Aqueous Extract of Rhubarb Promotes Hepatotoxicity via Facilitating PKM2-Mediated Aerobic Glycolysis in a Rat Model of Diethylnitrosamine-Induced Liver Cancer

Abstract

Objective: To identify the polar parts in Rhubarb that cause hepatotoxicity and explore the underlying mechanisms.

Methods: The rat model of liver cancer was established by gavage of diethylnitrosamine (DEN; 0.002 g/rat) for 14 weeks. Starting from the 11th week, Rhubarb granule (4 g/kg), aqueous, ethyl acetate and n-butanol extract of Rhubarb or Rhein equivalent to a dose of 4 g/kg Rhubarb granule were administered intragastrically for 4 consecutive weeks. Liver tissues from rats treated with DEN and Rhubarb granules were used for non-targeted metabolomics analysis. The correlation between pyruvate kinase isozyme type M2 (PKM2) expression level and the progress and prognosis of hepatocellular carcinoma (HCC) was evaluated through bioinformatics analysis based on TCGA database. Liver tissues and blood samples from rats treated with DEN and aqueous, ethyl acetate and n-butanol extract of Rhubarb were used for the screening of hepatotoxic polar parts of Rhubarb. The liver injuries were evaluated by the changes in pathology, liver function, and the expression levels of proliferating cell nuclear antigen (PCNA) and transforming growth factor beta1 (TGF-β1). The mechanism studies focus on PKM2 expression, and the metabolic reprogramming via detecting the activities of lactate dehydrogenase A (LDHA) and isocitrate dehydrogenase (ICDH). Furthermore, molecular docking analysis was performed to validate the target interaction between Rhein and PKM2, and the hepatotoxicity of Rhein was evaluated by testing liver function in the DEN-induced liver cancer model.

Results: The non-targeted metabolomics analysis revealed that Rhubarb promoted aerobic glycolysis in the rat model of DEN-induced liver cancer. And bioinformatics analysis revealed that high PKM2 expression was closely related to the progression and poor prognosis of HCC. In vivo studies indicated that the aqueous extract of Rhubarb, but not ethyl acetate and n-butanol extract, promoted the liver injuries induced by DEN. The mechanism study showed that the aqueous extract of Rhubarb increased the expression of PKM2 and promoted aerobic glycolysis. Moreover, Rhein had a strong binding affinity for PKM2 and aggravated liver injury in the DEN-induced liver cancer model.

Conclusion: Aqueous extract of Rhubarb promoted hepatotoxicity via facilitating PKM2-mediated aerobic glycolysis in the rat model of DEN-induced liver cancer.

Keywords: PKM2; Rhein; aerobic glycolysis; aqueous extract of rhubarb; hepatotoxicity.

Graphical abstract

Figure 1

The glucose metabolism analyzed by non-targeted metabolomics. (A) The relative amount of lactate in indicated groups. (B) The relative amount of succinate in indicated groups. The rat model of liver cancer was established by gavage of DEN (0.002 g/rat) for 14 weeks. Starting from the 11th week, Rhubarb granule (4 g/kg) was administered intragastrically for 4 consecutive weeks. Values are presented as mean ± SD (n = 8 for each group). *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

Figure 2

The association between PKM2 expression level and the progression and prognosis of HCC. (A) The PKM2 gene expression level in normal (n = 50) and primary tumor (n = 371) samples. (B) The PKM2 gene expression level in HCC with different cancer stages. (C) The PKM2 gene expression level in HCC with different tumor grades. (D) The survival of HCC patients with high and low/medium PKM2 expression. All data are from TCGA database. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Aqueous extract of Rhubarb promotes DEN-induced hepatotoxicity. Representative images of H&E staining (A) and Masson’s trichrome staining (B) of the liver sections from indicated groups. The activity of ALT (C) and AST (D) in indicated groups (Values are presented as mean ± SD; n ≥ 5 for each group). (E) Western blot of PCNA in the rat liver samples from indicated groups. Quantification of PCNA expression is depicted in (F). (G) Western blot of TGF-β1 in the rat liver samples. Quantification of TGF-β1 expression is depicted in (H). All samples were collected from rats at week 14 after initiation of DEN treatment. Western blot was performed at least three times and each repeat was performed as a separate, independent experiment. *p < 0.05, **p < 0.01, ns: non-significant.

Figure 4

Aqueous extract of Rhubarb promotes DEN-induced aerobic glycolysis in liver tissues. (A) Western blot of PKM2 in the rat liver samples from indicated groups. Quantification of PKM2 expression is depicted in (B). Western blot was performed at least three times and each repeat was performed as a separate, independent experiment. The activities of LDHA (C) and ICDH (D) in indicated groups (Values are presented as mean ± SD; n = 4 for each group). Liver tissue samples were collected from rats at week 14 after initiation of DEN treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

Figure 5

Rhein promotes DEN-induced hepatotoxicity. (A) Rhein interacts with PKM2 molecular docking verification. The activities of ALT (B) and AST (C) in indicated groups (Values are presented as mean ± SD; n = 5 for each group). Liver tissue samples were collected from rats at week 14 after initiation of DEN treatment. **p < 0.01, ***p < 0.001.