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中华妇幼临床医学杂志(电子版) ›› 2023, Vol. 19 ›› Issue (04) : 428 -436. doi: 10.3877/cma.j.issn.1673-5250.2023.04.008

论著

产前他克莫司治疗对先天性膈疝大鼠病理模型肺血管重构的影响
靳茜雅, 黄晓松, 谭诚, 蒋琴, 侯昉, 李瑶悦, 徐冰, 贾红慧, 刘文英†()   
  1. 电子科技大学附属医院·四川省人民医院，成都　610072
    德阳市人民医院，德阳　618000
  • 收稿日期:2022-12-10 修回日期:2023-05-04 出版日期:2023-08-01
  • 通信作者: 刘文英

Antenatal treatment of tacrolimus on pulmonary vascular remodeling in rat with experimental pathological model of congenital diaphragmatic hernia

Xiya Jin, Xiaosong Huang, Cheng Tan, Qin Jiang, Fang Hou, Yaoyue Li, Bing Xu, Honghui Jia, Wenying Liu†()   

  1. Sichuan Provincial People′s Hospital, University of Electronic Science & Technology of China, Chengdu 610072, Sichuan Province, China
    Deyang People′s Hospital, Deyang 618000, Sichuan Province, China
  • Received:2022-12-10 Revised:2023-05-04 Published:2023-08-01
  • Corresponding author: Wenying Liu
  • Supported by:
    Key Research and Development Project of Science & Technology Department of Sichuan Province(2021YFS0381)
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引用本文：

靳茜雅, 黄晓松, 谭诚, 蒋琴, 侯昉, 李瑶悦, 徐冰, 贾红慧, 刘文英. 产前他克莫司治疗对先天性膈疝大鼠病理模型肺血管重构的影响[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 428-436.

Xiya Jin, Xiaosong Huang, Cheng Tan, Qin Jiang, Fang Hou, Yaoyue Li, Bing Xu, Honghui Jia, Wenying Liu. Antenatal treatment of tacrolimus on pulmonary vascular remodeling in rat with experimental pathological model of congenital diaphragmatic hernia[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2023, 19(04): 428-436.

目的

探讨产前对合并肺发育不良及肺动脉高压(PAH)的先天性膈疝(CDH)大鼠病理模型，采取他克莫司(FK506)治疗的抗血管重构作用及其作用机制。

方法

选取17只健康、成年的无特定病原体(SPF)级Sprague Dawley(SD)大鼠为研究对象，均为8周龄，雌、雄鼠分别为12、5只。采用除草醚构建CDH大鼠病理模型。12只雌鼠受孕后，通过随机数字表法，根据不同处理方式处理分为FK506组(n＝3)、CDH组(n＝3)、CDH+FK506组(n＝3)和对照组(n＝3)。观察4组胎鼠肺组织发育情况，采取血管弹性纤维染色(EVG)，观察肺血管厚度；α-SMA和CD31免疫荧光双重染色，检测胎鼠新生血管生成；Western blotting法检测胎鼠肺组织中BMPR2、p-SAMD1、p-SMAD5蛋白的表达水平。本动物实验均经电子科技大学附属医院·四川省人民医院动物实验伦理委员会伦理审查通过[伦审(研)2022年第191号]。

结果

①4组胎鼠的双肺重量和双肺重量/胎鼠体重总体比较，差异均有统计学意义(H＝81.25、106.98，P均<0.001)，而4组胎鼠体重总体比较，差异无统计学意义(P>0.05)。②EVG染色结果显示，4组胎鼠肺动脉中膜厚度百分比(MT%)和肺泡面积百分比(S%)总体比较，差异均有统计学意义(F＝13.26、P＝0.006，F＝37.48、P<0.001)。其中，CDH组胎鼠MT%，较对照组增厚(P＝0.001)；CDH+FK506组大鼠肺动脉MT%，则较CDH组降低(P＝0.002)。③4组CDH胎鼠α-SMA和CD31阳性细胞增殖指数分别总体比较，差异均有统计学意义(F＝33.76、9.18，P<0.05)。进一步进行4组间两两多重比较的结果显示，CDH组分别和CDH+FK506组、对照组组间比较，差异均有统计学意义(P<0.05)。④FK506组、CDH+FK506组和对照组3组胎鼠BMPR2、p-SMAD1蛋白相对表达水平总体比较，差异均有统计学意义(F＝11.45、10.94，P<0.05)，而3组胎鼠p-SMAD5蛋白相对表达水平总体比较，差异无统计学意义(F＝0.01、P>0.05)。其中，CDH组胎鼠BMPR2、p-SMAD1表达水平，较对照组降低(P＝0.049、0.018)，CDH+FK506组BMPR2、p-SMAD1表达水平，则较CDH组升高(P＝0.010、0.023)。

