Inhibition of ERK1/2 by silymarin in mouse mesangial cells

The present study aimed to show that pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-1β] synergistically induce the production of nitric oxide (NO) production in mouse mesangial cells, which play an important role in inflammatory glomerular injury. We also found that co-treatment with cytokines at low doses (TNF-α; 5 ng/ml, IFN-γ; 5 ng/ml, and IL-1β; 1.25 U/ml) synergistically induced NO production, whereas treatment with each cytokine alone did not increase NO production at doses up to 100 ng/ml or 50 U/ml. Silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), attenuates cytokine mixture (TNF-α, IFN-γ, and IL-1β)-induced NO production. Western blot and RT-PCR analyses showed that silymarin inhibits inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Silymarin also inhibited extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) phosphorylation. Collectively, we have demonstrated that silymarin inhibits NO production in mouse mesangial cells, and may act as a useful anti-inflammatory agent.

Silibinin Inhibits LPS-Induced Macrophage Activation by Blocking p38 MAPK in RAW 264.7 Cells

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-alpha, and IL1beta. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.

Radicicol Inhibits iNOS Expression in Cytokine-Stimulated Pancreatic Beta Cells

Here, we show that radicicol, a fungal antibiotic, resulted in marked inhibition of inducible nitric oxide synthase (iNOS) transcription by the pancreatic beta cell line MIN6N8a in response to cytokine mixture (CM: TNF-alpha, IFN-gamma, and IL-1beta). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-kappaB/Rel, which plays a critical role in iNOS transcription, in a dose-related manner. Nitrite production in the presence of PD98059, a specific inhibitor of the extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) pathway, was dramatically diminished, suggesting that the ERK1/2 pathway is involved in CM-induced iNOS expression. In contrast, SB203580, a specific inhibitor of p38, had no effect on nitrite generation. Collectively, this series of experiments indicates that radicicol inhibits iNOS gene expression by blocking ERK1/2 signaling. Due to the critical role that NO release plays in mediating destruction of pancreatic beta cells, the inhibitory effects of radicicol on iNOS expression suggest that radicicol may represent a useful anti-diabetic activity.

Ape1/Ref-1 Stimulates GDNF/GFR alpha1-mediated Downstream Signaling and Neuroblastoma Proliferation

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor alpha1 (GFR alpha1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRalpha1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFR alpha1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRalpha1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFR alpha1-specific RNA experiments demonstrated that the downregulation of GFR alpha1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLC gamma-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFR alpha signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.