Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells.

Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox), and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47(phox) abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent.

Lu CY ，Yang YC ，Li CC ，Liu KL ，Lii CK ，Chen HW ... -  《-》

Andrographis paniculata diterpenoids and ethanolic extract inhibit TNFα-induced ICAM-1 expression in EA.hy926 cells.

Andrographis paniculata (A. paniculata), a traditional herb in Southeastern Asia, is used to treat inflammation-mediated diseases. The two major bioactive diterpenoids in A. paniculata are andrographolide (AND) and 14-deoxy-11,12-didehydroandrographolide (deAND). Because of the anti-inflammatory evidence for AND, we hypothesized that deAND might possess similar potency for inhibiting monocyte adhesion to the vascular endothelium, which is a critical event for atherosclerotic lesion formation. In the present study, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine cell viability. We evaluated the production of intracellular reactive oxygen species (ROS) by using DCFDA assay. We assayed the protein expression by using Western blot analysis, the mRNA expression by using RT-PCR, and the nuclear protein-DNA binding activity by using EMSA. We showed that pretreatment of EA.hy926 cells with A. paniculata ethanolic extract (APE), deAND, and AND significantly inhibited TNFα-induced ICAM-1 protein and mRNA expression, ICAM-1 promoter activity, and monocyte adhesion. TNFα-stimulated IKKβ phosphorylation, IκBα phosphorylation and degradation, p65 nuclear translocation, and NFκB nuclear protein-DNA binding activity were attenuated by pretreatment with APE, deAND, and AND. APE, deAND, and AND attenuated TNFα-induced Src phosphorylation and membrane translocation of the NOX subunits p47phox and p67phox. Both APE and AND induced protein expression of heme oxygenase 1 and the glutamate cysteine ligase modifier subunit and enhanced glutathione content. Pretreatment with AND and deAND inhibited TNFα-induced ROS generation. These results suggest that the mechanism by which APE, deAND, and AND down-regulates TNFα-induced ICAM-1 expression in EA.hy926 cells is via attenuation of activation of the IKK/IκB/NFκB pathway.

Lin HC ，Li CC ，Yang YC ，Chiu TH ，Liu KL ，Lii CK ，Chen HW ... -  《-》

Induction of heme oxygenase 1 and inhibition of tumor necrosis factor alpha-induced intercellular adhesion molecule expression by andrographolide in EA.hy926 cells.

Yu AL ，Lu CY ，Wang TS ，Tsai CW ，Liu KL ，Cheng YP ，Chang HC ，Lii CK ，Chen HW ... -  《-》

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways.

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.

Yang YC ，Lii CK ，Wei YL ，Li CC ，Lu CY ，Liu KL ，Chen HW ... -  《-》

Clopidogrel Protects Endothelium by Hindering TNFα-Induced VCAM-1 Expression through CaMKKβ/AMPK/Nrf2 Pathway.

Yang H ，Zhao P ，Tian S 《-》