温阳化浊通络方通过调控Sema3A/Nrp1通路促进系统性硬化病真皮微血管内皮细胞血管生成

doi:10.12122/j.issn.1673-4254.2026.06.12

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (6): 1323-1330.doi: 10.12122/j.issn.1673-4254.2026.06.12

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温阳化浊通络方通过调控Sema3A/Nrp1通路促进系统性硬化病真皮微血管内皮细胞血管生成

郭克磊¹^,²(), 王艺圆¹, 李颖利¹^,², 张红³, 韩立¹^,², 杜瑞娟¹^,², 欧阳竞锋⁴, 卞华¹^,²()

^1.南阳理工学院，张仲景国医国药学院，河南南阳 473004
^2.南阳理工学院，河南省张仲景方药与免疫调节重点实验室，河南南阳 473004
^3.南阳市中心医院风湿免疫科，河南南阳 473001
^4.中国中医科学院医学实验中心，北京 100700

收稿日期:2025-11-19 出版日期:2026-06-20 发布日期:2026-06-24
通讯作者: 卞华 E-mail:keleiguo318@126.com;biancrown@163.com
作者简介:郭克磊，博士，副教授，E-mail: keleiguo318@126.com
基金资助:
国家自然科学基金(82074415);中原英才计划-中原科技创新领军人才项目(234200510006);河南省科技攻关项目(202102310176);河南省科技攻关项目(232102311201);南阳市基础与前沿技术研究专项计划重点项目(23JCQY1006)

Wenyang Huazhuo Tongluo Formula promotes angiogenesis in systemic sclerosis dermal microvascular endothelial cells by regulating the Sema3A/Nrp1 pathway

Kelei GUO¹^,²(), Yiyuan WANG¹, Yingli LI¹^,², Hong ZHANG³, Li HAN¹^,², Ruijuan DU¹^,², Jingfeng OUYANG⁴, Hua BIAN¹^,²()

^1.ZHANG Zhongjing School of Chinese Medicine, Nanyang Institute of Technology, Nanyang 473004, China
^2.Henan Key Laboratory of ZHANG Zhong-jing Formulae and Herbs for Immunoregulation, Nanyang Institute of Technology, Nanyang 473004, China
^3.Department of Rheumatology Immunology, Nanyang Central Hospital, Nanyang 473001, China
^4.Experimental Research Center, Chinese Academy of Medical Sciences, Beijing 100700, China

Received:2025-11-19 Online:2026-06-20 Published:2026-06-24
Contact: Hua BIAN E-mail:keleiguo318@126.com;biancrown@163.com
Supported by:
National Natural Science Foundation of China(82074415)

摘要/Abstract

摘要：

目的探讨温阳化浊通络方（WHT）对系统性硬化病（SSc）真皮微血管内皮细胞血管生成的促进作用及潜在机制。方法 Wistar大鼠分别灌胃15、30、60 g/kg温阳化浊通络方及生理盐水制备低、中、高浓度含药血清和空白血清。体外培养人真皮微血管内皮细胞（HDMEC），经SSc患者血清处理建立SSc细胞模型，分为对照组、模型组、温阳化浊通络方低、中、高剂量组和EGCG组（Sema3A抑制剂），CCK-8检测细胞增殖，划痕检测细胞迁移，Matrigel体外成管实验检测血管生成，qRT-PCR及Western blotting检测Sema3A、Nrp1、VEGFA、CD31、α-SMA mRNA和蛋白表达水平。结果与对照组相比，模型组细胞增殖率和迁移率下降，α-SMA、Sema3A、VEGFA蛋白和mRNA表达水平增加，CD31及Nrp1蛋白和mRNA表达水平降低，血管生成受到抑制（P<0.05）；与模型组相比，低、中、高剂量含药血清组及EGCG组均可提高细胞存活率和迁移率，降低α-SMA、Sema3A、VEGFA蛋白和mRNA表达水平，促进CD31及Nrp1蛋白和mRNA表达水平，增强血管生成（P<0.05）。结论温阳化浊通络方可能通过调节Sema3A/Nrp1信号通路促进SSc HDMEC细胞增殖、迁移和血管生成。

关键词: 系统性硬化病, 温阳化浊通络方, Sema3A/Nrp1, 血管生成

Abstract:

Objective To investigate the effect of Wenyang Huazhuo Tongluo Formula (WHT) for promoting angiogenesis in systemic sclerosis (SSc) dermal microvascular endothelial cells and its possible mechanism. Methods Wistar rats were gavaged with 15, 30, or 60 g/kg WHT and normal saline for 7 consecutive days to prepare low-, medium-, or high-concentration WHT-medicated sera and blank serum, respectively. Human dermal microvascular endothelial cells (HDMECs) in routine culture were treated with the sera from SSc patients to establish an SSc cell model. The cells were treated with the blank serum, low-, medium-, or high-concentration WHT-medicated sera, or blank serum combined with EGCG (a Sema3A inhibitor). The changes in cell proliferation, migration and angiogenesis were assessed using CCK-8 assay, wound-healing assay and Matrigel tube formation assay, and the mRNA and protein expressions of Sema3A, Nrp1, VEGFA, CD31 and α-SMA were analyzed using qRT-PCR and Western blotting. Results HDMECs treated with the serum from SSc patients exhibited significantly reduced cell proliferation and migration rates, increased α-SMA, Sema3A and VEGFA protein and mRNA expression levels, decreased CD31 and Nrp1 protein and mRNA expression levels, and suppressed angiogenesis. In SSc serum-induced cells, treatments with low-, medium-, or high-concentration WHT-medicated sera or EGCG all significantly improved cell survival and migration rates, reduced α-SMA, Sema3A and VEGFA protein and mRNA expression levels, enhanced CD31 and Nrp1 protein and mRNA expressions, and promoted angiogenesis of the cells. Conclusion WHT promotes angiogenesis of SSc sera-induced HDMECs by modulating the Sema3A/Nrp1 signaling pathway.