结论

在除草醚诱导的CDH大鼠病理模型中，产前对其采取FK506干预，可以减轻CDH大鼠模型的肺血管重构，对缓解CDH大鼠的肺发育不良具有正面影响。

关键词: 疝, 横膈, 先天性, 高血压, 肺性, 他克莫司, 印迹法, 蛋白质, 肺动脉, BMPR2, p-SMAD1, p-SMAD5, 大鼠
Objective

To explore whether FK506 has anti-vascular remodeling effect and its mechanism on prenatal administration of tacrolimus (FK506) in rats with experimental pathological model of pulmonary arterial hypertension (PAH) associated with congenital diaphragmatic hernia (CDH).

Methods

Seventeen healthy, adult specific pathogen free (SPF)-grade Sprague Dawley (SD) rats were selected in this study, all of which were 8 weeks old, with 12 females and 5 males, respectively. The CDH rat model was made with nitrofen. After a successful pregnancy, they were randomly divided into FK506 group (n=3), CDH group (n=3), CDH+ FK506 group (n=3) and control group (n=3). The development of lung tissue in each group were observed. The vessel wall thickness of the pulmonary arteries was observed by elastic van gieson (EVG) staining, immunofluorescent double staining of α-SMA and CD31 for the detection of neo-vascularization. The expression levels of BMPR2, p-Smad1 and p-Smad5 in fetal rats lungs were determined using Western blotting. The study was performed with the approval of the Ethics Committee of Sichuan Provincial People′s Hospital, University of Electronic Science & Technology of China [Approval No.2022(19)].

Results

①The overall comparison of lung weight and lung weight/body weight of 4 groups showed statistically significant differences (H=81.25, 106.98; both P<0.001), while the overall comparison of body weight of 4 groups showed no statistical significance (P>0.05). EVG staining showed that the percentage of pulmonary artery media thickness (MT%) and alveolar area (S%) in 4 groups were statistically significant differences (F=13.26, P=0.006; F=37.48, P<0.001). Further, EVG staining showed that MT% in CDH group was greater than that in control group (P=0.001), The MT% of pulmonary artery in CDH+ FK506 group was lower than that in CDH group (P=0.002). ③Overall comparison of the proliferation indices of α-SMA and CD31-positive cells among 4 groups, respectively, showed statistically significant differences (F=33.76, 9.180; P<0.05). Further, The differences between CDH group and CDH+ FK506 group and control group were statistically significant (P<0.05). ④Western blotting analysis showed that the overall comparison of relative expression levels of BMPR2 and p-SMAD1 proteins in fetal rats of 3 groups had statistically significant differences (F=11.45, 10.94; P<0.05), while the overall comparison of the relative expression levels of p-SMAD5 protein in fetal rats of 3 groups did not show statistically significant difference (F=0.01, P>0.05). Further, the relative expression levels of BMPR2 and p-Smad1 in CDH group were lower than those in control group (P=0.049, 0.018), and the relative expression levels of BMPR2 and p-Smad1 in CDH+ FK506 group were higher than those in CDH group (P=0.010, 0.023).

Conclusions

Prenatal administration of FK506 can reduce pulmonary vascular remodeling in Nitrofen-induced CDH rat model, it has beneficial effects on alleviating lung hypoplasia in congenital diaphragmatic hernia.

