TAK1/AP-1-Targeted Anti-Inflammatory Effects of Barringtonia augusta Methanol Extract.

Barringtonia augusta methanol extract (Ba-ME) is a folk medicine found in the wetlands of Thailand that acts through an anti-inflammatory mechanism that is not understood fully. Here, we examine how the methanol extract of Barringtonia augusta (B. augusta) can suppress the activator protein 1 (AP-1) signaling pathway and study the activities of Ba-ME in the lipopolysaccharide (LPS)-treated RAW264.7 macrophage cell line and an LPS-induced peritonitis mouse model. Non-toxic concentrations of Ba-ME downregulated the mRNA expression of cytokines, such as cyclooxygenase and chemokine ligand 12, in LPS-stimulated RAW264.7 cells. Transfection experiments with the AP-1-Luc construct, HEK293T cells, and luciferase assays were used to assess whether Ba-ME suppressed the AP-1 functional activation. A Western blot assay confirmed that C-Jun N-terminal kinase is a direct pharmacological target of Ba-ME action. The anti-inflammatory effect of Ba-ME, which functions by ß-activated kinase 1 (TAK1) inhibition, was confirmed by using an overexpression strategy and a cellular thermal shift assay. In vivo experiments in a mouse model of LPS-induced peritonitis showed the anti-inflammatory effect of Ba-ME on LPS-stimulated macrophages and acute inflammatory mouse models. We conclude that Ba-ME is a promising anti-inflammatory drug targeting TAK1 in the AP-1 pathway.

Cucurbitacin-I induces hypertrophy in H9c2 cardiomyoblasts through activation of autophagy via MEK/ERK1/2 signaling pathway.

Cucurbitacin-I, a natural triterpenoids initially identified in medicinal plants, shows a potent anticancer effect on a variety of cancer cell types. Nevertheless, the cardiotoxicity of cucurbitacin-I has not heretofore been reported. In this study, the mechanisms of cucurbitacin-I-induced cardiotoxicity were examined by investigating the role of MAPK-autophagy-dependent pathways. After being treated with 0.1-0.3µM cucurbitacin-I for 48h, H9c2 cells showed a gradual decrease in the cell viabilities, a gradual increase in cell size, and mRNA expression of ANP and BNP (cardiac hypertrophic markers). Cucurbitacin-I concentration-dependent apoptosis of H9c2 cells was also observed. The increased apoptosis of H9c2 cells was paralleling with the gradually strong autophagy levels. Furthermore, an autophagy inhibitor, 3-MA, was used to block the cucurbitacin-I-stirred autophagy, and then the hypertrophy and apoptosis induced by 0.3µM cucurbitacin-I were significantly attenuated. In addition, cucurbitacin-I exposure also activated the MAPK signaling pathways, including ERK1/2, JNK, and p38 kinases. Interestingly, only the ERK inhibitor U0126, but not the JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580, weakened the induction of 0.3µM cucurbitacin-I in hypertrophy, autophagy and apoptosis. Our findings suggest that cucurbitacin-I can increase the autophagy levels of H9c2 cells, most likely, through the activation of an ERK-autophagy dependent pathway, which results in the hypertrophy and apoptosis of cardiomyocytes.

Regulation of Transforming Growth Factor ß-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

OBJECTIVE: Transforming growth factor ß-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). METHODS: The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1ß-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA). RESULTS: Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1ß-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination. CONCLUSION: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Picroside II Inhibits the MEK-ERK1/2-COX2 Signal Pathway to Prevent Cerebral Ischemic Injury in Rats.

