抑制Yes相关蛋白通过抑制上皮间质转化减轻CCl4诱导的小鼠肝纤维化

doi:10.12122/j.issn.1673-4254.2024.10.01

摘要/Abstract

摘要：

目的探索Yes相关蛋白（YAP）可否通过调控上皮间质转化影响肝纤维化的发生发展。方法将8周龄C57BL/6小鼠18只随机分为对照组、肝纤维化模型组和YAP抑制剂维替泊芬干预组，6只/组。肝纤维化模型采用四氯化碳（CCl₄）溶液腹腔注射8周造模；维替泊芬干预组在CCl₄基础上第7~9周采用维替泊芬腹腔注射干预。HE染色、Masson染色、肝脏生化学检测观察小鼠肝脏纤维化程度；转录组、蛋白组学测序及联合生信分析探明肝纤维化过程中上皮间质转化通路是否受YAP调控；免疫组化染色和Western blotting检测YAP及上皮间质转化关键基因E-cadherin、N-cadherin、Twist等表达变化。采集健康体检、慢性乙型肝炎、乙肝肝硬化患者血清各60例，酶联免疫吸附法检测其中YAP、N-cadherin、Vimentin、Twist血清表达水平。C57BL/6小鼠24只随机分为对照组、肝纤维化模型组、Twist抑制剂干预组和Twist抑制剂与YAP激动剂共同干预组，6只/组。HE染色、Masson染色、网状纤维染色观察小鼠肝脏纤维化程度，Western blotting检测各组α-SMA、YAP和Twist表达变化。结果小鼠肝组织病理学结果提示肝纤维化小鼠与对照组相比肝小叶结构破坏、假小叶形成，维替泊芬干预组纤维间隔变性，部分小叶结构恢复。随肝纤维化发生，血浆ALT、AST水平显著升高（P<0.01），维替泊芬干预组肝功能改善（P<0.01）。采用肝组织转录组、蛋白组测序及联合分析找到在肝纤维化形成和维替泊芬干预过程中同时在mRNA和蛋白水平差异表达的基因，发现了N-cadherin和Twist在3组间差异具有统计学意义（P<0.05），并进行PPI分析显示YAP与E-cadherin、N-cadherin存在关联。免疫组化和Western blotting结果提示N-cadherin、Twist、Vimentin随肝纤维化形成升高，E-cadherin在肝纤维化组织中表达下降（P<0.01）。抑制YAP可下调肝组织N-cadherin、Twist蛋白表达（P<0.01）。慢性乙型肝炎患者血清YAP、N-cadherin、Vimentin和Twist水平均随肝炎及肝硬化发生升高，在APRI>0.5或FIB-4>1.45患者中显著升高（P<0.01）。血清YAP在健康对照、肝炎、肝硬化患者中平均水平分别为4.09、5.69和5.36 ng/mL（P<0.01），其与N-cadherin、Vimentin、Twist水平均呈显著正相关，相关系数分别为0.626、0.435、0.526。采用Harmine抑制小鼠肝组织Twist表达，并在Harmine基础上予以YAP激动剂XMU-MP-1干预，肝组织病理学结果提示抑制Twist使肝纤维化小鼠肝组织炎症及纤维化程度减轻，同时激活YAP表达可再次加重胶原纤维沉积。Western blotting检测结果提示Harmine下调肝纤维化小鼠肝组织中α-SMA、YAP及Twist蛋白表达，同时激活YAP使肝组织α-SMA和YAP表达升高（P=0.079，P<0.05）。结论上皮间质转化是肝纤维化发生的重要机制之一，抑制YAP可通过减少上皮间质转化发生减轻肝纤维化。

关键词: 肝纤维化, Yes相关蛋白, 上皮间质转化, 血清标志物

Abstract:

Objective To explore whether Yes-associated protein (YAP) affects occurrence and progression of liver fibrosis by regulating epithelial-mesenchymal transition (EMT). Methods In a 8-week-old C57BL/6 mouse model of CCl₄-induced liver fibrosis, the effect of verteporfin (a YAP inhibitor) intervention was assessed with HE staining and by detecting liver biochemistry and expressions of YAP and EMT-related genes using immunohistochemistry and Western blotting. Transcriptome and proteomic sequencing and informatics analysis were used to investigate the main downstream pathways of YAP in liver fibrosis. Serum levels of YAP, N-cadherin, vimentin and Twist were examined in 60 healthy individuals, 60 patients with chronic hepatitis B (CHB), and 60 patients with HBV-related liver cirrhosis. In another 24 C57BL/6 mice, the effects of Twist inhibitor alone or in combination with harmine (a YAP activator) on CCl₄-induced liver fibrosis were evaluated by histopathological examination and Western blotting. Results The mouse models of liver fibrosis showed obvious structural damages of the liver lobes with formation of pseudolobules, and verteporfin treatment significantly improved these pathologies and lowered plasma ALT and AST levels of the mice. Transcriptome and proteomic sequencing and informatics analysis suggested that N-cadherin and Twist were differentially expressed in liver fibrosis in close correlation with YAP. Inhibition of YAP obviously downregulated hepatic N-cadherin and Twist protein expressions in the mice with liver fibrosis. In patients with CHB and liver cirrhosis, serum levels of YAP elevated obviously with the severity of liver fibrosis and were significantly correlated with N-cadherin, vimentin and Twist levels. In mice with liver fibrosis, inhibiting Twist effectively improved liver inflammation and fibrosis, while the combined treatment with YAP activator worsened hepatic collagen fiber deposition and increased hepatic YAP and α-SMA expressions. Conclusion EMT is an important pathogenic mechanism of liver fibrosis, and inhibiting YAP can alleviate liver fibrosis by reducing EMT.

