[Effect of Zhishi Xiebai Guizhi Decoction on phenotypic transformation of pulmonary artery smooth muscle cells in rats with hypoxic pulmonary hypertension]

Abstract

This study aims to investigate the effect of Zhishi Xiebai Guizhi Decoction on the phenotypic transformation of pulmonary artery smooth muscle cells(PASMCs) in rats with hypoxic pulmonary hypertension(HPH). Healthy SPF SD rats were randomly assigned to five groups: control group, hypoxia model group, hypoxia + low-dose Zhishi Xiebai Guizhi Decoction group(440 mg·kg~(-1)·d~(-1)), hypoxia + high-dose Zhishi Xiebai Guizhi Decoction group(880 mg·kg~(-1)·d~(-1)), and hypoxia + sildenafil group(30 mg·kg~(-1)·d~(-1)), with right rats in each group. Rats in the hypoxia model and hypoxia + drug groups were exposed to a hypobaric oxygen chamber with a simulated altitude of 5 000 m to induce the PH model. The control group was fed under standard conditions. The hypoxia + drug groups received Zhishi Xiebai Guizhi Decoction and sildenafil once daily for 28 days. After the experiment, the ratio of pulmonary acceleration time to pulmonary ejection time(PAT/PET) was measured in rats from each group using echocardiography. Additionally, the mean pulmonary artery pressure(mPAP) in rats from each group was determined through right heart catheterization. Hematoxylin-eosin(HE) staining was employed to observe pathological changes in lung tissue, while Western blot was utilized to detect the expression levels of proteins associated with the phenotypic transformation of pulmonary artery tissue. At the in vitro level, primary PASMCs were cultured using the type Ⅱ collagenase digestion method and identified through immunocytochemistry staining. SPF SD rats were administered Zhishi Xiebai Guizhi Decoction, and drug-containing serum was collected. Cell counting kit-8(CCK-8) was utilized to determine the optimal intervention concentration of drug-containing serum on PASMCs under hypoxic conditions. The cells were categorized into the normoxia control group, hypoxia control group, and hypoxia + drug-containing serum groups(10% and 15% drug-containing serum). 5-ethynyl-2'-deoxyuridine(EdU) was employed to assess cell proliferation in each group. Cell migration was evaluated through Transwell and cell scratch experiments, while Western blot was used to analyze the expression levels of proteins associated with the phenotypic transformation of PASMCs in each group. In vivo results indicated that the expression of PAT/PET and contraction-rela-ted proteins of pulmonary artery tissue was significantly decreased in the hypoxia model group compared to the control group, while the expression of mPAP, pulmonary vessel wall thickness, and synthetic-related proteins was significantly increased. Furthermore, compared to the hypoxia model group, the expression of PAT/PET and contraction-related proteins of the pulmonary artery tissue of rats in the drug groups increased, along with a significant decrease in mPAP, pulmonary vessel wall thickness, and synthetic-related proteins. In vitro results demonstrated that hypoxia induction significantly up-regulated the proliferation and migration abilities of PASMCs, as well as the expression of synthetic-related proteins while decreasing the expression of contraction-related proteins compared to the normoxia control group. Additionally, compared to the hypoxia control group, serum with Zhishi Xiebai Guizhi Decoction was found to inhibit the proliferation and migration of PASMCs induced by hypoxia, reduce the expression of synthetic-related proteins, and up-regulate the expression of contraction-related proteins. In conclusion, Zhishi Xiebai Guizhi Decoction could inhibit the excessive proliferation and migration of PASMCs in a hypoxic environment, providing a protective effect on rats with HPH. This mechanism may involve the regulation of protein expression levels associated with the phenotypic transformation of PASMCs.

Keywords: Zhishi Xiebai Guizhi Decoction; hypoxic pulmonary hypertension; phenotypic transformation; pulmonary artery smooth muscle cells.