烟雾暴露和低氧单独及联合作用对大鼠肺血管重建及蛋白激酶C-α表达的影响

Pulmonary vascular remodeling and protein kinase C-α expression in chronic smoke exposure and/or hypoxia rats

目的探讨烟雾暴露和低氧单独及联合作用对大鼠肺血管重建及蛋白激酶C-α(PKC-α)表达的影响。方法健康雄性W istar大鼠56只,随机分为正常对照组(C组)、烟雾暴露组(S4W组和S8W组)、低氧组(H4W组和H8W组)和烟雾暴露+低氧组(SH4W组和SH8W组),复制大鼠慢性烟雾暴露及低氧模型。右心导管法记录并测定平均肺动脉压(mPAP),分离心脏计算右心室肥厚指数(RVH I)。弹力纤维染色计算腺泡内肺动脉3种类型血管的构成比及肺动脉管壁骨架蛋白(α-SM-actin)染色显示肺血管重建,采用原位末端脱氧核苷酸转移酶标记(TUNEL)法检测细胞凋亡、增殖细胞核抗原(PCNA)染色检测细胞增殖、逆转录-聚合酶链反应(RT-PCR)检测PKC-αmRNA表达,免疫荧光及W estern b lot检测PKC-α蛋白表达。结果与C组大鼠mPAP[(14±4)mm Hg,1 mm Hg=0.133 kPa]比较,H4W、H8W、SH4W、SH8W组大鼠mPAP均显著增高[(31±7)(32±8)、(32±9)、(31±10)mm Hg,P均<0.01],而S4W、S8W组均无显著升高[(15±5)、(16±6)mm Hg,P均>0.05]。S4W、S8W、H4W、H8W、SH4W和SH8W组大鼠RVH I(0.258±0.024、0.394±0.021、0.374±0.020、0.414±0.019、0.434±0.023、0.442±0.020)、肌型动脉占血管总数的百分比[(33.5±6.8)%、(41.1±9.8)%、(35.9±6.6)%、(46.0±6.3)%、(42.9±6.5)%、(50.2±9.9)%]及α-SM-actin表达(53±15、75±14、56±11、82±17、83±17、98±16)均显著高于C组(P均<0.01),凋亡指数(2.5±1.0、3.8±1.4、2.3±1.1、3.3±1.1、3.5±1.4、4.8±1.4)和增殖指数(33.1±11.8、43.8±11.0、36.5±10.6、46.3±12.1、45.3±12.4、53.3±13.4)均高于C组(P均<0.01)。肺动脉PKC-αmRNA和蛋白的表达均增强(P均<0.01)。且SH4W、SH8W组各指标分别与H4W、H8W组比较,差异均有统计学意义(P均<0.01)。结论烟雾暴露和低氧均能诱导肺动脉平滑肌细胞凋亡与增殖,导致肺血管重建,二者协同作用能加重肺血管重建;激活PKC信号转导途径可能是其部分机制。

Objective To investigate pulmonary vascular remodeling and protein kinase C-α（ PKC-α） expression in chronic smoke exposure and/or hypoxia rats. Methods Fifty-six male Wistax rats were randomly divided into seven groups ： control group （ C group ） , smoke exposure groups （ S4w, S8w group ） , hypoxia groups（ H4w, H8w group） , smoke exposure plus hypoxia groups （ SH4w, SH8w group）. Wistar rats were exposed to cigarette smoke and/or hypoxia air [ O2 （ 10.0 ± 0. 5 ） % ] for 4 to 8 weeks. A method by fight cardic catheterization was used for measuring mean pulmonary artery pressure（mPAP）. Right ventricle （RV） ,left ventricle （ LV ） plus interventricular septurn （ S ） were split and weighed and right ventricular hypertrophy index（RVHI） was calculated. To evaluate vascular remodeling, alpha-smooth muscle actin （ α-SM-actin） staining and count of the percentage of muscularized small pulmonary arteries which was determined by morphometric analysis of histological sections were used. Pulmonary artery smooth muscle cell （PASMC） apoptosis was detected by in situ end labehng technique （TUNEL）, and proliferation by proliferating cell nuclear antigen （PCNA） staining. Reverse transcription-polymerase chain reaction （RT- PCR）, immunofluorescence staining and Western blot analysis were used for the detection of PKC-α mRNA and protein expression in pulmonary arteries. Results mPAP in H4w,H8w,SH4w,SH8w group[ （31 ±7）, （32 ±8） ,（32 ±9） ,（31 ±10） mm Hg, 1 mm Hg =0. 133 kPa] were higher than that in C group[ （14 ±4） mm Hg, all P 〈 0. 01 ] ,but mPAP in S4w and S8w group[ （15 ±5）, （16 ± 6）mm Hg] were not increased （all P 〉 0. 05）. In S4w, S8w, H4w, Haw, SH4w and SH8w group, RVHI （0. 258 ± 0. 024,0. 394 ± 0. 021,0. 374 ± 0. 020,0. 414 ±0. 019,0. 434 ±0. 023,0. 442 ±0. 020 ,respectively） ,the percentage of muscularized arteries [ （33.5 ±6. 8）%,（41.1 ±9. 8）% ,（35.9 ±6. 6）% ,（46.0 ±6. 3）% ,（42. 9 ±6. 5）% ,（50. 2 ±9. 9）%, respectively] and α-SM-actin expression（53 ± 15,75 ± 14,56 ± 11,82 ± 17,83 ± 17,98 ± 16, respectively） were increased significantly（ all P 〈0. 01 ）. PASMC apoptosis was increased and proliferation was markedly increased. Apoptotic indices（AI,2.5 ±1.0,3.8 ±1.4,2.3 ± 1.1,3.3 ± 1.1,3.5 ± 1.4,4.8 ±1.4, respectively） and proliferation indices（ PI,33.1 ± 11.8,43.8 ± 11.0,36. 5 ± 10. 6,46. 3 ± 12. 1,45. 3± 12. 4,53.3 ± 13.4, respectively） were higher than those in C group （ all P 〈 0. 01 ）. The expressions of PKC-α mRNA and protein were higher than those of C group（ all P 〈0. 01）. The differences were more significant between SH4w and H4w group,SH8w and H8w group （ all P 〈 0. 01 ）. Conclusions It is suggested that smoke is synergistic with hypoxia in aggravating pulmonary vascular remodeling. The possible mechanism is through PKC signaling transduction pathway activation.