Zhishi Xiebai Guizhi Decoction modulates hypoxia and lipid toxicity to alleviate pulmonary vascular remodeling of pulmonary hypertension in rats

Abstract

Background: Pulmonary hypertension (PH) is a severe cardio-pulmonary vascular disease, involves complex molecular mechanism especially during the pathological process of pulmonary vascular remodeling, brings a significant challenge to clinical treatment and thus resulting in high mortality rates. Classic Traditional Chinese medicine formula, Zhishi Xiebai Guizhi Decoction (ZXGD), holds therapeutic potential for PH. In present study, we sought to explore therapeutic potential of ZXGD against PH in rats.

Methods: We employed a combination methods of chemical profiling, echocardiographic, morphologic measurements, molecular biology, rats models and cultured pulmonary artery smooth muscle cells (PASMCs) to achieve this.

Results: Eighteen compounds were precisely identified in ZXGD using UHPLC-QTOF-MS/MS. Our data demonstrated ZXGD could alleviate PH by reducing pulmonary artery pressure and alleviating pulmonary vascular remodeling in rats. Specifically, ZXGD was found to intervene in abnormal expansion of PASMCs, thereby attenuating pulmonary vascular remodeling. ZXGD was also observed to modulate expressions of HIF-1α, ROS, and Nrf2 to alleviate hypoxia and oxidative stress. Additionally, ZXGD significantly regulated disorders in pro-inflammatory cytokines, thus mitigating inflammation. Furthermore, ZXGD decreased levels of decadienyl-L-carnitine and LDL-C, while elevating HDL-C and lipid droplet counts, thereby reducing cholesterol and lipid toxicity and preserving mitochondrial function. Importantly, inhibition of HIF-1α reversed expression of key pathological triggers for pulmonary vascular remodeling. Neohesperidin and naringin in ZXGD extract were identified as the primary contributors to its pharmacological effects against PH.

Conclusion: Altogether, our study empirically explored therapeutic potential and pharmacological mechanisms of ZXGD in treating PH, offering a groundwork for the development of novel anti-PH drugs.

Keywords: Chemical compounds; HIF-1α; Pulmonary hypertension; Pulmonary vascular remodeling; Zhishi Xiebai Guizhi Decoction.

Fig. 1

Mass spectrometry based chemical profiling method precisely identified 18 main compounds in ZXGD extract. A The base peak chromatogram (BPC) of ZXGD based on UHPLC-TOF–MS. B The extracted ion chromatograms (EICs) of 18 chemical components in ZXGD in both positive and negative ion modes. C Peaks 1–18 were identified and assigned as neohesperidin, naringin, adenosine, naringenin, adenine, limonin, isosakuranetin, xanthotoxol, eriocitrin, hesperitin, poncirin, scopoletin, syringic acid, esculetin, neoeriocitrin, magnoloside a, magnoloside b, lonicerin

Fig. 2

ZXGD modulated the apoptosis resistance and augmented proliferation of PASMCs in vitro. A, B Effects of ZXGD on viability of PASMCs treated with and without PDGF-BB were examined by MTT (n = 6). C–H Expression levels of Caspase 3, Caspase 8, Caspase 9, Bax, Bcl2 and PCNA in PASMCs (n = 6). **p < 0.01, ***p < 0.001 vs control, ^##p < 0.01, ^###p < 0.001 vs PDGF-BB. 5 represents 5 mg/ml, 10 represents 10 mg/ml

Fig. 3

ZXGD alleviated pulmonary arterial hypertension and inhibited pulmonary vascular remodeling in MCT induced PH rats. A Representative images of pulmonary flow were measured by pulsed-wave Doppler. D, E Statistical analysis of pulmonary acceleration time (PAT) and the ratio PAT to right ventricular ejection time (ET) (PAT/ET) (n = 4). B, C HE and α-SMA staining of pulmonary vessels in rats (magnification, ×200). F, G Quantitative analysis of wall thickness and positive staining of pulmonary vessels (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 vs control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs PH. 2.7 represents 2.7 g/kg, 5.4 represents 5.4 g/kg

Fig. 4

ZXGD ameliorated pulmonary vascular remodeling by modulating the proliferation and apoptosis proteins expression in vivo. A–F Expression levels of Caspase 3, Caspase 8, Caspase 9, Bax, Bcl2 and PCNA in lung tissues(n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs PH

Fig. 5

ZXGD inhibited hypoxia, oxidative stress and inflammation in rats with PH and PASMCs induced with PDGF-BB. A–E Expression of HIF-1α, Nrf2, IL-1β, IL-6 and IL-10 in lung tissue of rats were detected using Western blot (n = 3 or 6). F–I Concentration of IL-6, IL-1β, TNF-α and IL-10 in lung tissue of rats were examined by ELISA (n = 6). J Level of ROS in PASMCs was assessed with flow cytometry, and statistical data was obtained. **p < 0.01, ***p < 0.001 vs control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs PH or PDGF-BB

Fig. 6

ZXGD regulated lipid metabolism to reduce cell lipid toxicity in rats with PH. A–C, H Concentration of TC, LDL-C and HDL-C (n = 6) in serum as well as decadienyl-l-carnitine (n = 3) in lung tissue were examined by ELISA and Biochemical kit. D–F Expression levels of PPARγ, PLA2 and PLIN2 in lung tissue were detected using Western blot (n = 6). G Count of lipid droplets in PASMCs was assessed with Oil red O staining, and statistical data were obtained. *p < 0.05, **p < 0.01, ***p < 0.001 vs control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs PH or PDGF-BB

Fig. 7

ZXGD modulated HIF-1α mediated pulmonary vascular remodeling. A Expression level of HIF-1α in lung tissue of rats was tested with PCR (n = 3). B Expression level of HIF-1α in PASMCs transfected with siRNA (n = 6). C Effects of ZXGD on viability of PASMCs transfected with HIF-1α siRNA was examined by MTT (n = 6). D–G Levels of IL-6, IL-10, LDL-C and decadienyl-l-carnitine in PASMCs transfected with HIF-1α siRNA. H–K Expression level of HIF-1α, Caspase-3, PCNA, PLIN2 in PASMCs transfected with HIF-1α siRNA (n ≥ 3). *p < 0.05, ***p < 0.001 vs control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs PH or PDGF-BB

Fig. 8

Effect of neohesperidin and naringin on cell viability, HIF-1α, Caspase3, PLIN2 in PASMCs. A–D Effects of neohesperidin and naringin on PASMCs viability were observed by MTT (n = 6). E–J Expression levels of HIF-1α, Caspase3, PLIN2 in PASMCs were evaluated by Western Blot (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs PDGF-BB. 5 represents 5 µM, 10 represents 10 µM

Fig. 9

The pharmacological mechanism of ZXGD against pulmonary hypertension