FoxP1通过激活自噬抑制内皮细胞内皮-间充质转化的作用机制研究

doi:10.3877/cma.j.issn.2095-1221.2026.01.002

目的

探讨叉头盒转录因子P1 （FoxP1）介导自噬途径调控转化生长因子-β1（TGF-β1）诱导的内皮-间充质转化（EndMT）过程的作用机制。

方法

将人脐静脉内皮细胞分成对照、TGF-β1诱导EndMT模型组（TGF-β1）、干扰RNA阴性对照组（TGF-β1+si-NC）、FoxP1干扰RNA组（TGF-β1+si-FoxP1）、过表达空质粒阴性对照组（TGF-β1+oe-NC）、FoxP1过表达组（TGF-β1+oe-FoxP1）、FoxP1过表达联合自噬抑制剂组（TGF-β1+oe-FoxP1+3-MA）。Western blot检测内皮、间充质标志物及胶原蛋白表达；应用mCherry-EGFP-LC3双荧光系统检测自噬；通过划痕实验评估细胞迁移能力。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

Western blot结果显示，与对照比较，TGF-β1组内皮标志物VE-cadherin、CD31表达降低，间充质标志物α-SMA、Vimentin表达升高，胶原蛋白Collagen Ⅰ（3.08 ± 0.09比1.00 ± 0.08）、Collagen Ⅲ表达（3.14 ± 0.10比1.00 ± 0.05）升高，细胞迁移能力[（75.20 ± 4.30）%比（36.80 ± 2.60）%]、p62表达（2.24 ± 0.07比1.00 ± 0.07）增强，Beclin-1 （0.49 ± 0.03比1.00 ± 0.02）、LC3 Ⅱ/Ⅰ表达水平（0.17 ± 0.01比1.00 ± 0.08）降低（P均< 0.01）；与TGF-β1+oe-NC组相比，TGF-β1+oe-FoxP1组VE-cadherin和CD31表达上调，α-SMA、Vimentin表达下降，胶原蛋白Collagen Ⅰ（2.08 ± 0.10比4.38 ± 0.15）、Collagen Ⅲ表达（1.86 ± 0.07比3.60 ± 0.14）、细胞迁移能力[（46.66 ± 5.15）%比（77.56 ± 7.30）%]、p62表达（1.77 ± 0.09比2.24 ± 0.08）降低，Beclin-1 （0.82 ± 0.01比0.49 ± 0.03）、LC3 Ⅱ/Ⅰ表达水平（0.55 ± 0.02比0.20 ± 0.01）升高（P均< 0.01）。与TGF-β1+oe-FoxP1组相比，TGF-β1+oe-FoxP1+3-MA组Beclin-1 （1.60 ± 0.03比1.96 ± 0.02）、LC3 Ⅱ/Ⅰ表达（2.20 ± 0.04比3.88 ± 0.16）下降，p62 （0.70 ± 0.02比0.49 ± 0.05）、Collagen Ⅰ（0.80 ± 0.02比0.51 ± 0.03）、Collagen Ⅲ表达（0.70 ± 0.01比0.29 ± 0.02）升高（P均< 0.01），E-cadherin和CD31的表达均下调，α-SMA、Vimentin表达上调，细胞迁移能力[（58.63 ± 6.19）%比（40.84 ± 5.27）%]增强（P均< 0.01）。

结论

FoxP1过表达可激活自噬途径抑制TGF-β1诱导的EndMT过程，旨在为心肌纤维化的机制研究与治疗提供新的细胞学视角。

关键词: 心力衰竭, 内皮-间充质转化, FoxP1, 自噬

Objective

To explore the mechanismof forkhead box p1 (FoxP1) , a forkhead box transcription factor, mediates the autophagy pathway to regulate the process of transforming growth factor-β1 (TGF-β1) -induced endothelial to mesenchymal transition (EndMT) .

