摘要
基于多聚嘧啶片段结合蛋白1(PTBP1)/丙酮酸激酶M2(PKM2)通路,探讨君仁补肺益心颗粒(JRBF)对低氧性肺动脉高压(HPH)小鼠的治疗作用及分子机制。体内实验部分,将60只SPF级雄性C57BL/6小鼠随机分成正常组,模型组,西地那非组,JRBF低、中、高剂量组。每日给予药物灌胃后,进行常压低氧10%±0.5%造模8 h,连续4周,采用右心导管法检测各组小鼠右心室收缩压(RVSP),小动物B超机检测肺动脉加速时间(PAAT)、肺射血时间(PET)、三尖瓣环收缩期位移(TAPSE)和舒张期右心室前壁厚度(RVAWd),HE染色和Masson染色检测肺小动脉病理改变、心肌细胞面积和胶原沉积情况,Western blot检测小鼠肺组织中PTBP1、PKM2和PKM1表达,免疫荧光检测PTBP1和肺动脉平滑肌细胞(PASMCs)标志蛋白α-SMA的共定位表达,乳酸测定试剂盒检测肺组织和血清中乳酸含量。体外实验部分,低氧(1%,48 h)诱导人肺动脉平滑肌细胞(hPASMCs)建立模型,CCK-8实验筛选出JRBF含药血清最佳干预浓度(10%),进一步通过EdU、Transwell分别检测细胞增殖、迁移能力,Western blot检测小鼠肺组织中PTBP1、PKM2和PKM1表达,试剂盒检测乳酸生成。结果提示,JRBF显著改善HPH小鼠RVSP、PAAT/PET、TAPSE,减轻肺血管重塑、心肌细胞肥大及胶原纤维沉积;JRBF下调肺组织和PASMCs中PTBP1、PKM2表达,抑制乳酸蓄积;10%JRBF含药血清可抑制低氧诱导的hPASMCs增殖、迁移,降低细胞乳酸生成及PTBP1、PKM2表达。综上所述,JRBF可能通过抑制PTBP1/PKM2信号轴,调控糖酵解途径,从而拮抗HPH发生发展。
This study is aimed to investigate the therapeutic effects of Junren Bufei Yixin Granules(JRBF)on hypoxic pulmonary hypertension(HPH)and explore the therapeutic mechanism through the polypyrimidine tract-binding protein 1(PTBP1)/pyruvate kinase isozyme type M2(PKM2)signaling pathway.Sixty SPF-grade C57BL/6 male mice were randomized into normal,model,sildenafil,and low/medium/high-dose JRBF groups.HPH was induced by hypoxic exposure(10%O2,8 h/day)for 4 weeks.The right ventricular systolic pressure(RVSP)was measured by right cardiac catheterization.Echocardiography was employed record the pulmonary artery acceleration time(PAAT),pulmonary ejection time(PET),tricuspid annular plane systolic excursion(TAPSE),and right ventricular anterior wall thickness at diastole(RVAWd).The histopathological changes were observed by hematoxylin-eosin(HE)and Masson staining.Western blot was employed to measure the expression levels of PTBP1,PKM2,and PKM1 in the lung tissue.The immunofluorescence assay was employed to detect the co-localization of PTBP1 with the marker alpha-smooth muscle actin(α-SMA)of pulmonary arterial smooth muscle cells(PASMCs).The lactate assay kit was used to measure the lactate levels in the serum and lung tissue.In the cell experiments,the hypoxia model was established by exposing human pulmonary artery smooth muscle cells(hPASMCs)to 1%O2 for 48 h.The optimal(10%)JRBF concentration for intervention was determined by the cell-counting kit-8(CCK-8).The cell proliferation(EdU),migration(Transwell),PTBP1/PKM2/PKM1,and lactate production were analyzed.The results showed that JRBF significantly reduced the RVSP,improved the PAAT/PET ratio and TAPSE,and attenuated pulmonary arteriolar remodeling and myocardial fibrosis.Furthermore,JRBF suppressed PTBP1 and PKM2 expression and reduced the lactate accumulation in the lung tissue and PASMCs.The cell experiment results showed that the 10%JRBF-containing serum inhibited the proliferation and migration of hPASMCs,while decreasing the PTBP1/PKM2 expression and lactate production.In conclusion,JRBF mitigates HPH progression by suppressing the PTBP1/PKM2 axis and restoring the glycolytic flux.
作者
郑润锈
王石中
谭骏岚
曹闲雅
黄威
戴晴
宋岚
戴爱国
ZHENG Run-xiu;WANG Shi-zhong;TAN Jun-lan;CAO Xian-ya;HUANG Wei;DAI Qing;SONG Lan;DAI Ai-guo(School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Provincial Key Laboratory of Vascular Biology and Translational Medicine,Changsha 410208,China;Department of Respiratory Diseases,Medical School,Hunan University of Chinese Medicine,Changsha 410208,China;Department of Respiratory Medicine,the First Hospital of Hunan University of Chinese Medicine,Changsha 410021,China)
出处
《中国中药杂志》
2026年第2期543-552,共10页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82370069)
湖南省科技攻关项目-芙蓉实验室科技攻关项目(2023SK2089)
湖南省自然科学基金项目(2024JJ5299)
湖南省卫健委重大科研专项(R2023097)
2024年湖南省研究生科研创新项目(CX20240727)。
