Oroxylin A, a broad‑spectrum anticancer agent, relieves monocrotaline‑induced pulmonary arterial hypertension by inhibiting the Warburg effect in rats

Abstract

Pulmonary arterial hypertension (PAH) is a chronic and fatal disease characterized by pulmonary vascular remodeling, similar to the 'Warburg effect' observed in cancer, which is caused by reprogramming of glucose metabolism. Oroxylin A (OA), an active compound derived from Scutellaria baicalensis, which can inhibit glycolytic enzymes [hexokinase 2 (HK2), Lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase 1 (PDK1) by downregulating aerobic glycolysis to achieve the treatment of liver cancer. To the best of our knowledge, however, the impact of OA on PAH has not been addressed. Consequently, the present study aimed to evaluate the potential protective role and mechanism of OA against PAH induced by monocrotaline (MCT; 55 mg/kg). The mean pulmonary artery pressure (mPAP) was measured using the central venous catheter method; HE and Masson staining were used to observe pulmonary artery remodeling. Non‑targeted metabolomics was used to analyze the metabolic pathways and pathway metabolites in MCT‑PAH rats. Western Blot analysis was employed to assess the levels of glucose transporter 1 (Glut1), HK2), pyruvate kinase (PK), isocitrate dehydrogenase 2 (IDH2), pyruvate dehydrogenase kinase 1(PDK1), and lactate dehydrogenase (LDH) protein expression in both lung tissue samples from MCT‑PAH rats. The results demonstrated that intragastric administration of OA (40 and 80 mg/kg) significantly decreased mPAP from 43.61±1.88 mmHg in PAH model rats to 26.51±1.53 mmHg and relieve pulmonary artery remodeling. Untargeted metabolomic analysis and multivariate analysis indicated abnormal glucose metabolic pattern in PAH model rats, consistent with the Warburg effect. OA administration decreased this effect on the abnormal glucose metabolism. The protein levels of key enzymes involved in glucose metabolism were evaluated by western blotting, which demonstrated that OA could improve aerobic glycolysis and inhibit PAH by decreasing the protein levels of Glut1, HK2, LDH, PDK1 and increasing the protein levels of PK and IDH2. In conclusion, OA decreased MCT‑induced PAH in rats by reducing the Warburg effect.

Keywords: Warburg effect; glycolysis; metabolomics; oroxylin A; pulmonary arterial hypertension.

Figure 1.

OA alleviated the development of MCT-induced PAH in rats. (A) Timeline of OA treatment. (B) mPAP quantitative analysis. (C) Growth rate of rats. (D) Representative hematoxylin and eosin staining of pulmonary artery (scale bar, 50 µm). (E) Quantitative analysis of vascular remodeling. (F) Representative pulmonary artery after Masson staining (scale bar, 50 µm). (G) Quantitative analysis of vascular fibrosis. *P<0.05 vs. control. ^#P<0.05 vs. PAH, OA, oroxylin A; PAH, pulmonary arterial hypertension; mPAP, pulmonary arterial pressure; MCT, monocrotaline; DCA, dichloroacetate; w, week; WA, wall area.

Figure 2.

Metabolic analysis of OA-treated PAH lung samples by ultra performance liquid chromatography-mass spectrometry. Score plots of samples between control and PAH groups using (A) PCA and (B) OPLS-DA, PAH and OA80 using (C) PCA and (D) OPLS-DA models, PAH and OA40 using (E) PCA and (F) OPLS-DA models and PAH and DCA groups using (G) PCA and (H) OPLS-DA models. OA, oroxylin A; PAH, pulmonary arterial hypertension; PCA, principal component analysis; OPLS-DA, orthogonal partial least squares discriminant analysis; DCA, dichloroacetate.

Figure 3.

Analysis of metabolic pathways and metabolites of PAH affected by OA. (A) Metabolic pathway analysis between the Control group and the PAH group. (B) Hierarchical clustering of differential metabolites involved in glucose metabolic pathways for each group. OA, oroxylin A; PAH, pulmonary arterial hypertension; DCA, Dichloroacetate.

Figure 4.

Changes in metabolites in the Warburg effect of PAH treated with OA. (A) α-D-glucose. (B) D-glucose 6-phosphate. (C) Phosphoenolpyruvic, (D) pyruvic, (E) cis-aconitic, (F) isocitric, (G) succinic and (H) L-lactic acid. *P<0.05 vs. control. ^#P<0.05 vs. PAH. OA, oroxylin A; PAH, pulmonary arterial hypertension; DCA, dichloroacetate.

Figure 5.

Expression of glycolysis-associated proteins in lung tissue from PAH model rats treated with OA. Representative western blotting and quantitative analysis of (A) Glut1, (B) HK2, (C) PK, (D) LDH, (E) PDK1 and (F) IDH2. *P<0.05 vs. control. ^#P<0.05 vs. PAH. OA, Oroxylin A; PAH, pulmonary arterial hypertension; DCA, dichloroacetate; Glut1, glucose transporter 1; HK2, Hexokinase 2; PK. Pyruvate kinase; LDH, Lactate dehydrogenase; PDK1, Pyruvate dehydrogenase kinase 1; IDH2, Isocitrate dehydrogenase 2.