枳实薤白桂枝汤对低氧性肺动脉高压大鼠肺动脉平滑肌细胞表型转化的影响

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

探讨枳实薤白桂枝汤对低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)表型转化的影响。体内水平上,健康SPF级SD大鼠随机分为对照组、低氧模型组、低氧+枳实薤白桂枝汤低剂量组(440 mg·kg^(-1)·d^(-1))、低氧+枳实薤白桂枝汤高剂量组(880 mg·kg^(-1)·d^(-1))、低氧+西地那非组(30 mg·kg^(-1)·d^(-1)),每组8只。低氧模型组和低氧+药物组置于模拟海拔5000 m的低压氧舱构建HPH模型,对照组置于舱外常规饲养,低氧+药物组分别给予各自剂量的枳实薤白桂枝汤和西地那非,每天1次,连续28 d。实验结束后,超声心动图检测各组大鼠肺动脉加速时间和射血时间比值(pulmonary acceleration time/pulmonary ejection time,PAT/PET);右心导管术检测各组大鼠平均肺动脉压(mean pulmonary artery pressure,mPAP);苏木素-伊红(hematoxylin-eosin,HE)染色观察肺组织病理改变;蛋白免疫印迹(Western blot)法检测肺动脉组织表型转化相关蛋白表达。体外水平上,Ⅱ型胶原酶消化法培养原代PASMCs,免疫细胞化学染色鉴定PASMCs;SPF级SD大鼠给予枳实薤白桂枝汤干预后采集含药血清;cell counting kit-8(CCK-8)确定含药血清在低氧环境下对PASMCs的最佳干预浓度。将细胞分为常氧对照组、低氧对照组、低氧+含药血清组(10%含药血清、15%含药血清),5-ethynyl-2′-deoxyuridine(EdU)检测各组细胞增殖情况;Transwell、细胞划痕实验检测各组细胞迁移情况;Western blot检测各组PASMCs表型转化相关蛋白表达水平。体内结果表明,与对照组比较,低氧模型组PAT/PET、肺动脉组织收缩型相关蛋白表达显著下调,mPAP、肺血管壁厚度、合成型相关蛋白表达显著上调;与低氧模型组比较,药物组大鼠PAT/PET、肺动脉组织收缩型相关蛋白表达上调,mPAP、肺血管壁厚度、合成型相关蛋白显著下调。体外结果表明,与常氧对照组比较,低氧诱导可使PASMCs增殖迁移能力、合成型相关蛋白表达显著上调,收缩型相关蛋白表达显著下调;与低氧对照组比较,枳实薤白桂枝汤含药血清可抑制低氧诱导PASMCs的增殖迁移,下调合成型相关蛋白的表达,同时上调收缩型相关蛋白表达。综上,枳实薤白桂枝汤可以抑制低氧环境下PASMCs的过度增殖、迁移,从而对HPH大鼠具有保护作用,其机制可能是通过调控PASMCs表型转化相关蛋白的表达水平有关。

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 as-signed 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 5000 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 ac-celeration 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 expres-sion levels of proteins associated with the phenotypic transformation of pulmonary artery tissue.At the in vitro level,primary PASMCs were cultured using the typeⅡcollagenase 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 catego-rized into the normoxia control group,hypoxia control group,and hypoxia+drug-containing serum groups(10%and 15%drug-contai-ning 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,com-pared 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 nor-moxia control group.Additionally,compared to the hypoxia control group,serum with Zhishi Xiebai Guizhi Decoction was found to in-hibit the proliferation and migration of PASMCs induced by hypoxia,reduce the expression of synthetic-related proteins,and up-regu-late the expression of contraction-related proteins.In conclusion,Zhishi Xiebai Guizhi Decoction could inhibit the excessive prolifera-tion 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.