title: Ruscogenin suppresses mouse neutrophil activation: Involvement of protein kinase A pathway.
abstract: Ruscogenin, a natural steroidal sapogenin, presents in both food and medicinal plants. It has been found to exert significant anti-inflammatory activities. Considering that activation of neutrophil is a key feature of inflammatory diseases, this study was performed to investigate the inhibitory effect of ruscogenin and its underlying mechanisms responsible for neutrophil activation. Ruscogenin displayed potent antioxidative effects against Formyl-Met-Leu-Phe (FMLP)-induced extra- and intracellular superoxide generation in mouse bone marrow neutrophils, with IC50 values of 1.07±0.32 μM and 1.77±0.46 μM, respectively. Phorbol myristate acetate (PMA)-elicited extra- and intracellular superoxide generation were also suppressed by ruscogenin, with IC50 values of 1.56±0.46 μM and 1.29±0.49 μM, respectively. However, ruscogenin showed weak inhibition in NaF-induced response. Inhibition of superoxide generation was mediated neither by a superoxide-scavenging ability nor by a cytotoxic effect. Furthermore, ruscogenin inhibited the membrane translocation of p47phox and p67phox. It reduced FMLP-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and p21-activated kinase (PAK). The cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were increased by ruscogenin. Moreover, ruscogenin inhibited phosphorylation of protein kinase B (Akt), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). In addition, the inhibitory effects of ruscogenin on superoxide production and the phosphorylation of Akt, p38MAPK, and ERK1/2 were reversed by PKA inhibitor (H89), suggesting a PKA-dependent mechanism. In summary, our data suggest that ruscogenin inhibits activation of neutrophil through cPLA2, PAK, Akt, MAPKs, cAMP, and PKA signaling pathways. Increased PKA activity is associated with suppression of the phosphorylation of Akt, p38MAPK, and ERK1/2 pathways.
authors.affiliation: School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Y N
authors.initials: YN
authors.lastname: Lin
authors.affiliation: School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, PR China.
authors.forename: R
authors.initials: R
authors.lastname: Jia
authors.affiliation: School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Y H
authors.initials: YH
authors.lastname: Liu
authors.affiliation: School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Y
authors.initials: Y
authors.lastname: Gao
authors.affiliation: School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, PR China.
authors.forename: L L
authors.initials: LL
authors.lastname: Wang
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, PR China. Electronic address: junpingkou@cpu.edu.cn.
authors.forename: J P
authors.initials: JP
authors.lastname: Kou
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, PR China. Electronic address: boyangyu59@163.com.
authors.forename: B Y
authors.initials: BY
authors.lastname: Yu
mesh_terms: D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018375:Neutrophil Activation / Q000187:drug effects*; D054510:Phospholipases A2, Cytosolic / Q000378:metabolism; D013150:Spirostans / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism
chemical_list: D013150:Spirostans; D013481:Superoxides; C012541:ruscogenin; D054462:p21-Activated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054510:Phospholipases A2, Cytosolic
keywords: NADPH oxidase; Neutrophils; Ruscogenin; Signaling pathways; Superoxide anion