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. 2022 Sep 20;42(9):1324-1334.
doi: 10.12122/j.issn.1673-4254.2022.09.08.

[Changes of YAP activity at the early stage of nonalcoholic steatohepatitis and its spatiotemporal relationship with ductular reaction in mice]

[Article in Chinese]
Y Liu  1 , J Liang  1 , W Zeng  1 , Y Wang  1
Affiliations

[Changes of YAP activity at the early stage of nonalcoholic steatohepatitis and its spatiotemporal relationship with ductular reaction in mice]

[Article in Chinese]
Y Liu et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To explore the changes in Yes-associated protein (YAP) activity at the early stage of nonalcoholic steatohepatitis (NASH) and the spatiotemporal relationship between YAP and ductular reaction (DR).

Methods: Male C57BL/6J mouse models of NASH were established by feeding with a methionine- and choline-deficient (MCD) diet or a thioacetamide (TAA) diet for 12 weeks. At different time points during the feeding, liver histology of the mice was observed with HE and Masson trichrome staining. The mRNA expressions of YAP and its _target genes (Ctgf, Cyr61, Acta2) were determined by qPCR, and the total protein expression level of YAP was measured with immunoblotting. The expression and distribution of YAP and the markers of DR (K19 and Sox9) were observed with immunohistochemical staining.

Results: At the early stage of NASH induced by MCD diet (1 to 4 weeks), the mRNA expression of YAP and its _target genes and the total protein expression of YAP increased significantly (P < 0.01). The number of YAP-positive hepatocytes reached the peak level of 90.8 (cells per ×400 field of view) at week 2 and then decreased to 30.8 at week 4 (P < 0.001); YAP-positive ductular cells appeared near the portal area, where DR began to occur. From 8 to 12 weeks, numerous K19/Sox9-positive DR cells were observed in the hepatic lobules around the central vein (P < 0.01), while only a few YAP-positive hepatocytes were present in the liver tissue (P > 0.05), and the number of YAP-positive ductular cells gradually increased with time (P < 0.001). At the early stage of NASH induced by TAA diet (3 days to 2 weeks), the mRNA expression of YAP and its _target genes and the total protein expression of YAP increased significantly (P < 0.05), and the number of YAP-positive hepatocytes reached the peak of 69.2 at week 2 and then decreased to 55.2 at week 4 (P < 0.001); YAP-positive ductular cells first appeared at the initial location of DR near the central vein. From 6 to 12 weeks, numerous K19/Sox9-positive DR cells occurred in the hepatic lobules around the central vein (P < 0.01). While the number of YAP-positive hepatocytes decreased (P < 0.001), the number of YAP-positive ductular cells continued to increase (P < 0.001).

Conclusion: During the development of NASH, YAP activation occurs earlier than DR but they are spatiotemporally correlated. YAP activation in hepatocytes may participate in DR by promoting hepatocyte dedifferentiation.

目的: 探究非酒精性脂肪性肝炎（NASH）早期Yes-相关蛋白（YAP）活性变化特点及其与胆管反应（DR）发生的时空关系。

方法: 通过蛋氨酸胆碱缺乏（MCD）饮食、硫代乙酰胺（TAA）饮食喂养雄性C57BL/6J小鼠12周，构建NASH小鼠模型。通过HE、Masson染色评估肝脏病理学改变，qPCR技术检测YAP及其靶基因（Ctgf、Cyr61、Acta2）的mRNA表达情况；免疫印记检测YAP的总蛋白表达水平；免疫组化检测YAP、DR标志（K19和Sox9）表达与分布情况。

结果: 在MCD饮食诱导的NASH疾病早期（1~4周），肝组织YAP及其靶基因Ctgf、Cyr6、Acta2的mRNA表达量和YAP总蛋白表达量均增加（P < 0.01）。YAP阳性肝细胞数量在第2周达到峰值90.8/×400视野后，第4周减少至30.8/×400视野（P < 0.001），并在汇管区附近（即DR发生初始位置）观察到YAP阳性胆管样细胞。8~12周时可在中央静脉周围肝小叶中观察到大量的K19/Sox9阳性DR细胞（P < 0.01），同时肝组织中仅有少量YAP阳性肝细胞（P > 0.05），且YAP阳性胆管样细胞数量逐渐增加（P < 0.001）。在TAA饮食诱导的NASH疾病早期（3 d~2周），肝组织YAP及其靶基因Ctgf、Cyr6、Acta2的mRNA表达量和YAP总蛋白表达量均增加（P < 0.05）。YAP阳性肝细胞数量在2周达到峰值69.2/×400视野；4周减少至55.2/×400视野（P < 0.001），并且在中央静脉附近（即DR发生初始位置）观察到YAP阳性胆管样细胞。6~12周时可在中央静脉周围肝小叶中观察到大量的K19/Sox9阳性DR细胞（P < 0.01），同时YAP阳性肝细胞数量持续减少（P < 0.001），YAP阳性胆管样细胞数量持续增加（P < 0.001）。

结论: NASH损伤肝脏组织中，YAP激活早于DR发生，YAP活性变化特点及其分布与DR发生分布具有时空相关关系，同时肝细胞内的YAP激活似乎可以诱导肝细胞转分化为胆管样细胞，参与DR发生。

Keywords: YAP; ductular reaction; nonalcoholic steatohepatitis; spatiotemporal relationship.

