Laboratory of Animal Disease Model, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, 625014, Sichuan, China.
Hepatol Int. 2018 Jan;12(1):26-36. doi: 10.1007/s12072-017-9841-y. Epub 2018 Jan 12.
This study aimed to investigate the mechanism of the interaction between Yes-associated protein (YAP) and transforming growth factor-β (TGF-β)/Smad signaling pathways in the development of non-alcoholic fatty liver disease (NAFLD).
Serum samples of monkeys with biopsy-proven NAFLD and healthy normal monkeys were used to measure fasting plasma glucose (FPG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG) and albumin (ALB) with the BECKMAN CX5 PRO. Hematoxylin-eosin staining (H&E) was used for pathologic analysis, Masson trichrome staining was used to assess for fibrosis staging, and Oil Red O staining was used to detect lipid droplet deposition. According to an NAFLD activity score of < 4 points and > 4 points, the samples were divided into groups: the steatosis group and fibrosing NASH group. Furthermore, monkeys with a fibrosis stage < 2 were assigned to the mild fibrosis group, while monkeys with fibrosis stage ≥ 2 were assigned to the significant fibrosis group. Moreover, the fibrosis stage was subdivided as follows: stages 1a, 1c and 2-3. Immunohistochemistry and real-time quantitative PCR were used to quantify protein and gene expression, respectively.
In the present study, 54 monkeys with NAFLD and 23 normal monkeys were recruited. Serum FPG and TG levels were higher in fibrosing NASH monkeys compared with simple steatosis and normal monkeys, and differences between simple steatosis and normal monkeys were not statistically significant (p > 0.05). YAP increased in NAFLD, which mainly localized in the nuclei of hepatocytes, perivascular cells and bile duct cells; the accumulation of YAP correlated with the severity of hepatocyte injury. Compared with normal monkeys, the expression of TGF-β, α-smooth muscle actin (α-SMA), Drosophila mothers against decapentaplegic protein 3 (Smad3) and connective tissue growth factor (CTGF) in the liver of simple steatosis monkeys significantly increased (p < 0.01). Compared with simple steatosis monkeys, the expression of TGF-β, α-SMA, Smad3 and CTGF in fibrosing NASH significantly increased (p < 0.01). However, the expression of Drosophila mothers against decapentaplegic protein 7 (Smad7) in the liver of fibrosing NASH monkeys significantly decreased (p < 0.01). With the severity of liver fibrosis, the expression of TGF-β, α-SMA, Smad3 and CTGF gradually increased, and the difference was statistically significant (p < 0.01). However, there was no significant difference in the expression of Smad3 between fibrosis stage 1a and 1c. Compared with normal monkeys, the expression of Smad7 in the liver of monkeys with fibrosis significantly decreased (p < 0.01), but was significantly higher at fibrosis stage 1c than at fibrosis stage 1a and 2.
The YAP and TGF-β signaling pathways and the interaction between them promote the development and progression of NAFLD.
本研究旨在探讨 Yes 相关蛋白（YAP）与转化生长因子-β（TGF-β）/Smad 信号通路在非酒精性脂肪性肝病（NAFLD）发展中的相互作用机制。
采用贝克曼 CX5 PRO 检测活检证实的 NAFLD 猴和健康正常猴的血清样本中的空腹血糖（FPG）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）、甘油三酯（TG）和白蛋白（ALB）。苏木精-伊红（H&E）染色用于病理分析，Masson 三色染色用于评估纤维化分期，油红 O 染色用于检测脂质滴沉积。根据 NAFLD 活动评分<4 分和>4 分，将样本分为两组：脂肪变性组和纤维化 NASH 组。此外，将纤维化分期<2 的猴分为轻度纤维化组，纤维化分期≥2 的猴分为显著纤维化组。此外，纤维化分期进一步分为 1a、1c 和 2-3 期。免疫组织化学和实时定量 PCR 分别用于定量蛋白和基因表达。
本研究共纳入 54 只 NAFLD 猴和 23 只正常猴。与单纯脂肪变性和正常猴相比，纤维化 NASH 猴的血清 FPG 和 TG 水平升高，而单纯脂肪变性和正常猴之间的差异无统计学意义（p>0.05）。YAP 在 NAFLD 中增加，主要定位于肝细胞、血管周细胞和胆管细胞的核内；YAP 的积累与肝细胞损伤的严重程度相关。与正常猴相比，单纯脂肪变性猴肝组织中 TGF-β、α-平滑肌肌动蛋白（α-SMA）、果蝇母源抑制物 Decapentaplegic 蛋白 3（Smad3）和结缔组织生长因子（CTGF）的表达显著增加（p<0.01）。与单纯脂肪变性猴相比，纤维化 NASH 猴 TGF-β、α-SMA、Smad3 和 CTGF 的表达显著增加（p<0.01）。然而，纤维化 NASH 猴肝组织中果蝇母源抑制物 Decapentaplegic 蛋白 7（Smad7）的表达显著降低（p<0.01）。随着肝纤维化程度的加重，TGF-β、α-SMA、Smad3 和 CTGF 的表达逐渐增加，差异具有统计学意义（p<0.01）。然而，Smad3 在纤维化 1a 期和 1c 期之间的表达差异无统计学意义。与正常猴相比，纤维化猴肝组织中 Smad7 的表达显著降低（p<0.01），但在纤维化 1c 期明显高于纤维化 1a 期和 2 期。
YAP 和 TGF-β 信号通路及其相互作用促进了 NAFLD 的发生和发展。
