Huang Weijuan, Li Lin, Tian Xiaopeng, Yan Jinjin, Yang Xinzheng, Wang Xinlong, Liao Guozhen, Qiu Genquan
Department of Scientific Research, Xi'an Medical College, Xi'an, Shaanxi 710061, P.R. China.
State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat‑sen University, Guangzhou, Guangdong 510060, P.R. China.
Mol Med Rep. 2015 Apr;11(4):2569-77. doi: 10.3892/mmr.2014.3026. Epub 2014 Dec 1.
Previous studies have shown that Astragalus and Paeoniae Radix Rubra extract (APE) is capable of protecting against liver fibrosis in rats. The hypothesis of the present study was that APE exerts its anti‑fibrotic effect by mediating the transforming growth factor β (TGF‑β)/Smad signaling pathway. In order to investigate this hypothesis, a series of assays were designed to detect the effects of APE on cell proliferation, cell invasion and the activation of hepatic stellate cells (HSCs). In addition, the effects of APE on the TGF‑β/Smad signaling pathway were explored, with the aim of elucidating the underlying mechanisms. HSCs were initially isolated from normal rat liver. A number of assays were then employed in order to evaluate the effects of APE on the function of these cells. Cell proliferation was investigated using an MTT assay and cell invasion was observed with the use of transwell invasion chambers. Collagen synthesis was measured with a 3H‑proline incorporation assay and expression of α‑smooth muscle actin was used to determine the extent of HSC activation. Protein expression induced by TGF‑β1 in HSCs was investigated by western blot and immunofluorescence analyses. Plasminogen activator inhibitor type1 (PAI‑1) and urokinase‑type plasminogen activator (uPA) transcriptional activity was measured using reverse transcription polymerase chain reaction. The results demonstrated that APE (5‑80 µg/ml) significantly inhibited fetal bovine serum‑induced cell proliferation in a dose‑dependent manner. Cell invasion and activation of HSCs induced by TGF‑β1 were disrupted by treatment with APE in a dose‑dependent manner. TGF‑β1 was observed to increase the phosphorylation of Smad2/3, while APE administered at higher doses produced inhibitory effects on Smad2/3 phosphorylation. In addition, administration of APE abrogated the TGF‑β1‑induced reduction in Smad‑7 expression in a dose‑dependent manner. The results further indicated that APE treatment not only reduced PAI‑1 expression, but also increased uPA expression in a dose‑dependent manner. In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF‑β1/Smad pathway.
先前的研究表明，黄芪和赤芍提取物（APE）能够保护大鼠免受肝纤维化的影响。本研究的假设是，APE通过介导转化生长因子β（TGF-β）/Smad信号通路发挥其抗纤维化作用。为了研究这一假设，设计了一系列试验来检测APE对细胞增殖、细胞侵袭和肝星状细胞（HSC）激活的影响。此外，还探讨了APE对TGF-β/Smad信号通路的影响，以阐明其潜在机制。HSC最初从正常大鼠肝脏中分离出来。然后采用了多种试验来评估APE对这些细胞功能的影响。使用MTT试验研究细胞增殖，并使用Transwell侵袭小室观察细胞侵袭。用3H-脯氨酸掺入试验测量胶原蛋白合成，并使用α-平滑肌肌动蛋白的表达来确定HSC激活的程度。通过蛋白质印迹和免疫荧光分析研究TGF-β1诱导的HSC中蛋白质表达。使用逆转录聚合酶链反应测量纤溶酶原激活物抑制剂1型（PAI-1）和尿激酶型纤溶酶原激活物（uPA）的转录活性。结果表明，APE（5-80μg/ml）以剂量依赖性方式显著抑制胎牛血清诱导的细胞增殖。APE处理以剂量依赖性方式破坏了TGF-β1诱导的HSC细胞侵袭和激活。观察到TGF-β1增加Smad2/3的磷酸化，而高剂量施用的APE对Smad2/3磷酸化产生抑制作用。此外，APE的施用以剂量依赖性方式消除了TGF-β1诱导的Smad-7表达降低。结果进一步表明，APE处理不仅降低了PAI-1表达，还以剂量依赖性方式增加了uPA表达。总之，APE对HSC的细胞增殖、侵袭和激活具有抑制作用，这些作用的潜在机制可能涉及TGF-β1/Smad途径。
