Tanshinone IIA Inhibits Angiotensin II-Induced Cell Proliferation in Rat Cardiac Fibroblasts

Abstract

Tanshinone IIA extracted from danshen, a popular medicinal herb used in traditional Chinese medicine, exhibits cardio-protective effects. However, the mechanism of its cardioprotective effect is not well established. The aims of this study were to examine whether tanshinone IIA may alter angiotensin II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pre-treated with tanshinone IIA and stimulated with Ang II, cell proliferation and endothelin-1 (ET-1) expression were examined. The effect of tanshinone IIA on Ang II-induced reactive oxygen species (ROS) formation, and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition, the effect of tanshinone IIA on nitric oxide (NO) production, and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. The increased cell proliferation and ET-1 expression by Ang II (100 nM) were partially inhibited by tanshinone IIA. Tanshinone IIA also inhibited Ang II-increased ROS formation, and ERK phosphorylation. In addition, tanshinone IIA was found to increase the NO generation, and eNOS phosphorylation. N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of tanshinone IIA on Ang II-induced cell proliferation. The results suggest that tanshinone IIA prevents cardiac fibroblast proliferation by interfering with the generation of ROS and involves the activation of the eNOS-NO pathway.

Tanshinone IIA Inhibits High Glucose-Induced Collagen Synthesis via Nuclear Factor Erythroid 2-Related Factor 2 in Cardiac Fibroblasts

“…Proliferation was assessed by quantifying 5-bromo-2'-deoxyuridine (BrdU) incorporation [5]. Cells were removed from the culture dish by addition of trypsin and then centrifuged.…”

“…Total RNA was extracted from cardiac fibroblasts using the TRIzol method according to the protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA, USA), and used to synthesize single-stranded complementary (c) DNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) as previously described [5]. TGF-β1 mRNA was quantified using TaqMan Gene Expression Master Mix (Applied Biosystems) with specific primers in an ABI 7300 Real-Time PCR System (Applied Biosystems).…”

“…Western blot analysis Nuclear protein was prepared as previously described [4]. Western blot analysis was performed as previously described [5]. Whole-cell extracts were obtained in a RIPA buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 1.0% Triton X-100, 1% Deoxycholate, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail; Complete, Roche Diagnostics GmbH, Germany).…”

“…The determination of intracellular reactive oxygen species production was based on the oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) to a fluorescent 2′,7′-dichlorofluorescein (DCF) as previously described [5]. DCFH was added at a final concentration of 10 μM and incubated for 30 min at 37°C.…”

“…A series of our studies demonstrated tanshinone IIA prevents doxorubicininduced cardiomyocyte apoptosis [2], attenuates H 2 O 2 -induced injury [3]and induces heme oxygenase-1 (HO-1) expression by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in vascular endothelial cells [4], inhibits angiotensin II-induced cell proliferation via antioxidant activity in rat cardiac fibroblasts [5]. However, the effects and mechanisms of tanshinone IIA on cardio-protection are yet to be delineated.…”