The objective of this study is to explore the neuroprotective effect and mechanism of picroside II on ERK1/2-COX2 signal transduction pathway after cerebral ischemic injury in rats. Focal cerebral ischemic models were established by inserting monofilament threads into the middle cerebral artery in 200 Wistar rats. Twenty four rats were randomly selected into control group, while the other rats were randomly divided into six groups: model group, picroside group, lipopolysaccharide (LPS) with picroside group, U0126 with picroside group, LPS group, and U0126 group with each group containing three subgroups with ischemia at 6, 12, and 24 h. Neurobehavioral function in the rats was evaluated by modified neurological severity score points (mNSS) test; structure of neurons was observed using hematoxylin-eosin (HE) staining; apoptotic cells were counted using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; expressions of phosphorylated mitogen/extracellular signal-regulated kinase kinas1/2 (pMEK1/2), phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2), and cyclooxygenase (COX2) in the cortex were determined using immunohistochemistry (IHC) and Western blot (WB); and real-time PCR was used to determine the level of COX2 mRNA. The neurological behavioral malfunction appeared in all rats with middle cerebral artery occlusion (MCAO). In the model group, neuron damage was extensive, while the neurobehavioral function score, apoptotic cell index, expression of pMEK1/2, pERK1/2, and COX2 and the level of COX2 mRNA increased significantly when compared to the control group. The peak COX2 mRNA level was in ischemia 12 h, prior to the peak in COX2 protein expression. In the picroside and U0126 groups, the neurological behavioral function was improved, and the number of apoptotic cells and the expression of pMEK1/2, pERK1/2, and COX2 decreased significantly when compared to the model group. In the LPS with picroside group, at ischemia 6 h neuron damage was extensive, and pMEK1/2, pERK1/2, and COX2 expression were much higher than in the model group. But at ischemia 12 and 24 h, the expression of pMEK1/2, pERK1/2, and COX2 decreased slightly, and the neurobehavioral function also improved slightly. In LPS group, neuron damage was extensive, pMEK1/2, pERK1/2, and COX2 expression was still at a high level, and COX2 mRNA peak arrived at ischemic 12 h. Picroside II downregulates COX2 expression after MCAO by inhibiting MEK-ERK1/2 in rats to protect neurons from apoptosis and inflammation.

[Study on effect of fushenkeli on expression of TAK1 in human renal tubular epithelial cells and its possible mechanism].

OBJECTIVE: To investigate the effects of fushenkeli on the expression of TAK1 in the proliferation of the renal tubular epithelial cells induced by TGF-beta1 and its possible mechanism. METHOD: Human renal tubular epithelial (HK-2) cells were divided into five groups:blank control group, TGF-beta1 group (5 microg x L(-1)), intervention group 1 (5 microg x L(-1) of TGF-beta1 + 100 mg x L(-1) of fushenkeli), intervention group 2 (5 microg x L(-1) of TGF-beta1 + 500 mg x L(-1) of fushenkeli) and intervention group 3 (5 microg x L(-1) of TGF-beta1 + 1 g x L(-1) of fushenkeli). HK-2 proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Type IV collagen in the supernatants of the cultured HK-2 was detected by ELISA at 12, 24, 48 hours respectively. The protein and mRNA expressions of TAK1 was measured by Western blot and real-time quantitative PCR. RESULT: 1) The cell proliferation and the expression of type IV collagen were increased compared with the control group (P<0.05, P<0.01), but they were decreased in intervention group. 2) The expressions of protein and mRNA of TAK1 in TGF-beta1 group were upregulating significantly compared with control group (P<0.01), but they were downregulating in intervention group, especially in intervention group 3. CONCLUSION: Fushenkeli could inhibits TAK1 expression induced by TGF-beta1 in the proliferation of HK-2 cell.

An N-methyl-D-aspartate antagonist, MK-801, preferentially reduces electroconvulsive shock-induced phosphorylation of p38 mitogen-activated protein kinase in the rat hippocampus.

Electroconvulsive shock (ECS) activates the mitogen-activated protein kinase (MAPK) family in the rat hippocampus, but the signaling pathways for this activation are not well understood. We investigated whether N-methyl-D-aspartate (NMDA) receptor mediated signaling is involved in the phosphorylation-activation of the MAPK family. The NMDA receptor antagonist, MK-801, dose-dependently reduced ECS-induced phosphorylation of p38 and its upstream kinase MKK6 up to 1 mg/kg. MK-801 also reduced the phosphorylation of ERK1/2 and MEK1, but only at high dosage, 2 mg/kg. Moreover, the reduction in the phosphorylation of p38 and MKK6 was greater than that of ERK1/2 and MEK1. Our results suggest that ECS activates p38 and ERK1/2 partly through an NMDA receptor-mediated signaling system in the rat hippocampus and that NMDA receptor mediated signaling is more responsible for the activation of the MKK6-p38 pathway than the MEK1-ERK pathway.