3-Bromopyruvate Alleviates the Development of Monocrotaline-Induced Rat Pulmonary Arterial Hypertension by Decreasing Aerobic Glycolysis, Inducing Apoptosis, and Suppressing Inflammation
Pulmonary arterial hypertension (PH) is a progressive and life-threatening disease characterized by pulmonary vasoconstriction and remodeling, ultimately leading to right heart failure and death. Despite advances in treatment options, PH remains relatively incurable, and its prognosis remains unfavorable. Recent studies have highlighted the role of metabolic dysregulation, particularly the Warburg effect (aerobic glycolysis), in the pathogenesis of PH. The Warburg effect, which involves the conversion of glucose to lactate even in the presence of oxygen, drives cellular hyperproliferation and apoptosis resistance, contributing to the development of PH.
This study investigates the potential therapeutic effects of 3-bromopyruvate (3-BrPA), a glycolysis inhibitor, in a monocrotaline (MCT)-induced rat model of PH. The findings demonstrate that 3-BrPA significantly alleviates PH by reducing aerobic glycolysis, inducing apoptosis, and suppressing inflammation. The results suggest that 3-BrPA could be a promising therapeutic strategy for PH treatment.
Background and Rationale
PH is a complex disease with limited therapeutic options. Current treatments primarily focus on vasodilation to alleviate symptoms and improve quality of life. However, these approaches do not address the underlying pathological mechanisms, such as pulmonary vascular remodeling and metabolic dysregulation. Emerging evidence suggests that metabolic pathways, particularly aerobic glycolysis, play a critical role in the development of PH. The Warburg effect, initially described in cancer cells, has been implicated in the hyperproliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs) in PH.
3-BrPA, a well-known glycolysis inhibitor, has shown promise as an anti-cancer agent by targeting metabolic enzymes such as hexokinase 2 (HK-2) and inducing apoptosis in cancer cells. Given the similarities between PH and cancer biology, particularly the reliance on aerobic glycolysis, this study aimed to explore the effects of 3-BrPA on PH development and its underlying mechanisms.
Methods
Animal Model and Experimental Design
Adult male Sprague-Dawley rats were used to establish the MCT-induced PH model. PH was induced by a single intraperitoneal injection of MCT (60 mg/kg). Control animals received an equivalent volume of 0.9% NaCl. For the 3-BrPA treatment, MCT-induced PH rats were injected intraperitoneally with 3-BrPA (2 mmol/L) or phosphate-buffered saline (PBS) every other day for four weeks. Hemodynamic and metabolic parameters, including right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), pulmonary arteriolar remodeling, lactate levels, and glucose consumption, were evaluated.
Hemodynamic and Metabolic Measurements
RVSP was measured using a catheter inserted into the right ventricle via the jugular vein. Glucose and lactate levels were detected in serum samples using a lactate- and glucose-measuring instrument. Glucose consumption was calculated as the difference between glucose levels at baseline and four weeks post-MCT injection.
Histological and Immunohistochemical Analysis
Lung and heart tissues were processed for histological and immunohistochemical analysis. Hematoxylin and eosin (H&E) staining was used to assess pulmonary arteriolar remodeling and right ventricular hypertrophy. Immunohistochemistry and immunofluorescence were performed to evaluate the expression of HK-2, glucose transporter protein-1 (GLUT1), and CD68, a macrophage marker. Western blotting was used to measure the expression of apoptosis-related proteins, including cytochrome C (Cyto C) and cleaved caspase-3 (Casp 3).
Apoptosis Assays
Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay and a Cyto C release apoptosis assay kit. The TUNEL assay was used to detect apoptotic cells in lung tissue, while the Cyto C release assay measured the translocation of Cyto C from mitochondria to the cytosol.
Results
Metabolic Characteristics of MCT-Induced PH Rats
MCT-induced PH rats exhibited significant increases in RVSP, RVHI, and pulmonary vascular remodeling four weeks post-MCT injection. Lactate production and glucose consumption were significantly elevated in MCT rats compared to controls, indicating increased aerobic glycolysis. The expression of HK-2, a key enzyme in glycolysis, was also upregulated in the lungs of MCT rats.
