Puerarin suppresses production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through regulating MAPK phosphorylation, O-GlcNAcylation and NF-κB translocation

Abstract

Abstract. Microglial cells play a critical role in mediating central nervous system inflammatory processes. Activated microglial cells induced by proinflammatory factor, such as lipopolysaccharide (LPS), release many kinds of neurotoxic cytokines including reactive oxygen species (ROS) which contributes to the pathogenesis of neurodegenerative diseases. Puerarin, extracted from kudzu root, possesses the characteristic of neuroprotection, antioxidation and anticancer. In the present study, we observed that LPS induced over-production of nitric oxide (NO) and increased the level of intracellular ROS in N9 microglial cells, but it was inhibited by puerarin. Furthermore, treatment with puerarin on N9 cells suppressed the over-expression of inducible nitric oxide synthase (iNOS) induced by LPS which is implicated in intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) level, phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathway. We also observed that the enhanced phosphorylation of p38, JNK and ERK1/2 in N9 cells induced by LPS were inhibited by puerarin, otherwise the down-regulation of O-GlcNAcylation level of protein in N9 cell induced by LPS was up-regulated by pretreatment with puerarin. These results indicate that puerarin effectively inhibits microglia activation induced by LPS through inhibiting expression of iNOS, production of NO and ROS which was mediated via regulating O-GlcNAcylation, phosphorylation of MAPK and NF-κB translocation. IntroductionMicroglial cells, macrophage-like cells in the central nervous system, participate in CNS immune response through releasing a variety of factors, including trophic factors and chemokines, to promote neuroprotection (1). Whereas under chronic inflammatory environment, excessive activation of microglia cells secrete cytotoxic substances and neurotoxic cytokines, such as reactive oxygen species (ROS) and nitric oxide (NO) which contribute to the pathogenesis of neurodegenerative diseases (2). Lipopolysaccharide (LPS), which consist in outer membrane of gram-negative bacteria as potent pro-inflammatory agents, induce microglial cell inflammatory injury through releasing proinflammatory mediators such as NO generated by inducible nitric oxide synthase (iNOS) (3,4). NO is considered as a main risk factor of progressive damage in neurodegenerative diseases (5,6). Microglia cell inflammatory response to LPS is involved in speedy regulation of many signal transduction molecules. The regulation of a signal pathway, such as mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB), is implicated in major molecular mechanisms of LPS-induced NO and ROS production in microglial cells (4,(6)(7)(8).O-Linked O-GlcNAcylation of protein, a single monosaccharide N-acetylglucosamine (GlcNAc) attached to the hydroxyl groups of specific serine or threonine residues, is an important post-translational modification on nuclear and cytoplasmic proteins (9,10). More than 500 proteins have been identified to be O-GlcNAcylated...

O-GlcNAcylation in immunity and inflammation: An intricate system (Review)

“…Moreover, OGT plays a key role in the inflammatory responses of macrophages. It has been observed that in N9 microglial cells, in response to LPS, the enhanced expression of iNOS, NO and ROS is mediated via the downregulation of OGT and protein O-GlcNAcylation, or via the upregulation of MAPKs phosphorylation and NF-κB translocation (101). In addition, overexpression of OGT exhibits inhibitory effects on the LPS-driven activation of NF-κB and iNOS through modulation of histone acetylation either directly or indirectly (102).…”

Advances in Neuroprotective Ingredients of Medicinal Herbs by Using Cellular and Animal Models of Parkinson’s Disease

“…In a latest study by Li et al, treatment with puerarin to 6-OHDA-lesioned rats was observed to significantly increase the protein expression of DJ-1 and superoxide dismutase-2 in the SN [88]. In recent report by Zheng et al, puerarin was found to suppress LPS-induced release of iNOS and phosphorylation of MAPKs in N9 cells [89]. Similar in vitro effects of puerarin are also evident in 6-OHDA-induced neurotoxicity to PC12 cells, wherein puerarin inhibits the MPP + -induced phosphorylation of JNK [90].…”

Plants‐Derived Neuroprotective Agents: Cutting the Cycle of Cell Death through Multiple Mechanisms

“…Puerarin ( 22 ) was also reported to prevent the dysfunction of the proteasomal system thus preventing the accumulation of conjugated proteins [ 209 ]. It was shown to suppress the LPS-induced release of iNOS and phosphorylation of MAPKs in N9 cells [ 210 ] and inhibits the MPP+-induced phosphorylation of JNK [ 211 ]. In 6-OHDA-lesioned rats, 23 significantly increased the protein expression of DJ-1 and SOD-2 in the substantia nigra [ 212 ] and increased the expression of the glial cell-derived neurotrophic factor in the striatum of rats administered with 6-OHDA [ 213 ].…”