正肝方对二乙基亚硝胺诱导的肝癌大鼠的抗癌作用及机制：基于激活Hippo/YAP通路

doi:10.12122/j.issn.1673-4254.2025.04.15

摘要/Abstract

摘要：

目的探讨正肝方通过调控Hippo/YES-相关蛋白同源癌蛋白（YAP）信号通路对二乙基亚硝胺（DEN）诱导的肝癌大鼠模型的抗癌作用及对肝癌潜在的抑制机制。方法将80只SD大鼠随机分为正常对照组（n=10）、造模组（n=70）。造模组通过连续12周腹腔注射DEN（50 mg/kg）建立肝癌模型。建模成功后，随机分为5组：模型对照组、阳性对照组（槐耳颗粒，4 g/kg）、正肝方低、中、高剂量组（2、4、8 g/kg），n=10。各组接受相应药物干预，1次/d，持续17周。正常对照组和模型组仅灌喂等量纯水。观察大鼠生存状况和生存率，并测定体质量、肝脏指数、脾脏指数和胸腺指数。观测大鼠肝脏组织的形态变化。通过HE染色观察组织病理变化。利用免疫组化技术检测组织中YAP及p-YAP的表达水平。Western blotting检测组织中Hippo/YAP通路中YAP、p-YAP、MST1、LATS1和p-LATS1等相关蛋白表达。结果与正常对照组相比，模型对照组大鼠生存状况较差，生存率降低，体质量、肝脏指数、脾脏指数升高（P<0.01），胸腺指数降低（P<0.05）。正肝方治疗后，肝脏指数、脾脏指数降低（P<0.05），胸腺指数升高（P<0.01）。大鼠肝脏组织形态及HE染色结果表明，模型组肝小叶结构受损，肝细胞排列紊乱，正肝方治疗后这些病理变化得到改善。免疫组化结果显示，与模型对照组相比，在正肝方低、中、高剂量组及阳性对照组中，YAP的阳性表达水平逐渐减弱，且与正肝方的剂量呈负相关（P<0.01）；而p-YAP的阳性表达则逐渐增强（P<0.01），且与正肝方的剂量呈正相关（P<0.01）。Western blotting结果显示，与模型对照组相比，在接受正肝方治疗的各剂量组中，随着药物浓度的增加，YAP蛋白表达水平随着药物剂量的增加而降低（P<0.001），p-YAP的阳性表达水平逐渐升高，尤其在高剂量组中这一变化更为明显（P<0.001）；正肝方各剂量组及槐耳颗粒组治疗组的组织中MST1蛋白表达明显升高（P<0.05）。同时，正肝方用药组的组织中，LATS1和p-LATS1蛋白的水平也明显升高，且与正肝方的剂量呈正相关。结论正肝方可以通过调节激活Hippo信号通路的关键蛋白，上调MST1蛋白表达，激活LATS1，活化的LATS1进而调控下游靶点YAP表达，将其磷酸化，降低其含量，从而抑制到肝癌细胞的增殖，诱导细胞凋亡的作用。

关键词: 正肝方, 肝癌, Hippo/YAP通路, 作用机制, 二乙基亚硝胺

Abstract:

Objective To investigate the inhibitory effect of Zheng GanDecoction (ZGF) on tumor progression in a rat model of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and explore the possible mechanism. Methods Seventy SD rats were subjected to regular intraperitoneal injections of DEN (50 mg/kg) for 12 weeks to induce HCC tumorigenesis, with another 10 rats receiving saline injections as the normal control. After successful modeling, the rats were randomized into 5 groups (n=10) for daily treatment with distilled water ( model group), Huaier Granules (4 g/kg; positive control group), or ZGF at low, medium, and high doses (2, 4, and 8 g/kg, respectively) via gavage for 17 weeks. Body weight changes of the rats were monitored, and after completion of the treatments, the rats were euthanized for measurement of liver, spleen and thymus indices and morphological and histopathological examinations of the liver tissues using HE staining. The expressions of YAP, p-YAP, MST1, LATS1 and p-LATS1 in the liver tissues were detected using immunohistochemistry and Western blotting. Results Compared with the normal control rats, the rat models with DEN-induced HCC exhibited much poorer general condition with a significantly reduced survival rate, increased body weight and liver and spleen indices, and a lowered thymus index. ZGF treatment obviously reduced liver and spleen indices, increased the thymus index, and improved pathologies of the liver tissues of the rat models. Immunohistochemistry and Western blotting showed a dose-dependent reduction of YAP expression and an increment of p-YAP expression in ZGF-treated rats, which also exhibited significantly upregulated hepatic expressions of MST1, LATS1 and p-LATS1. Conclusion ZGF inhibits DEN-induced HCC in rats by activating the Hippo/YAP pathway via upregulating MST1 and LATS1 expression, which promotes YAP phosphorylation and degradation to suppress proliferation and induce apoptosis of the tumor cells.

Key words: Zheng Gan Decoction, hepatocellular carcinoma, Hippo/YAP pathway, therapeutic mechanism, diethylnitrosamine

宋添力, 王一民, 孙童, 刘绪, 黄胜, 冉云. 正肝方对二乙基亚硝胺诱导的肝癌大鼠的抗癌作用及机制：基于激活Hippo/YAP通路[J]. 南方医科大学学报, 2025, 45(4): 799-809.