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Figures

图 1
图 1
MCD组小鼠肝脏病理学改变以及病理评分 HE staining of mouse liver tissue in control group (A) and MCD (methionine- and choline-deficient) group at 1 week (B), 2 weeks (C), 4 weeks (D), 8 weeks (E), and 12 weeks (F) and histology score (G) and fibrosis score (H) of mice in MCD group (Green arrow: Steatosis; blue arrow: Hepatocyte ballooning; black arrow: Inflammation. Original magnification: ×200). **P < 0.01, ***P < 0.001 vs control.
图 2
图 2
TAA组小鼠肝脏病理学改变以及病理评分 HE staining of mouse liver tissue in control group (A) and TAA (thioacetamide) group at 3 days (B), 2 weeks (C), 4 weeks (D), 6 weeks (E), 12 weeks (F) and histology score (G) and fibrosis score (H) of mice in TAA group (Green arrow: Steatosis; Black arrow: Inflammation. ×200). **P < 0.01, ***P < 0.001 vs control.
图 3
图 3
MCD组NASH早期(1~4周)小鼠肝组织中YAP的表达与分布 Expression and distribution of YAP in the liver tissue of the mice at 1 week to 4 weeks of NASH development in MCD group. YAP immunohistochemical staining of the liver tissue in control group (A) and MCD group at 1 week (B), 2 weeks (C), and 4 weeks (D). G: Number of YAP-positive hepatocytes at the early stage (1-4 weeks) of NASH in MCD group (×100) (A-D), (×400) (E, F). ***P < 0.001 vs control.
图 4
图 4
TAA组NASH早期(3 d~2周)小鼠肝组织中YAP的表达与分布 Expression and distribution of YAP in the liver tissue of the mice at the early stage (3 days to 2 weeks) of NASH in TAA group. YAP immunohistochemical staining of liver tissue in control group (A) and TAA group at 3 days (B), 1 week (C), and 2 weeks (D). G: Changes in the number of YAP-positive hepatocytes at the early stage (3 days to 2 weeks) of NASH in TAA group (×100) (A-D), (×400) (E, F). ***P < 0.001 vs control.
图 5
图 5
NASH早期小鼠肝组织YAP及其靶基因mRNA表达水平 The mRNA expression levels of YAP and its _target genes in the liver tissue of the mice at the early stage of NASH in MCD (A) and TAAgroup (B). *P < 0.05, **P < 0.01, ***P < 0.001 vs control.
图 6
图 6
NASH疾病早期小鼠肝组织YAP总蛋白表达水平 Protein expression level of YAP in the liver tissue of the mice at the early stage of NASH in MCD (A) and TAAgroup (B). ***P < 0.001 vs control.
图 7
图 7
MCD组NASH小鼠肝组织K19/Sox9阳性DR细胞的表达与分布 Expression and distribution of K19/Sox9-positive DR cells in the liver tissue of the mice in MCD group. K19/Sox9 immunohistochemical staining of liver tissue in control group (A, E) and MCD group at 4 weeks (B, F), 8 weeks (C, G), and 12 weeks (D, H). I, J: Changes of the number of K19/Sox9-positive DR cells in MCD group (×200). **P < 0.01, ***P < 0.001 vs control.
图 8
图 8
TAA组NASH小鼠肝组织K19/Sox9阳性DR细胞的表达与分布 Expression and distribution of K19/Sox9-positive DR cells in the liver tissue of the mice in TAA group. A-H: K19/Sox9 immunohistochemical staining of liver tissue in control group (A, E) and TAA group at 4 weeks (B, F), 6 weeks (C, G), 12 weeks (D, H). I, J: Number of K19/Sox9-positive DR cells in TAAgroup (×200). **P < 0.01, ***P < 0.001 vs control.
图 9
图 9
MCD组NASH疾病进展过程中，YAP激活与DR发生的时空和数量关系 Spatiotemporal and quantitative relationship between YAP activation and DR during NASH development in MCD group. A-D: YAP immunohistochemical staining of the liver tissue in control group (A) and MCD group at 4 weeks (B), 8 weeks (C), and 12 weeks (D). E: The number of YAP-positive hepatocytes first increased and then decreased, followed by the amplification of YAP-positive ductular cells and the development of DR in MCD group. Black arrow: YAP-positive ductular cells (×200). **P < 0.01, ***P < 0.001 vs control.
图 10
图 10
TAA组NASH疾病进展过程中，YAP激活与DR发生的时空和数量关系 Spatiotemporal and quantitative relationship between YAP activation and DR during NASH development in TAA group. A-D: YAP immunohistochemical staining of the liver tissue in control group (A) and TAA group at 4 weeks (B), 6 weeks (C), and 12 weeks (D). E: Number of YAP-positive hepatocytes first increased and then decreased, followed by the amplification of YAP-positive ductular cells and the development of DR in TAA group. Black arrow: YAP-positive ductular cells (×200). **P < 0.01, ***P < 0.001 vs control.
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