Gynura Rhizoma containing pyrrolizidine alkaloids induces the hepatic sinusoidal obstruction syndrome in mice via upregulating fibrosis-related factors

Abstract

Recently, hepatic sinusoidal obstruction syndrome (HSOS) caused by herbal preparations containing pyrrolizidine alkaloids (PAs), such as Gynura Rhizoma (Tusanqi), has gained global attention. However, the lack of a reliable and reproducible animal model has greatly hampered mechanistic studies. Therefore, we aimed to establish a reproducible HSOS mouse model and investigate the hepatotoxic mechanism. The model was established by intragastrical administration of Gynura Rhizoma extract, i.e., 1.0 g extract/kg per day (equal to 16.7 g crude drug/kg per day based on extraction rate and 49.1 mg PA/kg per day based on the total PA content in the extract determined) for 40 successive days. Then, the mice were sacrificed, and their blood samples and livers were collected for analyses. Using hematoxylin-eosin (HE) and Masson staining, scanning electron microscopy imaging, clinical biomarkers, and other assays, we showed that the HSOS was successfully induced in our mouse model. Furthermore, we detected the key factors involved in liver fibrosis in the mice, revealing significantly increased hydroxyproline concentration; elevated expression of α-smooth muscle actin (α-SMA) and fibrosis-related genes such as Collagen-1, Collagen-3, Mmp2, Mmp13, Timp1, Timp3, and Activin, upregulated Smad3 phosphorylation, and increased serum TGF-β levels. Moreover, pro-inflammatory cytokines, including Tnf-α, Il-1β, and Il-6, were also increased in the model. All these results demonstrate the key roles of the TGF-β-Smad3 and inflammatory signaling pathways in this Gynura Rhizoma-induced HSOS mouse model, suggesting that blockade of fibrosis and/or inflammation should be an effective treatment for HSOS.

Keywords: Gynura Rhizoma; hepatic sinusoidal obstruction syndrome; herbal-induced liver injury; inflammation; liver fibrosis; pyrrolizidine alkaloid.

Fig. 1

Evaluation of the HSOS mouse model established by 40 successive days of administration of Gynura Rhizoma, i.e., 1.0 g total extract/kg per day (equal to 16.7 g crude drug/kg per day based on the extraction rate and 49.1 mg PAper daykg per day based on the total PA content in the extract). a Images of liver tissues (bar is 1 cm). b HE and Masson staining of liver tissues (400× magnification, bar is 100 μm). c Serum ALT and AST activities and TBIL levels. d Scanning electron microscopy images of liver tissues (40,000× magnification, bar is 4 μm). Yellow arrows indicate dilatation of sinusoids, enlarged and damaged fenestrae, and severe congestion. e MMP9 immunohistochemical staining of liver tissues (400× magnification, bar is 100 μm). Red arrows indicate MMP9-positive cells in the hepatic sinusoid and central vein. Values are expressed as the mean ± SD (n = 12). Statistics were analyzed by paired t-test. ***P < 0.001 vs. control group. Six fields were randomly selected and observed for each sample

Fig. 2

Evidence of hepatic fibrosis in the HSOS mouse model induced by Gynura Rhizoma. a Hepatic level of hydroxyproline. b Hepatic protein level of α-SMA. c Percentage of collagen-positive cells in liver tissue. d IHC staining for desmin and α-SMA and Sirus red staining of collagen (400× magnification, bar is 50 μm). Six fields were randomly selected and observed for each sample. e Hepatic mRNA expression of fibrosis-related genes. Values are expressed as the mean ± SD (n = 6–12). Statistics were analyzed by unpaired t-test. *P < 0.05, ***P < 0.001 vs. control group

Fig. 3

Key role of TGF-β-Smad3 signal pathway in the HSOS mouse mode induced by Gynura Rhizoma. a Protein concentration of TGF-β in serum. b Hepatic mRNA expression of Activin and Follistatin. c Immunohistochemical staining for p-Smad3 (400× magnification, bar is 50 μm). The bar represents 50 μm. Red arrows indicate p-Smad3-positive cells. Six fields were randomly selected and observed for each sample. d Hepatic mRNA expression of Collagen-Iα1 and Collagen-Iα2. Values are expressed as the mean ± SD (n = 6–12). Statistics were analyzed by unpaired t-test. **P < 0.01, ***P < 0.001 vs. control group

Fig. 4

Hepatic mRNA expression of proinflammatory cytokines. Values are expressed as the mean ± SD (n = 6). Statistics were analyzed by paired t-test. *P < 0.05, ***P < 0.001 vs. control group