Key words: liver fibrosis, Yes-associated protein, epithelial mesenchymal transition, biomarkers

赵文, 阮何静, 汪思远, 程羽哲, 雷淼, 赵久法, 刘传苗. 抑制Yes相关蛋白通过抑制上皮间质转化减轻CCl₄诱导的小鼠肝纤维化[J]. 南方医科大学学报, 2024, 44(10): 1839-1849.

Wen ZHAO, Hejing RUAN, Siyuan WANG, Yuzhe CHENG, Miao LEI, Jiufa ZHAO, Chuanmiao LIU. Inhibiting Yes-associated protein alleviates CCl₄ liver fibrosis in mice by reducing epithelial mesenchymal transition[J]. Journal of Southern Medical University, 2024, 44(10): 1839-1849.

图/表 6

图1 小鼠肝组织病理表现及血清AST、ALT水平

Fig.1 Liver histopathology and serum levels of ALT and AST in mice. A: HE staining (Original magnification: ×200), Masson's trichrome staining (×100), and immunohistochemical staining of α-SMA (×200). B, C: Serum levels of ALT and AST. D: Quantitative data of immunohistochemistry for α-SMA in liver tissues. *P<0.05, **P<0.01 vs control group; ##P<0.01 vs CCl4 group.

图2 小鼠肝组织转录组及蛋白组测序的差异基因及蛋白分析

Fig.2 Transcriptomic analysis of and proteomic analysis of differentially expression mRNAs and proteins in the liver tissue of mice. A, B: Venn diagram showing differentially expressed genes (DEGs) between the groups. C, D: Volcano plot showing differentially expressed proteins (DEPs). The red and green dots indicate significantly upregulated and downregulated genes, respectively. E, F: Venn diagram showing differentially expressed mRNAs and proteins between control and CCl4 groups. G, H: Venn diagram showing differentially expressed mRNAs and proteins between the CCl4 and verteporfin (VP) treatment groups. I: Bubble chart of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs. J: Heat map of the DEGs of cell adhesion molecules pathway. K: Expressions of YAP, N-cadherin and Twist mRNAs in the 3 groups. L: Protein-protein interaction (PPI) network of the DEGs.

图3 小鼠肝组织YAP及EMT关键蛋白表达

Fig.3 Expression of YAP and EMT-related proteins in the liver tissue of the mice. A: Immunohistochemical staining of YAP, N-cadherin, Twist and vimentin (×400). B-E: Quantitative data of immunohistochemical staining for YAP, N-cadherin, Twist and vimentin. F: Western blotting of hepatic YAP, N-cadherin, E-cadherin and Twist protein expressions. G: Quantification of the protein expression levels. *P<0.05, **P<0.01 vs control group; ##P<0.01 vs CCl4 group.

表1 健康体检者、慢性乙型肝炎和乙肝肝硬化患者临床特征

Tab.1 General characteristics of healthy controls and patients with CHB and HBV-related liver cirrhosis

Parameters	Healthy controls (n=60)	CHB (n=60)	Liver cirrhosis (n=60)	F/Z/χ²	P
Male [n (%)]	40 (66.7)	30 (50.0)	41 (68.3)	5.217	0.074
Age (year)	50.1±0.5	46.6±1.1	51.0±1.3	5.377	0.095
WBC (10⁹/L)	6.17 (5.38, 7.15)	5.48 (4.54, 6.56)	4.21 (2.72, 5.43)	32.55	<0.001
PLT (10⁹/L)	232 (200, 275)	187 (155, 227)	89 (48, 134)	54.95	<0.001
ALB (g/L)	45.8 (42.8, 47.4)	46.5 (44.6, 48.2)	32.4 (28.0, 36.2)	104.93	<0.001
ALT (U/L)	22 (17, 32)	18 (15, 25)	32 (18, 56)	19.42	<0.001
AST (U/L)	21 (18, 25)	22 (19, 26)	47 (34., 76)	72.43	<0.001
GGT (U/L)	26 (18, 41.5)	15 (12, 20)	47 (28, 109)	71.92	<0.001
APRI	0.24 (0.17, 0.30)	0.29 (0.23, 0.47)	1.80 (0.90, 2.89)	93.61	<0.001
FIB-4	0.96 (0.80, 1.19)	1.29 (0.96, 1.69)	5.06 (3.35, 10.02)	97.86	<0.001

图4 患者血清YAP、N-cadherin、vimentin及Twist表达水平及相关性分析

Fig.4 Plasma YAP, N-cadherin, vimentin and Twist levels in healthy controls and patients with CHB and HBV-related liver cirrhosis. A-D: Distribution of plasma YAP, N-cadherin, vimentin and Twist levels in the 3 groups. E-H: Distribution of plasma YAP, N-cadherin, vimentin and Twist levels in patients with APRI <0.5, =0.5-1.5, and >1.5. I-L: Distribution of plasma YAP, N-cadherin, vimentin and Twist levels in patients with FIB-4<1.45, 1.45-3.25, and >3.25. M-O: Correlation analysis of serum YAP level with serum N-cadherin, vimentin and Twist levels. *P<0.05, **P<0.01.

图5 激活YAP逆转抑制Twist引起的肝纤维化改善

Fig.5 Activation of YAP reverses Twist inhibition-induced improvement of liver fibrosis in mice. A: HE staining (×200), Masson's trichrome staining (×100), and Reticulum fiber staining (×100). B: Western blotting of protein levels of hepatic YAP, Twist, and α-SMA in mice. C: Quantification of protein expression levels. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs CCl4 group; &P<0.05 vs Harmine group.

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抑制Yes相关蛋白通过抑制上皮间质转化减轻CCl₄诱导的小鼠肝纤维化

Inhibiting Yes-associated protein alleviates CCl₄ liver fibrosis in mice by reducing epithelial mesenchymal transition

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