Tianli SONG, Yimin WANG, Tong SUN, Xu LIU, Sheng HUANG, Yun RAN. Zheng Gan Decoction inhibits diethylnitrosamine-induced hepatocellular carcinoma in rats by activating the Hippo/YAP signaling pathway[J]. Journal of Southern Medical University, 2025, 45(4): 799-809.

图/表 14

表1 正肝方颗粒剂配方表

Tab.1 Formulation of Zheng Gan Decoction Granules

Name of medicine	Drug specification (g/bag)	Mass of corresponding tablets (g)	Mass of drinkable tablets of punch (g)	Drug lot number
Huangqi	4.00	10.0	2.5	A2092661
Danshen	2.50	5.0	2.0	A2080131
Baihuasheshecao	2.83	15.0	15.3	A301B311
Gouqizi	4.17	5.0	1.2	A2080181
Biejia	1.67	10.0	6.0	A2120021
Nvzhenzi	2.31	6.0	2.6	A2021132
Sanlen	0.56	5.0	9.0	A209A321
Ezhu	0.63	5.0	8.0	A210B515
Chognlou	1.43	5.0	3.5	A2072911
Chuangxiong	1.67	5.0	1.0	A2039431

图1 DEN造模大鼠肝脏形态图

Fig.1 Morphology of the liver of rats with DEN-induced hepatocellular carcinoma (HCC).

图2 各组大鼠生存状态评分

Fig.2 Survival status score of the rats in each group.

图3 各组大鼠生存率

Fig.3 Survival rate of the rats in each group.#P<0.05 vs model group.

图4 各组大鼠体质量变化趋势

Fig.4 Body weight changes of the rats in each group. ##P<0.01 vs model group.

表2 各组大鼠肝脏指数、脾脏指数、胸腺指数情况

Tab.2 Liver index, spleen index and thymus index of the rats in each group

Group	Liver index (%)	Spleen index (%)	Thymus index (%)
Normal control	2.34±0.14^##	0.16±0.02^##	0.05±0.01
Model	8.23±1.32**	0.47±0.12**	0.03±0.03*
Low dose	7.95±0.96**	0.53±0.18**	0.05±0.02
Middle dose	5.97±0.55*	0.42±0.07	0.06±0.02^##
High dose	5.45±0.55^#	0.31±0.08^#	0.05±0.02^#
Active control	6.27±0.55*	0.44±0.05	0.05±0.01

图5 各组大鼠肝脏形态学观察

Fig.5 Histomorphologic changes of rat livers in each group. A: Normal control group. B: Model. C: Low dose group. D: Middle dose group. E: High dose group. F: Positive control group.

图6 各组大鼠肝脏病理学变化

Fig.6 Effects of Zheng Gan Decoction on histopathological changes in the liver of the rats in each group (HE staining).A: Normal control group. B: Model. C: Low dose group. D: Middle dose group. E: High dose group. F: Positive control group.

图7 正肝方对DEN致PLC大鼠肝脏组织YAP表达的影响

Fig.7 Effect of Zheng Gan Decoction on YAP expression in liver tissues of rats with DEN-induced HCC (immunohistochemistry). A: Normal control group. B: Model. C: Low dose group. D: Middle dose group. E: High dose group. F: Positive control group.

图8 正肝方对DEN致PLC大鼠肝脏组织p-YAP表达的影响

Fig.8 Effect of Zheng Gan Decoction on YAP expression in liver tissues of rats with DEN-induced HCC (immunohistochemistry0). A: Normal control group. B: Model. C: Low dose group. D: Middle dose group. E: High dose group. F: Positive control group.

图9 各组大鼠肝脏组织免疫组化染色YAP、p-YAP的表达比较

Fig. 9 Comparison of immunohistochemically detected YAP expression in rat liver tissue among the groups. **P<0.01, ****P<0.0001 vs control group; ##P<0.01, ###P<0.001, ####P<0.0001 vs model group.

图10 正肝方对DEN诱导肝癌大鼠肝组织中YAP及其磷酸化蛋白水平的影响

Fig. 10 Effect of Zheng Gan Decoction on expression levels of YAP and its phosphorylated proteins in the liver tissues of rats with DEN-induced HCC. *P<0.05, **P<0.01, ***P<0.001 vs control group; #P<0.05, ###P<0.001 vs model group.

图11 正肝方对DEN诱导肝癌大鼠肝组织中MST1蛋白水平的影响

Fig.11 Effect of Zheng Gan Decoction on MST1 protein levels in liver tissues of rats with DEN-induced HCC. *P<0.05 vs control group; ##P<0.01 vs model group.

图12 正肝方对DEN诱导肝癌大鼠肝组织中LATS1及其磷酸化蛋白水平的影响

Fig.12 Effect of Zheng Gan Decoction on expression levels of LATS1 and its phosphorylated proteins in liver tissues of rats with DEN-induced HCC. *P<0.05 vs control group; ##P<0.01 vs model group.

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