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

doi:10.12122/j.issn.1673-4254.2024.10.01

Abstract

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

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.

Figures/Tables 6

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.

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.

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.

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

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.

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

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