Hypertension, myocardial infarction, and diabetes mellitus can cause cardiac fibroblast proliferation, transform into myofibroblasts and secrete much extracellular matrix that deposits in the region of myocardial interstitial tissue, leading to myocardial fibrosis and eventually resulting in severely damaged cardiac function[1]. Therefore, regulation of fibroblast proliferation and phenotypic transformation can prevent and treat myocardial fibrosis and improve the prognosis of heart disease[13]. Tanshinone ⅡA is the liposoluble active component of traditional Chinese medicine salvia miltiorrhiza. It can effectively inhibit the proliferation of hepatic stellate cells and renal interstitial fibroblasts, and reverse or postpone hepatic and renal fibrosis[14-16]. Recent studies[4-5] demonstrate that tanshinone ⅡA can block rat cardiac fibroblast proliferation and phenotypic transformation, reduce extracellular matrix production and inhibit myocardial fibrosis[4-5]. Based on this, the present study further investigated the possible mechanism underlying tanshinone ⅡA inhibition of fibroblast proliferation and phenotypic transformation.
Evidence[2, 17] exists that fibroblast proliferation and phenotypic transformation are primarily related to excessive activation of TGF-β signal pathway. Following extracellular activation of TGF-β, Smads protein mediates intracellular signal transmission. Smads are a kind of highly conserved protein family, with a molecular weight of 42-60 kDa. They can be divided into three categories based on structural and functional characteristics: receptor-regulated Smads (it was Smad2/3 that was related to TGFβ-1 signal transduction), common mediator (primarily Smad4), and inhibitory Smads (consisting of Smad6 and Smad 7). Smad2/3 can promote fibroblast proliferation and phenotypic transformation as well as synthesis and secretion of extracellular matrix, playing a key role in the TGF-β1 signal transduction[18-19]. Fibronectin, the primary component of extracellular matrix, exhibits strong ability to bind fibrin, fibrinogen, and collagen. Its synthesis and expression level were obviously positively related to tissue fibrosis[20].
Results from this study demonstrated that TGF-β1 induces cardiac fibroblasts to express phosphorylated Smad2/3 expression in a time-dependent manner, which is consistent with the effects in promoting fibronectin expression. This suggests that TGF-β1/Smads signal pathway exists in cardiac fibroblasts. Tanshinone ⅡA pretreatment can inhibit TGF-β1-induced phosphorylated Smad2/3 and fibronectin expression in a dose-dependent manner: 10-6 mol/L tanshinone ⅡA pretreatment hardly produced effects on phosphorylated Smad2/3 and fibronectin expression, while 10-5 and 10-4 mol/L tanshinone ⅡA pretreatment could obviously decrease these expressions. Following 10-5 and 10-4mol/L tanshinone ⅡA pretreatment, phosphorylated Smad2/3 protein expression was decreased by 40.5% and 55.4%, respectively, while fibronectin mRNA expression was decreased by 32.7% and 42.9%, respectively and fibronectin protein expression was decreased by 45.8% and 57.1%, respectively. This indicates that tanshinone ⅡA can hinder TGF-β1-induced Smad2/3 phosphorylation and inhibit extracellular matrix synthesis and secretion.
This esperiment was a cytological in vitro experiment, so tanshinone ⅡA solution was used. To investigate whether tanshinone ⅡA effects on TGF-β1/Smads signal pathway in a dose dependent manner, this study used three concentrations of tanshinone ⅡA (10-6, 10-5, and 10-4 mol/L) based on existing reports[13-15], but whether these concentrations are the optimal doses remains unclear. In addition, TGF-β1 signal transduction is very complex. Results from this study revealed that tanshinone vA could inhibit TGF-β1-induced Smad2/3 phosphorylation, but the precise effect target of tanshinone IIA remains uncertain. Tanshinone IIA effecting on Smad2/3 directly or via upstream or downstream pathway needs further investigation.
Taken together, the present study observed the effects of tanshinone ⅡA on signal transduction pathway of TGF-β1, one of most important factors known to promote myocardial fibrosis, to further study the molecular mechanism underlying tanshinone ⅡA against myocardial fibrosis. Results preliminarily confirm that tanshinone ⅡA can a part block TGF-β1 intracellular signal transduction, inhibit cardiac fibroblast proliferation and phenotypic transformation, and reduce TGF-β1-induced extracellular matrix synthesis, showing a good prospect in clinical prevention and treatment of myocardial fibrosis.