Effects of 3-BrPA on Glycolysis
Treatment with 3-BrPA significantly reduced lactate production and glucose consumption in MCT-induced PH rats, demonstrating its inhibitory effect on aerobic glycolysis. These findings suggest that 3-BrPA effectively targets the metabolic shift associated with PH.
Effects of 3-BrPA on Hemodynamic Parameters and Body Weight
3-BrPA treatment significantly attenuated the MCT-induced increase in RVSP, indicating a reduction in pulmonary arterial systolic pressure. Although body weight (BW) decreased in both PBS- and 3-BrPA-treated MCT rats, the reduction was more pronounced in the 3-BrPA group, suggesting a potential metabolic impact of the treatment.
Effects of 3-BrPA on Pulmonary Arteriolar Remodeling
H&E staining revealed that 3-BrPA treatment significantly reduced pulmonary arteriolar remodeling in MCT rats. The percent medial thickness (% MT) of pulmonary arterioles was markedly decreased in 3-BrPA-treated rats compared to PBS-treated controls, demonstrating the anti-remodeling effects of 3-BrPA.
Effects of 3-BrPA on Right Ventricular Hypertrophy
3-BrPA treatment significantly reduced right ventricular hypertrophy in MCT rats, as evidenced by decreased RVHI and RV/BW ratios. Cardiomyocyte diameter in the right ventricle was also reduced in 3-BrPA-treated rats, further supporting the protective effects of 3-BrPA against ventricular hypertrophy.
Effects of 3-BrPA on HK-2 and GLUT1 Expression
Western blotting and immunohistochemical analysis showed that 3-BrPA downregulated the expression of HK-2 and GLUT1 in the lungs of MCT rats. These findings suggest that 3-BrPA inhibits glycolysis by targeting key metabolic enzymes and glucose transporters.
Effects of 3-BrPA on Apoptosis
3-BrPA treatment increased the expression of cleaved Casp 3 and promoted the release of Cyto C into the cytosol, indicating activation of the mitochondrial apoptotic pathway. The TUNEL assay confirmed that 3-BrPA significantly increased apoptosis in lung tissue, further supporting its pro-apoptotic effects in PH.
Effects of 3-BrPA on Inflammation
Immunofluorescence analysis revealed that 3-BrPA treatment reduced macrophage infiltration in the lungs of MCT rats, as indicated by decreased CD68-positive cells. These findings suggest that 3-BrPA suppresses inflammation, a key feature of PH pathogenesis.
Discussion
This study demonstrates that 3-BrPA effectively alleviates MCT-induced PH by targeting multiple pathological processes, including aerobic glycolysis, pulmonary vascular remodeling, right ventricular hypertrophy, apoptosis resistance, and inflammation. The findings highlight the potential of 3-BrPA as a novel therapeutic strategy for PH.
The Warburg effect, characterized by increased aerobic glycolysis, plays a critical role in the hyperproliferation and apoptosis resistance of PASMCs in PH. 3-BrPA, a glycolysis inhibitor, was shown to reduce lactate production and glucose consumption in MCT rats, indicating its ability to counteract the metabolic shift associated with PH. The downregulation of HK-2 and GLUT1 by 3-BrPA further supports its inhibitory effects on glycolysis.
In addition to its metabolic effects, 3-BrPA promotes apoptosis by activating the mitochondrial apoptotic pathway. The increased expression of cleaved Casp 3 and the release of Cyto C into the cytosol suggest that 3-BrPA induces apoptosis in PASMCs, thereby reducing pulmonary vascular remodeling. The TUNEL assay confirmed the pro-apoptotic effects of 3-BrPA in lung tissue.
Furthermore, 3-BrPA was shown to suppress inflammation in MCT rats, as evidenced by reduced macrophage infiltration. Inflammation is a key driver of PH pathogenesis, and the anti-inflammatory effects of 3-BrPA further contribute to its therapeutic potential.
Conclusion
In conclusion, this study provides compelling evidence that 3-BrPA alleviates MCT-induced PH by targeting aerobic glycolysis, inducing apoptosis, and suppressing inflammation. The findings suggest that 3-BrPA could be a promising therapeutic strategy for PH, addressing multiple pathological processes underlying the disease. Further research is needed to explore the clinical potential of 3-BrPA in PH treatment.
doi.org/10.1097/CM9.0000000000000577
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