Key words: systemic sclerosis, Wenyang Huazhuo Tongluo Formula, Sema3A/Nrp1, angiogenesis

郭克磊, 王艺圆, 李颖利, 张红, 韩立, 杜瑞娟, 欧阳竞锋, 卞华. 温阳化浊通络方通过调控Sema3A/Nrp1通路促进系统性硬化病真皮微血管内皮细胞血管生成[J]. 南方医科大学学报, 2026, 46(6): 1323-1330.

Kelei GUO, Yiyuan WANG, Yingli LI, Hong ZHANG, Li HAN, Ruijuan DU, Jingfeng OUYANG, Hua BIAN. Wenyang Huazhuo Tongluo Formula promotes angiogenesis in systemic sclerosis dermal microvascular endothelial cells by regulating the Sema3A/Nrp1 pathway[J]. Journal of Southern Medical University, 2026, 46(6): 1323-1330.

图/表 7

表1 qRT-PCR引物序列

Tab.1 Primer sequences for qRT-PCR

Gene	GenBank ID	Primer sequence (5'→3')	Length (bp)
Sema3A	NM_006080.3	Forward: TTTCCACTAAGCAGCAAC Reverse: ATCCCAAGCACAGTAAGG	134
Nrp1	NM_001244973.2	Forward: CCCCAAACCACTGATAACTC Reverse: TACCCAACATTCCAGAGCAA	125
VEGFA	NM_001171623.2	Forward: CATCTTCAAGCCATCCTG Reverse: AAATGCTTTCTCCGCTCT	246
CD31	NM_000442.5	Forward: CCTGCGGTATTCAAAGACAA Reverse: TGGGACCAGATCCTTCATTCAC	214
α-SMA	NM_001141945.3	Forward: GGTGATGGTGGGAATGGG Reverse: GCAGGGTGGGATGCTCTT	190
GAPDH	NM_002046.7	Forward: AACGGATTTGGTCGTATTG Reverse: GGAAGATGGTGATGGGATT	242

图1 WHT对HDMEC增殖的影响

Fig.1 Effect of Wenyang Huazhuo Tongluo Formula (WHT) on proliferation of systemic sclerosis (SSc) serum-induced human dermal microvascular endothelial cells HDMECs (n=3). △△P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

图2 WHT对HDMEC迁移的影响

Fig.2 Effect of WHT on migration of SSc serum-induced HDMECs. A: Cell migration detected by wound-healing assay (Original magnification: ×40). B: Cell migration rate of each group (n=3). △△P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

图3 WHT对HDMEC血管生成的影响

Fig.3 Effect of WHT on angiogenesis of SSc serum-induced HDMECs. A: Angiogenesis of the cells evaluated by Matrigel tube formation assay (×100). B, C: Nb branches and tube length of each group (n=3). △△P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

图4 WHT对CD31、α-SMA蛋白表达的影响

Fig.4 Effect of WHT on protein expression levels of CD31 and α-SMA in SSc serum-induced HDMECs. A: Western blotting for CD31 and α‑SMA in HDMECs. B, C: Quantitative analysis of CD31 (B) and α-SMA(C) protein expressions (n=3). △P<0.05, △△P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

图5 WHT对Sema3A、Nrp1、VEGFA、CD31、α-SMA mRNA表达的影响

Fig.5 Effect of WHT on mRNA expression levels of Sema3A (A), Nrp1 (B), VEGFA (C), CD31 (D) and α-SMA (E) in SSc serum-induced HDMECs (n=3). △△P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

图6 WHT对Sema3A、Nrp1、VEGFA蛋白表达的影响

Fig.6 Effect of WHT on protein expression levels of Sema3A, Nrp1 and VEGFA in SSc serum-induced HDMECs. A: Western blotting for Sema3A, Nrp1 and VEGFA in HDMECs. B-D: Quantitative analysis of Sema3A (B), Nrp1 (C) and VEGFA (D) protein expressions (n=3). △P<0.05, △△P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

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温阳化浊通络方通过调控Sema3A/Nrp1通路促进系统性硬化病真皮微血管内皮细胞血管生成

Wenyang Huazhuo Tongluo Formula promotes angiogenesis in systemic sclerosis dermal microvascular endothelial cells by regulating the Sema3A/Nrp1 pathway

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