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

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.

Cordyceps polysaccharide ameliorates airway inflammation in an ovalbumin-induced mouse model of asthma via TGF-ß1/Smad signaling pathway.

Allergic asthma is a chronic inflammatory disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), airway inflammation, and mucus overproduction. Cordyceps polysaccharide (CPS) is one of the main bioactive compounds of Cordyceps militarisis, a traditional Chinese medicine. In this study, we established a mouse model of asthma using ovalbumin (OVA) challenge and evaluated the potential regulatory effect of CPS (25, 50, and 100 mg/kg) on asthmatic mice. These results showed that the asthmatic mice treated with CPS suppressed the secretion of eotaxin, IL-4, IL-5, IL-13, and IFN-γ in the blood and bronchoalveolar lavage fluid (BALF), and decreased serum IgE levels compared to the vehicle-treated mice. CPS also alleviated inflammatory cell infiltration, goblet cell hyperplasia, and the increases of inflammatory cells in the mouse model of asthma. In addition, OVA-induced AHR was inhibited by CPS treatment. Further analyses of protein expression revealed that CPS inhibited the activation of transforming growth factor ß1 (TGF-ß1)/Smad pathway in mice with asthma. These findings indicated that CPS might serve as a potential therapeutic agent for the management of allergic asthma.

Synergistic effect of vitamin D and low concentration of transforming growth factor beta 1, a potential role in dermal wound healing.

Dermal wound healing, in which transforming growth factor beta 1 (TGFß1) plays an important role, is a complex process. Previous studies suggest that vitamin D has a potential regulatory role in TGFß1 induced activation in bone formation, and there is cross-talk between their signaling pathways, but research on their effects in other types of wound healing is limited. The authors therefore wanted to explore the role of vitamin D and its interaction with low concentration of TGFß1 in dermal fibroblast-mediated wound healing through an in vitro study. Human dermal fibroblasts were treated with vitamin D, TGFß1, both, or vehicle, and then the wound healing functions of dermal fibroblasts were measured. To further explore possible mechanisms explaining the synergistic effect of vitamin D and TGFß1, targeted gene silencing of the vitamin D receptor was performed. Compared to either factor alone, treatment of fibroblasts with both vitamin D and low concentration of TGFß1 increased gene expression of TGFß1, connective tissue growth factor, and fibronectin 1, and enhanced fibroblast migration, myofibroblast formation, and collagen production. Vitamin D receptor gene silencing blocked this synergistic effect of vitamin D and TGFß1 on both collagen production and myofibroblast differentiation. Thus a synergistic effect of vitamin D and low TGFß1 concentration was found in dermal fibroblast-mediated wound healing in vitro. This study suggests that supplementation of vitamin D may be an important step to improve wound healing and regeneration in patients with a vitamin D deficiency.

Daidzein stimulates collagen synthesis by activating the TGF-ß/smad signal pathway.

BACKGROUND/OBJECTIVES: The objective of this study was to investigate the effects of daidzein on collagen metabolism and its underlying mechanism in cultured skin fibroblast and nude mouse skin. METHODS: Skin fibroblasts were exposed to different concentrations of daidzein (0.5-50 µg/mL) for 24 h or 48 h, respectively. Female nude mice were treated topically with 200 µg/mL daidzein once a day for 6 weeks. Cell viability and cell cycle were determined by MTT and flow cytometer. The transcriptional activity of collagen type I was evaluated and the expression of procollagen, matrix metalloproteinase-1 (MMP1) and MMP2 were measured by real-time polymerase chain reaction. A Western blot analysis was applied to detect the levels of phosphorylated-Smad2 and Smad3. RESULTS: In the daidzein-treated cells the expression of type I procollagen increased markedly while the expressions of MMP1, and MMP2 was significantly inhibited. Additionally, the mouse skin showed more collagen deposition after daidzein treatment. The levels of transforming growth factor (TGF)-ß, phosphorylated-smad2 and smad3 were also higher in the daidzein treated skin fibroblasts than in the controls. CONCLUSIONS: The results showed that daidzein treatment can increase skin collagen synthesis and inhibit collagen degradation in vitro and in vivo. It seems that TGF-ß/smad signalling pathways play an important role in daidzein-induced collagen accumulation.

Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-ß1/Smad signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Shensong Yangxin Capsule (SSYX), a traditional Chinese herbal medicine, has long been used clinically to treat arrhythmias in China. However, the effect of SSYX on interstitial fibrosis in diabetic cardiomyopathy is unknown. The objective of this study was to investigate the effects of SSYX on myocardial fibrosis in diabetic rats. MATERIALS AND METHODS: The antifibrotic effect of SSYX was investigated in streptozocin (STZ)-induced diabetic rats with high fat-diet (HFD). Fasting blood glucose, heart weight/body weight (HW/BW) ratio, total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were measured. Echocardiography and histology examination were carried out to evaluate heart function. Expressions of Smad7, TGF-ß1, collagen I (col-1), collagen III (col-3), MMP-2, MMP-9 and α-SMA mRNA in heart tissues were measured by real time polymerase chain reaction (PCR). TGF-ß1, Smad2/3, p-Smad2/3 and Smad7 protein levels were measured by western blot analysis. Proliferation of cardiac fibroblast was detected via immunofluorescence. RESULTS: SSYX markedly decreased HW/BW ratio and improved the impaired cardiac function of type-2 diabetes mellitus (T2DM) rats. Transmission electron microscopy (TEM), haematoxylin and eosin (HE) and Masson staining results showed that SSYX attenuated cardiac fibrosis and collagen deposition in T2DM rats. Moreover, mRNA levels of TGF-ß1, col-1, col-3, MMP-2, MMP-9 and α-SMA were downregulated, whereas Smad7 expression was upregulated after treatment with SSYX in rats with cardiac fibrosis. Furthermore, SSYX decreased protein levels of TGF-ß1 and p-Smad2/3, and increased Smad7 expression. CONCLUSION: TGF-ß1/Smad signaling is involved in the cardiac fibrosis in diabetic cardiomyopathy and SSYX inhibits fibrosis and improves cardiac function via suppressing this pathway. Therefore, SSYX might be considered as an alternative therapeutic remedy for diabetic cardiomyopathy.

In vitro suppression of quercetin on hypertrophy and extracellular matrix accumulation in rat glomerular mesangial cells cultured by high glucose.

Quercetin's protective effects on the glomerulosclerosis of diabetic nephropathy (DN) in rat mesangial cells were investigated. The cell cycles, type IV collagen and laminin, TGF-ß(1) mRNA, Smad 2/3 and Smad 7, and activities of cell antioxidases were measured. Compared with the high glucose group, quercetin may decrease the cell percentages of G(0)/G(1) phase, Smad 2/3 expression, laminin and type IV collagen, and TGF-ß(1) mRNA level significantly. The antioxidant capacity, the cell percentages of S phase and Smad 7 expression was significantly increased by quercetin. These results suggest that quercetin is a protective agent against glomerulosclerosis in DN.