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

doi:10.3877/cma.j.issn.1673-5250.2023.04.008

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

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

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

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

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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