immunomodulatory activities in macrophages. Copyright Â