Study On Anti-inflammatory Mechanism Of Arctigenin And Neuro-protective Mechanism Of Ampelopsin

Arctigenin, a bioactive constituent from dried seeds of Arctium lappa L. Arctigenin is a common herbal medicinal preparation in China,which has been reported to exhibit anti-inflammatory,antioxidant, antitumor, neuroprotective effectes. Moreover, Arctigenin also inhibited the replication of human immunodeficiency virus. In China, Arctigenin has been used clinically as a therapeutic agent to treat inflammation several years. Previous studies demonstrated that Arctigenin suppressed the activation of NF-κB stimulated by LPS. Up to date, resports on the mechanism underlying the anti-inflammatory effect of Arctigenin are limited to study on the inhibition the activation of NF-κB. Thus, in the present study, we study another potent mechanism underlying the anti-inflammatory effect of Arctigenin.The JAK-STAT signaling pathway may be activated by diverse ligands, including cytokines and growth factors, mediates the immune and inflammatory response. The JAK family consists of four members, namely, JAK1, JAK2, JAK3, and TYK2, whereas in macrophages JAK2 is dominantly expressed. The stimulation of cells with suitable ligands, induces receptor oligomerization and causes the local aggregation of associated JAKs, which results in their activation by phosphorylation. JAKs are activated by tyrosine transphosphorylation, which allows them to phosphorylate their major downstream targets-STATs. When tyrosine is phosphorylated by JAKs, STATs are translocated to the nucleus to regulate the transcription of target genes,such as iNOS and COX-2.Cytokine-derived immune signaling is strictly regulated with respect to both magnitude and duration. Suppressor of cytokine signaling (SOCS) family proteins are negative feedback regulators of the JAK-STAT signal pathway. The SOCS family consists of eight members, cytokine-induced SH2 protein (CIS) and SOCS1-7, SOCS protein inhibits JAK-STAT signaling by directly interacting with JAK family proteins or with phosphorylated tyrosine residues in the cytokine receptors.Arctigenin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are still unknown. In the current study,we foud that Arctigenin effectively inhibited iNOS (mRNA, protein) gene expression and enzyme activtiy. It's anti-inflammatory effects appear in part to be due to the suppression of the ROS-dependent JAK-STAT inflammatory signaling pathway. Like as N-acetyl-L-cysteine, Arctigenin markedly inhibited the phosphorylation of STAT1 and 3, thus blocked their translocation to the nucleus. In the iNOS-luciferase assays, we foud the gene promoter activitie of iNOS was significantly increased by LPS stimulated. However, pretreated Arctigenin suppressed the report gene activity of iNOS by dose-dependent manner. At the same time, cells expressing dominant negative mutant STAT1 (STAT1YF) or dominant negative mutant STAT3 (STAT3YF) also reduced gene promoter activitie, meaning that STAT1 or STAT3 is a major molecule in regulating iNOS gene expression. and consequently inhibited expression of iNOS. To determine whether the anti-inflammatory effect of Arctigenin is obviously due to inhibition of STAT1 or STAT3, we tested other STAT responsive inflammatory genes such as IL-1β, IL-6, and MCP-1. Cells incubated with Arctigenin showed a dose-dependent reduction in IL-1β, IL-6, and MCP-1 expression in LPS-activated RAW264.7 cells. Furthermore, our results demonstrated that Arctigenin suppressed JAK2 phosphorylation, but not JAK1. In addition, we further show that induction of SOCS3 is likely to be one of the mechanisms underlying the Arctigenin-mediated inhibition of JAK-STAT signaling. We observed Arctigenin induced SOCS3 transcripts but not SOCS1 in RAW264.7 cells,meaning that SOCS3 contributes to the inhibition of the JAK-STAT pathway by Arctigenin. Taken together, these data suggest Arctigenin suppresses macrophages produce IL-1β, IL-6, and MCP-1 which these processes require the inhibition of the ROS-dependent JAK2-STAT1/3 signaling pathways. Ampelopsin, the most abudant flavonoids in Rattan Tea, has been proven to exert effects of antibacterial, antioxidation and hepatoprotective activities. Previous study have revealed that Ampelopsin can rescue hela, red blood and lymphocyte cells from H2O2-induced oxidative cell death in vitro and demonstrated that this effect was associated with its itself effect of eliminating ROS and preventing lipid peroxidation. These finding suggest that Ampelopsin may has neuroprotective effect in neurodegenerate diseases. Although investigators demonstrated that ampelopsin have broad biological activities, no evidence has been reported to prove the direct effect of ampelopsin on neuronal-like cells, PC 12 cells, and the mechanisms of this effect.In the present study, we first explored the effect of Ampelopsin on H2O2-induced oxidative cell death in PC 12 cells. Pre-treatment with Ampelopsin for 1 h significantly reduced the H2O2-induced viability loss, apoptotic rate and attenuated H2O2-mediated ROS production. In addition, Ampelopsin inhibited H2O2-induced lowered membrane potential, decreased cleaved PARP, Caspase-3. We also demonstrated that Ampelopsin could prevent the activation of p38MAPK kinase but not JNK and ERK pathways induced by H2O2. These findings suggest that Ampelopsin could reduce H2O2-induced cell apoptosis in part through inhibiting p38 signal. To elucidate the plausible signal transduction pathways involved in the Ampelopsin-induced HO-1 expression, we examined the phosphorylation of severalupstream kinases. Upon Ampelopsin treatment, concentration- and time- related increases in the phosphorylation of Akt and ERK1/2 were observed in PC 12 cells.Next, we explored the underlying mechanisms involved in the propective effects of Ampelopsin on the H2O2-induced cytotoxicty in PC 12 cells. We for the first time have revealed pretreated Ampelopsin 12 h increased antioxidant enzyme heme oxygenase-1 (HO-1) expression in concentration-dependent manners. In the same experiment condition, we also demonstrated that pretreated 15μg/ml Ampelopsin different times can also induce HO-1 expression in time-dependent manner. We found that Ampelopsin can activate ERK and Akt signal in concentration-dependent and time-dependent manners, which were correlated with its protective effect against H2O2-induced injury. To determine whether such activation of Akt and ERK1/2 could contribute to the Ampelopsin -mediated protection against the cytotoxic effect of H2O2, pharmacological inhibitors of these kinases were utilized. Western blot results showed that Ampelopsin-mediated cytoprotection against H2O2 cytotoxicity was attenuated by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), upstream of Akt and also by U0126, an inhibitor of MEK1/2, upstream of ERK1/2. Same to Western blot results, the MTT assay also demonstrated that LY294002 and U0126 could block the neuroprotective of Ampelopsin.Taken together, these findings suggest that Ampelopsin can induce HO-1 expression through activation of ERK signal pathways, thereby protecting the PC12 cells from H2O2-induced oxidative cell death.