Baicalein potently inhibits Rho kinase activity and suppresses actin stress fiber formation in angiotensin II-stimulated H9c2 cells.

Baicalein is a flavonoid (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran4-one) and an active principle in Scutellaria baicalensis. The present study was performed to investigate the mechanisms underlying the anti-fibrotic effects of baicalein with a focus on Rho kinase (ROCK) inhibition. The effect of baicalein on ROCK activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The underlying mechanisms of baicalein were examined using angiotensin II-stimulated H9c2 cells. Rho kinase (ROCK1 and ROCK2) studies using IMAP-TR-FRET showed that baicalein possesses potent ROCK inhibitory activity with IC50 values of 6.55 and 2.82 µM, respectively. Pretreatment with baicalein (for 2 h) concentration-dependently decreased the angiotensin II-induced phosphorylation of myosin phosphatase (MYPT) and myosin light chain (MLC). Furthermore, baicalein also concentration-dependently suppressed actin stress fiber formation in angiotensin II-stimulated H9c2 cells. These results suggest that baicalein potently inhibits ROCK and that by so doing it modulates actin stress fiber formation. These anti-fibrotic effects of baicalein explain, at least in part, its pharmacology and mode of action.

Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKKß inhibition in RAW 264.7 cells.

AIM OF THE STUDY: The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with the extract of Euonymus alatus (EEA), and specially focused on nuclear factor κB (NF-κB) signaling pathway by targeting the IκB kinase ß (IKKß). MATERIALS AND METHODS: The effect of EEA for IKKß activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The effect of EEA on lipopolysaccharide (LPS)-induced NF-κB activation in murine macrophage RAW 264.7 cells with western blotting and immunofluorescent staining was evaluated. RESULTS: IKKß studies based on IMAP-TR-FRET showed that EEA possesses a potent IKKß inhibitory activity with IC(50) value of 11.83µg/ml. EEA (10, 30µg/ml) also attenuated the LPS-induced IκBα phosphorylation/degradation, NF-κB translocation and subsequent NO synthesis in RAW 264.7 cells. CONCLUSIONS: These results suggest that EEA abrogates LPS-induced NF-κB signaling pathway by targeting the IKKß in RAW 264.7 cells and these properties may provide a molecular basis for understanding the inhibitory effects of EEA on LPS-mediated inflammation.

Melanin-concentrating hormone-1 receptor binding activity of pheophorbides isolated from Morus alba leaves.

The time-resolved fluorescence technique based on melanin-concentrating hormone (MCH) receptor subtype-1 (MCH-1 receptor) binding assay was adopted to carry out a bioassay-guided fractionation of the methanol extract of Morus alba leaves. This fractionation and purification led to the isolation of two compounds identified as pheophorbide a methyl ester and 13(2)(S)-hydroxypheophorbide a methyl ester. These active pheophorbides exhibited potent inhibitory activity in binding of europium-labeled MCH to the human recombinant MCH-1 receptor (IC(50) value; 4.03 and 0.33 microM, respectively). Besides binding activity, the pheophorbides inhibited MCH-mediated extracellular signal-regulated kinase (ERK) phosphorylation in Chinese hamster ovary cells expressing human MCH-1 receptor. These results suggest that pheophorbide a methyl ester and 13(2)(S)-hydroxypheophorbide a methyl ester act as modulators of MCH-1 receptor and MCH-mediated ERK signaling.

Melanin-concentrating hormone-1 receptor antagonism and anti-obesity effects of ethanolic extract from Morus alba leaves in diet-induced obese mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Korea, Morus alba leaves have been traditionally administered as natural therapeutic agent for the alleviating dropsy and diabetes. AIM OF THE STUDY: The present study was performed to evaluate melanin-concentrating hormone receptor subtype 1 (MCH1) antagonism of the ethanol extract of Morus alba leaves (EMA) and its anti-obesity effect in diet-induced obese (DIO) mice. MATERIALS AND METHODS: The binding affinity of EMA for the MCH1 receptor with europium-labeled MCH (Eu-MCH), the function of recombinant MCH1 receptors expressed in CHO cells, and the anti-obesity effects in DIO mice were evaluated. RESULTS: MCH1 receptor binding studies showed, EMA exhibited a potent inhibitory activity with IC50 value of 2.3+/-1.0 microg/ml. EMA (10-100 microg/ml) also inhibited the intracellular calcium mobilization with the recombinant MCH1 receptors expressed in CHO cells. In an anti-obesity study with DIO mice, longterm oral administrations of EMA for 32 consecutive days produced a dose-dependent decrease in body weight and hepatic lipid accumulation. CONCLUSIONS: These results suggest that chronic treatment with EMA exerts an anti-obesity effect in DIO mice, and its direct MCH1 receptor antagonism may contribute to decrease body weight.

Antihypertensive, vasorelaxant, and antioxidant effect of root bark of Ulmus macrocarpa.

The present study was performed to evaluate the cardiovascular effects of ethanolic extract from the root bark of Ulmus macrocarpa (RBUM) in rats. The effects of RBUM on the vascular response of isolated rat aorta and the blood pressure of spontaneously hypertensive rats (SHRs) were evaluated. In addition, its antioxidant activity in H9c2 cells was investigated. In the free radical scavenging assay using 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH), RBUM exhibited significant scavenging activity with an EC50 value of 14.3 microg/ml. RBUM also induced resistance to hydrogen peroxide-mediated oxidative insult in H9c2 myocardial cells. In isolated rat aortic preparations, RBUM exhibited potent vascular relaxant effect with an EC50 value of 1.9 microg/ml. This relaxation was significantly inhibited by denudation of the endothelial layer, pretreatment with NG-nitro-L-arginine methyl ester (10 microM), raising extracellular K+ (45 mM), and pretreatment with tetraethylammonium (10 mM). In an antihypertensive study with SHRs, long-term administration with RBUM (100 mg/kg) for 42 d decreased systolic blood pressure (approximately 20 mmHg). In SHRs after 42 d of treatment, RBUM recovered aortic relaxation to acetylcholine and sodium nitroprusside, and attenuated lipid peroxidation in liver of SHRs. These results suggest that chronic treatment with RBUM exerts antihypertensive effects in SHRs, and its direct vasorelaxant and antioxidant properties may contribute to reduce elevated blood pressure.

Cardiovascular effects of lignans isolated from Saururus chinensis.

Saururus chinensis has been widely used as a traditional medicine for the treatment of beriberi, hypertension, pneumonia, edema, jaundice and gonorrhea. However, there is only limited information on the cardiovascular effects of S. chinensis extract or its single compounds. The present study was performed to investigate the effects of active lignans isolated from the extract of S. chinensis on vascular responses and heart functions. The vasorelaxant activity-guided fractionation of roots extract of S. chinensis led to the isolation of eight lignans as active principles. These lignans produced concentration-dependent relaxations of the endothelium-intact aortic preparations of rat aorta. Particularly, saucerneol ( 1), saucerneol D ( 2) and machilin D ( 8) exhibited distinctive vasorelaxant activity (EC (50) values: 2.2, 12.7 and 17.8 microM, respectively), which were significantly inhibited by removal of functional endothelium or pretreatment with N(G)-nitro-L-arginine methyl ester. Saucerneol ( 1) and saucerneol D ( 2) caused a significant decrease in left ventricular pressure, +dP/dt (max) and heart rate in isolated hearts. These results suggest that several lignans including saucerneol ( 1), saucerneol D ( 2) and machilin D ( 8), isolated from the ethanol extract of the roots of S. chinensis, have significant cardiovascular effects such as vasorelaxant and negative inotropic actions.