Mu Y P, Zhang X, Fan W W, Li X W, Chen G F, Chen J M, Zhang H, Liu P
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine; Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Zhonghua Gan Zang Bing Za Zhi. 2017 Aug 20;25(8):575-582. doi: 10.3760/cma.j.issn.1007-3418.2017.08.005.
The Notch signaling pathway is closely related to biliary fibrosis. Previous studies have shown that Astragaloside (AS) can prevent the progression of cholestatic liver fibrosis. The purpose of this study is to observe the effect of AS on the regulation of Notch signaling pathway in biliary fibrosis. Cholestatic liver fibrosis was established by common bile duct ligation (BDL) in rats. Two weeks after BDL, the rats were randomly divided into a model group (i.e., BDL), an Astragalosides group (AS), and a sorafenib (SORA) positive control group and treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Protein and gene expression were determined by immunostaining, immunoblotting and RT-PCR, respectively. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, -4, Jagged (JAG)1, Delta like (DLL)-1, -3, -4, Hes1, Numb and RBP-Jκ. Statistical analysis of variance analysis, q test, < 0.05 showed that the difference was statistically significant. (1) AS significantly reduced the deposition of collagen and the Hyp content of liver tissue (500.15 ± 86.10 vs. 625.72 ± 105.62, = 0.031), and inhibited the activation of hepatic stellate cells. (2) AS significantly decreased the protein and mRNA expressions of transforming growth factor (TGF)-β1 (1.02±0.15 vs. 1.89±0.36, = 0.007; 1.17±0.18 vs. 1.68±0.29, = 0.013, respectively) and α-smooth muscle actin (α-SMA, 0.41±0.11 vs. 0.72±0.16, = 0.003; 1.71±0.57 vs. 2.68±0.46, = 0.008, respectively) compared with BDL group. In contrast, AS significantly enhanced expression of the Smad 7 protein compared with the BDL group (0.72±0.008 vs. 0.33±0.001, = 0.005). AS also reduced biliary epithelial cell proliferation. AS reduced the mRNA levels of CK7, CK8 and CK18 (1.31±0.39 vs. 2.63±0.82, = 0.009; 0.71±0.09 vs. 0.87±0.08, = 0.031; 2.56±0.32 vs. 3.41±0.39, = 0.010, respectively) and reduced the positive areas of CK19 and OV6 (62 337.17±21 873.38 vs. 22 5472.67±26 933.63, = 0.000; 92 237.43±15 894.11 vs. 171 298.13±61 761.37, = 0.000, respectively). (3) The mRNA expression of Notch-2, -3, -4 and JAG1 were significantly reduced in the AS group compared to the BDL group (1.07±0.19 vs. 1.51±0.28, = 0.044; 0.99±0.24 vs. 1.18±0.10, = 0.043; 1.36±0.42 vs. 3.40±0.44, = 0.048; 2.62±0.43 vs. 3.73±0.83, = 0.046, respectively). In contrast, the mRNA level of Numb was clearly enhanced after AS treatment (0.90±0.05 vs. 0.75±0.11, = 0.019). In addition, consistent with the mRNA levels, the protein expressions of Notch-2, -3, -4 and JAG1 were reduced significantly (1.27±0.18 vs. 1.71±0.26, = 0.004; 0.99±0.11 vs. 4.38±0.60, = 0.001; 1.76±0.32 vs. 4.01±0.74, = 0.002; 1.62±0.33 vs. 2.74±0.63, = 0.002) and the Numb protein level was increased significantly (1.50±0.15 vs. 0.85±0.11, = 0.001) in AS group compared with BDL group. AS may prevent cholestatic liver fibrosis via inhibition of the Notch signaling pathway, thereby inhibiting the abnormal proliferation of biliary epithelial cells. Results indicate that AS may be a potential treatment for cholestatic liver disease.
Notch信号通路与胆汁性肝纤维化密切相关。既往研究表明，黄芪甲苷（AS）可阻止胆汁淤积性肝纤维化的进展。本研究旨在观察AS对胆汁性肝纤维化中Notch信号通路调控的影响。通过大鼠胆总管结扎（BDL）建立胆汁淤积性肝纤维化模型。BDL术后两周，将大鼠随机分为模型组（即BDL组）、黄芪甲苷组（AS组）和索拉非尼（SORA）阳性对照组，并进行3周治疗。通过组织染色测定胆管增殖和肝纤维化情况。分别通过免疫染色、免疫印迹和RT-PCR测定蛋白质和基因表达。通过分析Notch-1、-2、-3、-4、锯齿状蛋白（JAG）1、Delta样蛋白（DLL）-1、-3、-4、Hes1、Numb和RBP-Jκ的表达评估Notch信号通路的激活情况。方差分析、q检验的统计学分析显示，P<0.05表明差异具有统计学意义。（1）AS显著降低肝组织胶原沉积和羟脯氨酸含量（500.15±86.10 vs. 625.72±105.62，P=0.031），并抑制肝星状细胞激活。（2）与BDL组相比，AS显著降低转化生长因子（TGF）-β1的蛋白质和mRNA表达（分别为1.02±0.15 vs. 1.89±0.36，P=0.007；1.17±0.18 vs. 1.68±0.29，P=0.013）以及α-平滑肌肌动蛋白（α-SMA，分别为0.41±0.11 vs. 0.72±0.16，P=0.003；1.71±0.57 vs. 2.68±0.46，P=0.008）。相反，与BDL组相比，AS显著增强Smad 7蛋白表达（0.72±0.008 vs. 0.33±0.001，P=0.005）。AS还减少胆管上皮细胞增殖。AS降低CK7、CK8和CK18的mRNA水平（分别为1.31±0.39 vs. 2.63±0.82，P=0.009；0.71±0.09 vs. 0.87±0.08，P=0.031；2.56±0.32 vs. 3.41±0.39，P=0.010），并减少CK19和OV6的阳性面积（分别为62 337.17±21 873.38 vs. 225 472.67±26 933.63，P=0.000；92 237.43±15 894.11 vs. 171 298.13±61 761.37，P=0.000）。（3）与BDL组相比，AS组中Notch-2、-3、-4和JAG1的mRNA表达显著降低（分别为1.07±0.19 vs. 1.51±0.28，P=0.044；0.99±0.24 vs. 1.18±0.10，P=0.043；1.36±0.42 vs. 3.40±0.44，P=0.048；2.62±0.43 vs. 3.73±0.83，P=0.046）。相反，AS治疗后Numb的mRNA水平明显升高（0.90±0.05 vs. 0.75±0.11，P=0.019）。此外，与mRNA水平一致，与BDL组相比，AS组中Notch-2、-3、-4和JAG1的蛋白质表达显著降低（分别为1.27±0.18 vs. 1.71±0.26，P=0.004；0.99±0.11 vs. 4.38±0.60，P=0.001；1.76±0.32 vs. 4.01±0.74，P=0.002；1.62±0.33 vs. 2.74±0.63，P=0.002），且Numb蛋白水平显著升高（1.50±0.15 vs. 0.85±0.11，P=0.001）。AS可能通过抑制Notch信号通路来预防胆汁淤积性肝纤维化，从而抑制胆管上皮细胞的异常增殖。结果表明，AS可能是胆汁淤积性肝病的一种潜在治疗方法。