Methods

The human umbilical vein endothelial cells were divided into the following groups: control group (Blank) , TGF-β1-induced EndMT model group (TGF-β1) , small interfering RNA negative control group (TGF-β1+si-NC) , FoxP1 small interfering RNA group (TGF-β1+si-FoxP1) , overexpressing RNA negative control group (TGF-β1+oe-NC) , FoxP1 overexpressing group (TGF-β1+oe-FoxP1) , and FoxP1 overexpression combined with an autophagy inhibitor group (TGF-β1+oe-FoxP1+3-MA) . Western blot were used to detect the expression of endothelial、mesenchymal markers and collagen. The mCherry-EGFP-LC3 dual fluorescence system was used to detect autophagy status. Cell migration ability was assessed through scratch assay. The independent samples t-test was used for the comparisons between two groups, while for comparisons among multiple groups, one-way analysis of variance (ANOVA) was employed, and the LSD-t method was used for pairwise comparisons.

Results

Western blot results showed that, compared to the control group, the expression of endothelial markers VE-cadherin and CD31 were decreased in the TGF-β1 group, while the expression of mesenchymal markers α-SMA,Vimentin and collagen proteins Collagen Ⅰ (3.08 ± 0.09 vs 1.00 ± 0.08) , Collagen Ⅲ (3.14 ± 0.10 vs 1.00 ± 0.05) were increased. In addition, the cell migration ability [ (75.20 ± 4.30) %vs (36.80 ± 2.60) %] and the expression of p62 (2.24 ± 0.07 vs 1.00 ± 0.07) were increased in TGF-β1 group when compared to control group, while the expression levels of Beclin-1 (0.49 ± 0.03比1.00 ± 0.02) and LC3 Ⅱ/Ⅰ (0.17 ± 0.01 vs 1.00 ± 0.08) were decreased (all P < 0.01) . Compared to the TGF-β1+oe-NC group, the expression of VE-cadherin, CD31 and Beclin-1 (0.82 ± 0.01 vs 0.49 ± 0.03) , LC3 Ⅱ/Ⅰ (0.55 ± 0.02 vs 0.20 ± 0.01) (all P < 0.01) were increased in the TGF-β1+oe-FoxP1 group, while the expression of α-SMA, Vimentin and collagen proteins Collagen Ⅰ (2.08 ± 0.10 vs 4.38 ± 0.15) , Collagen Ⅲ (1.86 ± 0.07 vs 3.60 ± 0.14) as well as p62 (1.77 ± 0.09 vs 2.24 ± 0.08) , were decreased, along with areduced cell migration ability [ (46.66 ± 5.15) %比(77.56 ± 7.30) %] (all P < 0.01) . Compared to the TGF-β1+oe-FoxP1 group, the expression of Beclin-1 (1.60 ± 0.03 vs 1.96 ± 0.02) , LC3 Ⅱ/Ⅰ (2.20 ± 0.04 vs 3.88 ± 0.16) and E-cadherin, CD31 were decreased in the TGF-β1+oe-FoxP1+3-MA group, while the expression of p62 (0.70 ± 0.02 vs 0.49 ± 0.05) , Collagen I (0.80 ± 0.02 vs 0.51 ± 0.03) , CollagenⅢ (0.70 ± 0.01 vs 0.29 ± 0.02) and α-SMA, Vimentin were increased, along with the enhanced cell migration ability [ (58.63 ± 6.19) %比(40.84 ± 5.27) %] (all P < 0.01) .

Conclusion

Overexpression of FoxP1 can activate the autophagy pathway to inhibit the TGF-β1-induced EndMT process,while could provide a novel cytological perspective for the mechanistic research and therapeutic approaches to myocardial fibrosis.

