交感神经递质通过促进肺动脉平滑肌细胞增殖参与野百合碱诱导肺动脉高压模型大鼠的肺血管重塑  

Sympathetic neurotransmitters involvement in pulmonary vascular remodeling via promoting proliferation of pulmonary artery smooth muscle cells in monocrotaline induced pulmonary arterial hypertension in rats

作　　者：吴水碧 董文鹏 周静 林敏[1] 葛圣林[1] Wu Shuibi;Dong Wenpeng;Zhou Jing;Lin Min;Ge Shenglin(Department of Cardiovascular Surgery,the First Afiliated Hospital of Anhui Medical University,Hefei 230031,China;Department of Cardiovascular Surgery,the Third People's Hospital of Chengdu,Chengdu 610014,China)

机构地区：[1]安徽医科大学第一附属医院心脏大血管外科,合肥230031 [2]成都市第三人民医院心脏大血管外科,成都610014

出　　处：《中华实验外科杂志》2023年第5期823-827,共5页Chinese Journal of Experimental Surgery

基　　金：安徽省高等学校自然科学研究项目(KJ2018A0180);安徽医科大学第一附属医院临床研究启动计划(LCYJ2021YB016)。

摘　　要：目的探讨野百合碱诱导的大鼠肺动脉高压(PAH)模型中肺中小血管周围去甲肾上腺素(NE)和神经肽Y(NPY)的水平及其在肺血管重塑的作用。方法将20只6~8周龄的SpragueDawley(SD)大鼠(安徽医科大学实验动物中心提供)采用抽签法随机分为对照组(Control组)9只和野百合碱诱导肺动脉高压组(MCT-PAH组)11只。通过腹腔注射野百合碱(MCT,50mg/kg)构建PAH模型。通过血流动力学参数、苏木精-伊红(HE)染色评价PAH模型。酶联免疫吸附试验(ELISA)、免疫荧光及蛋白质印迹法评价NE和NPY表达。提取大鼠肺动脉组织做原代肺动脉平滑肌细胞(PASMCs)培养,通过细胞计数试剂盒(CCK-8)、流式细胞术检测PASMCs的增殖能力,两组之间比较采用t检验。结果与Control组比较,HE染色显示MCT-PAH组大鼠右心室心肌排列紊乱,间隙增宽,周围可见胶原纤维沉积,远端肺小动脉呈向心性肥厚,管壁增厚,管腔明显变窄;血浆NE[(100.32±8.95)nmol/L比(124.69±20.66)nmol/L,t=-2.892,P<0.05]和NPY水平[(914.15±181.51)ng/L(1115.86±138.91)ng/L,t=-2.388,P<0.05]、肺组织酪氨酸羟化酶(TH,0.366±0.01比0.865±0.111,t=-7.713,P<0.01)和NPY表达(0.798±0.080比1.044±0.126,t=-2.860,P<0.05)以及PASMCs周围的TH(25.75±1.11比34.45±6.07,t=-3.167,P<0.05)和NPY的平均荧光强度(40.10±3.20比49.47±2.28,t=-5.677,P<0.01)以及TH(1.86±2.13比18.96±4.81,t=-7.886,P<0.01)和NPY相对荧光强度均增加(5.92±1.59比58.93±13.93,t=-8.474,P<0.01)。体外实验结果显示与对照组比较,NE组[(100.00±6.74)%比(112.90±2.01)%,t=3.178,P<0.05]和NPY组[(100.00±14.17)%比(164.20±7.88)%,t=6.856,P<0.01]在浓度为100nmol/L时PASMCs的细胞活力最高,CFSE标记的PASMCs增殖指数(NE组:2.678±0.867比2.783±0.898,t=-4.657,P<0.05;NPY组:2.878±1.044比3.095±1.098,t=-4.035,P<0.05)和分裂指数(NE组:2.435±1.084比2.553±1.083,t=-5.953,P<0.01;NPY组:2.608±0.978比2.960±1.166,t=-2.470,P<0.01)均明显高于对照组。结论MCT-PAH大鼠全身和局�Objective To evaluate the level of norepinephrine and neuropeptide Y(NPY)at the pulmonary sympathetic vascular junction and the mechanism in pulmonary vascular remodeling in monocrotaline induced pulmonary arterial hypertension(MCT-PAH)rats.Methods A lottery method was used to randomly divide 206-to 8-week-old Sprague Dawley(SD)rats into 9 rats in the control group and 11 rats in MCT-PAH group.PAH model was induced by intraperitoneal injection of monocrotaline(MCT,50 mg/kg).The PAH model was evaluated by hemodynamic parameter measurements and hematoxylin and eosin(HE)staining.Enzyme linked immunosorbent assay(ELISA),immunofluorescence and Westren blotting were used to detect the expressions of NE and NPY.Pulmonary artery smooth muscle cells(PASMCs)were obtained from SD rat pulmonary artery tissue for primary culture.The proliferation of PASMCs was evaluated by cell counting kit-8(CCK-8)and flow cytometry.The t-test was used to compare between the two groups.Results Compared with the control group,HE staining showed disturbed right ventricular myocytes with widened gaps and collagen fiber deposition around them and distal small pulmonary arteries with centripetal hypertrophy,wall thickening and lumen narrowing.There were significant increases in plasma NE[(100.32±8.95)nmol/L vs.(124.69±20.66)nmol/L,t=-2.892,P<0.05]and NPY[(914.15±181.51)ng/L vs.(1115.86±138.91)ng/L,t=-2.388,P<0.05]levels.The expression levels of tyrosine hydroxylase(TH,0.366±0.016 vs.0.865±0.111,t=-7.713,P<0.01)and NPY(0.798.±0.080 vs.1.044±0.126,t=-2.860,P<0.05)in lung tissue and mean fluorescence intensity of TH(25.75±1.11 vs.34.45±6.07,t=-3.167,P<0.05)and NPY(40.10±3.20 vs.49.47±2.28,t=-5.677,P<0.01)and relative fluorescence intensities of TH(1.86±2.13 vs.18.96±4.81,t=-7.886,P<0.01)and NPY(5.92±1.59 vs.58.93±13.93,t=-8.474,P<O.O1)around distal small PASMCs.Compared with the control group,the cell viability of PASMCs was significantly increased at a concentration of 100 nmol/L NE[(100.00±6.74)%vs.(112.90±2.01)%,t=3.178,P<0.05]a

关 键 词：肺动脉高压 交感神经 去甲肾上腺素 神经肽Y 肺动脉平滑肌细胞 

分 类 号：R544.1[医药卫生—心血管疾病]