Qingda granule attenuates cardiac fibrosis via suppression of the TGF-ß1/Smad2/3 signaling pathway in vitro and in vivo.

ABSTRACT

Cardiac fibrosis plays an important role in hypertension-related contractile dysfunction and heart failure. Qingda granule (QDG), derived from the Qingxuan Jiangya decoction, has been used clinically for more than 60 years to treat hypertension. However, the effect of QDG on hypertensive cardiac fibrosis remains largely unknown. The objective of this study was to investigate the effect of QDG on cardiac fibrosis and explore the underlying mechanism in vivo and in vitro. For in vivo experiments, 30 male spontaneously hypertensive rats were randomly divided into groups that received no QDG or one of three doses (0.45, 0.9 or 1.8 g/kg/day). Positive-control animals received valsartan (VAL, 7.2 mg/kg/day). Treatments were administered by gavage for 10 weeks. All three doses of QDG and VAL led to significantly lower blood pressure than in SHR animals. Besides, all three doses of QDG and VAL attenuated pathological changes in SHR animals. However, only intermediate, high concentrations of QDG and VAL led to significantly lower left ventricle ejection fraction and left ventricle fractional shortening than in SHR animals. Therefore, the minimum and effective QDG dose (intermediate concentration of QDG) was selected for subsequent animal experiments in this study. Our results showed that intermediate concentration of QDG also significantly mitigated the increases in levels of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), collagen III, transforming growth factor-ß1 (TGF-ß1) and in the ratio of phospho-Smad2/3 to total Smad2/3 protein in cardiac tissue, based on immunohistochemistry, Western blotting, and Masson staining. For in vitro experiments, primary cardiac fibroblasts were stimulated with 100 nM angiotensin II in the presence or absence of QDG. And we tested different concentrations of QDG (3.125, 6.25, 12.5, 25, 50 µg/mL) in the cell viability experiment. Our results showed that 3.125, 6.25 and 12.5 µg/mL of QDG treatment for 24 h didn't affect the cell viability of cardiac fibroblasts. Consistently, QDG at 6.25 or 12.5 µg/mL significantly reduced cell viability and down-regulated α-SMA in primary cardiac fibroblasts were stimulated with 100 nM angiotensin II. Therefore, QDG at 12.5 µg/mL was chosen for the following cell experiment. Our results showed that QDG at 12.5 µg/mL alleviated the increase of PCNA, collagen Ⅲ, TGF-ß1 expression, and the ratio of phospho-Smad2/3 to total Smad2/3 protein. Our studies in vitro and in vivo suggest that QDG reduces blood pressure and cardiac fibrosis as well as protecting cardiac function, and that it exerts these effects in part by suppressing TGF-ß1/Smad2/3 signaling.