Key words: Heart failure, Endothelial-to-mesenchymal transition, FoxP1, Autophagy

表1 引物序列信息

基因	引物序列（5'→3'）	预期扩增长度（bp）
FoxP1	上游TGGCATCTCATAAACCATCAGC	134
	下游GGTCCACTCATCTTCGTCTCAG
GAPDH	上游ACAGCCTCAAGATCATCAGC	104
	下游GGTCATGAGTCCTTCCACGAT

表1 引物序列信息

基因	引物序列（5'→3'）	预期扩增长度（bp）
FoxP1	上游TGGCATCTCATAAACCATCAGC	134
	下游GGTCCACTCATCTTCGTCTCAG
GAPDH	上游ACAGCCTCAAGATCATCAGC	104
	下游GGTCATGAGTCCTTCCACGAT

图1 光学显微镜下观察对照和TGF-β1诱导后内皮细胞形态（×200）注：a图为对照组诱导后内皮细胞形态；b图为TGF-β1诱导后内皮细胞形态改变

图2 Western blot检测内皮标志物VE-cadherin、CD31和成纤维细胞标志物α-SMA、Vimentin、胶原蛋白Collagen Ⅰ/ Ⅲ的表达注：^**P < 0.01，n = 3

图3 倒置显微镜下观察对照和TGF-β1诱导后细胞迁移能力（×200）注：TGF-β1组细胞迁移能力增强；^**P < 0.01，n = 3

图4 FoXP1过表达或敲低效率验证注：a图为RT-qPCR检测FoxP1过表达效率；b图为Western blot检测FoxP1过表达效率；c图为RT-qPCR检测FoxP1敲低效率；d图为Western blot检测FoxP1敲低效率；^**P < 0.01，n = 3

图5 Western blot检测内皮标志物VE-cadherin、CD31和成纤维细胞标志物α-SMA、Vimentin的表达注：a、b图分别为Western blot检测过表达FoXP1及敲低FoXP1对内皮标志物VE-cadherin、CD31和成纤维细胞标志物α-SMA、Vimentin表达的影响；^**P < 0.01，n = 3

图6 倒置显微镜下观察过表达FoXP1和敲低FoXP1后各组细胞迁移能力（×200）注：a图为过表达FoXP1对细胞迁移的影响，可见TGF-β1+oe-FoxP1组细胞迁移能力减弱；b图为敲低FoXP1对细胞迁移的影响，可见TGF-β1+si-FoxP1组细胞迁移能力增强；^**P < 0.01，n = 3

图7 FoxP1过表达通过促进自噬抑制TGF-β1诱导的EndMT注：a图为Western blot检测自噬标志蛋白Beclin-1、p62及LC3 Ⅱ/Ⅰ表达水平，^**P < 0.01，n = 3；b图为倒置荧光显微镜观察mCherry-EGFP-LC3共转染后各组细胞自噬结果（×400）

图8 干扰FoxP1通过抑制自噬促进TGF-β1诱导的EndMT注：a图为Western blot检测自噬标志蛋白Beclin-1、p62及LC3 Ⅱ/Ⅰ的表达水平；b图为倒置荧光显微镜观察mCherry-EGFP-LC3共转染后各组细胞自噬结果（×400）；^**P < 0.01，n = 3

图9 Western blot检测自噬相关蛋白、内皮标志物、成纤维细胞标志物和胶原蛋白表达水平注：a图为自噬相关蛋白Beclin-1、LC3 Ⅱ/Ⅰ、p62水平；b图为内皮标志物VE-cadherin、CD31、成纤维细胞标志物α-SMA、Vimentin和胶原蛋白CollagenⅠ、Collagen Ⅲ的蛋白水平；^**P < 0.01，n = 3

图10 倒置荧光显微镜观察mCherry-EGFP-LC3共转染后各组细胞自噬结果（× 400）注：与TGF-β1+oe-FoxP1组相比，TGF-β1+oe-FoxP1+3-MA组黄色荧光斑点增多，红色荧光斑点减少

图11 倒置显微镜下观察各组细胞迁移能力（× 200）注：与TGF-β1+oe-FoxP1组相比，TGF-β1+oe-FoxP1+3-MA组细胞迁移能力增强；^**P < 0.01，n = 3

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The mechanism of FoxP1 inhibits endothelial-mesenchymal transition in endothelial cells via autophagy activation

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The mechanism of FoxP1 inhibits endothelial-mesenchymal transition in endothelial cells via autophagy activation