摘要
目的探究低氧诱导小鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)游离铁减少的机制及其在PASMCs增殖和肺血管重塑中的作用。方法①体外培养原代小鼠PASMCs,分为常氧组(C)、常氧柠檬酸铁铵组(C+FAC)、常氧雷帕霉素组(C+R)、低氧组(H)、低氧柠檬酸铁铵组(H+FAC)、低氧雷帕霉素组(H+R)。C组、C+FAC组和C+R组置于常氧(21%O_(2))环境中培养48 h,H组、H+FAC组和H+R组置于低氧(1%O_(2))环境中培养48 h。C+FAC组和H+FAC组使用柠檬酸铁铵(ferric ammonium citrate,FAC)处理,C+R组和H+R组使用雷帕霉素(rapamycin)处理。检测各组细胞增殖情况,细胞内游离铁含量,铁调节蛋白(iron regulatory proteins,IRPs)、转铁蛋白受体(transferrin receptor,TFR)、铁转运蛋白(ferroportin,FPN)和铁蛋白重链(ferritin heavy chain,FTH)等铁稳态调节蛋白表达水平。②将24只C57/BL6N小鼠(雄性,6周龄,体质量18~22 g)分4组:常氧组(C-4W)、常氧高铁饮食组(C+HID-4W)、低氧组(H-4W)和低氧高铁饮食组(H+HID-4W)(n=6);H-4W组与H+HID-4W组置于模拟海拔5000 m低压低氧环境中4周。检测各组小鼠右心功能,右心室收缩压,以及肺血管重塑变化。结果与C组相比,H组PASMCs增殖活性显著增强(P<0.01),细胞内游离铁含量显著减少(P<0.01),FTH的蛋白水平显著上调(P<0.01)。与H组相比,H+FAC组PASMCs内游离铁含量显著增加(P<0.01),细胞增殖活性显著降低(P<0.01)。与H组相比,H+R组PASMCs内游离铁含量显著增加(P<0.01),细胞增殖活性显著降低(P<0.05),FTH的蛋白水平显著降低(P<0.05)。与H-4W组相比,H+HID-4W组小鼠的右心功能显著改善(P<0.01),右心室收缩压显著降低(P<0.05),右心肥厚显著减轻(P<0.01),肺动脉增厚显著抑制(P<0.01)。结论低氧通过抑制铁蛋白自噬途径,介导游离铁减少,进而促进PASMCs增殖以及肺动脉重塑。
Objective To investigate the mechanism of hypoxia-induced free iron reduction in mouse pulmonary artery smooth muscle cells(PASMCs)and its role in the proliferation of PASMCs and pulmonary vascular remodeling.Methods①Primary mouse PASMCs were cultured and divided into normoxic group(C),normoxic ferric ammonium citrate group(C+FAC),normoxic rapamycin group(C+R),hypoxic group(H),hypoxic ferric ammonium citrate group(H+FAC)and hypoxic rapamycin group(H+R).The C,C+FAC and C+R groups were incubated in normoxic(21%O_(2))enviroment for 48 h.The H,H+FAC and H+R groups were incubated in hypoxic(1%O_(2))environment for 48 h.The C+FAC and H+FAC groups were treated with ferrous ammonium citrate(FAC),and the C+R and H+R groups were treated with rapamycin(R).Cell proliferation was detected with CCK-8 assay and EdU proliferation assay.The content of intracellular free iron was measured by FerroOrange staining.The protein levels of iron regulatory proteins(IRPs),transferrin receptor(TFR),ferroportin(FPN)and ferritin heavy chain(FTH)were detected with Western blotting.②Twenty-four C57/BL6N mice(male,6 weeks old,weighting 18~22 g)were divided into normoxic group(C-4W),normoxic high-iron diet group(C+HID-4W),hypoxic group(H-4W)and hypoxic high-iron diet group(H+HID-4W)(n=6).The H-4W and H+HID-4W groups were placed in a low-pressure hypoxic environment at a simulated altitude of 5000 m for 4 weeks.Right heart function,right ventricular systolic pressure,and pulmonary vascular remodeling in each group were observed and measured.ResultsCompared with C group,significantly enhanced proliferation(P<0.01),reduced intracellular free iron content(P<0.01),and up-regulated protein level of FTH were observed in the PASMCs of H group(P<0.01).Compared with H group,the free iron content was obviously increased(P<0.01)and cell proliferation was decreased in H+FAC group(P<0.01).The H+R group had notably increased free iron content(P<0.01),decreased cell proliferation(P<0.05),and reduced protein level of FTH when compared with H group(P<0.05).Co
作者
印承杨
陈德伟
张二龙
徐刚
高钰琪
YIN Chengyang;CHEN Dewei;ZHANG Erlong;XU Gang;GAO Yuqi(Institute of Medicine and Equipment for High Altitude Region,College of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Key Laboratory of Extreme Environmental Medicine of Ministry of Education,College of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Department of High Altitude Physiology and Pathology,College of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2023年第12期1249-1258,共10页
Journal of Army Medical University
基金
国家自然科学基金重点项目(81830062)。
关键词
低氧性肺动脉高压
铁蛋白自噬
肺动脉平滑肌细胞
铁稳态
细胞增殖
hypoxic pulmonary hypertension
ferritinophagy
pulmonary artery smooth muscle cells
iron homeostasis
cell proliferation
