Zheng Gan Decoction inhibits diethylnitrosamine-induced hepatocellular carcinoma in rats by activating the Hippo/YAP signaling pathway

doi:10.12122/j.issn.1673-4254.2025.04.15

Abstract

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

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.

Figures/Tables 14

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

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

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

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

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

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

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.

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.

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.

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.

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.

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.

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.

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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