Key words: Hernias, diaphragmatic, congenital, Hypertension, pulmonary, FK506, Blotting, western, Pulmonary artery, BMPR2, p-SMAD1, p-SMAD5, Rats
表1 4组胎鼠的双肺重量、胎鼠体重及双肺重量/胎鼠体重总体及组间多重比较
组别 胎鼠数(只) 双肺重量(mg，±s) 胎鼠体重(g, ±s) 双肺重量/胎鼠体重
①FK506组 33 167.0(156.2，179.5) 5.8±0.6 0.029(0.027，0.030)
②CDH组 42 99.6(85.32，120.5) 5.7±0.6 0.018(0.015，0.021)
③CDH+FK506组 38 99.7(82.00，117.7) 5.5±0.7 0.018(0.016，0.021)
④对照组 45 155.6(117.9，118.0) 5.4±0.9 0.029(0.025，0.030)
总体比较        
统计量   H＝81.25 F＝3.23 H＝106.98
P值   <0.001 0.240 <0.001
多重比较的P值        
① vs ④   0.318 0.055 >0.999
② vs ④   <0.001 0.108 <0.001
③ vs ②   >0.999 0.753 >0.999
图1 产前给予FK506对CDH胎鼠肺组织结构和血管厚度的影响(图1A~1D分别为对照组、FK506组、CDH组和CDH+FK506组，胎肺组织HE染色，高倍)注：FK506为他克莫司，CDH为先天性膈疝，HE为苏木精-伊红，EVG为血管弹性纤维染色
表2 4组CDH胎鼠左侧肺组织中膜厚度百分比和肺泡面积百分比总体及组间多重比较(±s)
组别 胎鼠数(只)a MT%(%) S%(%)
①FK506组 3 40.2±12.0 12.8±5.8
②CDH组 3 60.3±10.5 22.1±5.8
③CDH+FK506组 3 40.5±7.6 37.1±4.0
④对照组 3 36.2±2.7 36.7±7.0
总体比较      
F值   13.26 37.48
P值   0. 006 <0.001
多重比较的P值      
① vs ④   0.923 >0.999
② vs ④   0.001 <0.001
② vs ③   0.002 0.022
图2 对照组、CDH组和CDH+FK506组EVG染色图(图2A~2C：对照组、CDH组和CDH+FK506组，EVG染色，高倍)注：FK506为他克莫司，CDH为先天性膈疝，HE为苏木精-伊红，EVG为血管弹性纤维染色
表3 4组CDH胎鼠肺动脉中膜平滑肌层标志物α-SMA和CD31阳性细胞增殖指数总体及组间多重比较(±s)
组别 胎鼠数(只)a α-SMA CD31
①FK506组 3 28.7±1.5 26.7±5.2
②CDH组 3 71.5±8.9 48.0±2.2
③CDH+FK506组 3 48.3±6.6 25.6±9.3
④对照组 3 38.1±0.7 29.1±5.3
总体比较      
F值   33.76 9.18
P值   <0.001 0.006
多重比较的P值      
① vs ④   0.446 >0.999
② vs ④   <0.001 0.029
② vs ③   <0.001 0.011
图3 α-SMA和CD31免疫荧光染色图像[图3A~3D：对照组、FK506组、CDH组和CDH+FK506组α-SMA免疫荧光染色(绿色)；图3E~3H：对照组、FK506组、CDH组和CDH+FK506组CD31免疫荧光染色(红色)]注：α-SMA为α平滑肌肌动蛋白，CDH为先天性膈疝，FK506为他克莫司
表4 3组胎鼠BMPR2、p-SMAD1、p-SMAD5蛋白相对表达水平总体及组间多重比较(±s)
组别 胎鼠数(只) BMPR2 p-SMAD1 p-SMAD5
①CDH组 3 0.56±0.14 0.48±0.09 1.01±0.46
②CDH+FK506组 3 1.20±0.17 0.97±0.19 0.96±0.54
③对照组 3 1.00±0.19 1.00±0.16 1.00±0.35
总体比较        
F值   11.45 10.94 0.01
P值   <0.001 0.010 <0.991
多重比较的P值        
① vs ③   0.049 0.018 >0.999
① vs ②   0.010 0.023 0.999
图4 Western blotting检测对照组、CDH组和CDH+FK506组胎鼠肺组织中BMPR2、p-SMAD1、p-SMAD5蛋白电泳图注：CDH为先天性膈疝，FK506为他克莫司
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