低分子量硫酸葡聚糖的合成及其在抑制TGF-β1诱导的A549上皮-间充质转化中的作用

doi:10.5246/jcps.2025.03.016

中国药学(英文版) ›› 2025, Vol. 34 ›› Issue (3): 210-222.DOI: 10.5246/jcps.2025.03.016

低分子量硫酸葡聚糖的合成及其在抑制TGF-β1诱导的A549上皮-间充质转化中的作用

宋国豪¹, 符林波², 莫伊点², 耿彤彤², 吕禧敏², 孟祥豹², 王恒¹^,^*(), 李忠堂²^,^*(), 李中军²^,^*()

^1. 石河子大学药学院/新疆植物药资源利用教育部重点实验室/红花产业研究院, 新疆石河子 832003
^2. 北京大学药学院, 天然药物及仿生药物全国重点实验室, 北京 100191

收稿日期:2024-12-08 修回日期:2025-01-28 接受日期:2025-02-16 出版日期:2025-03-31 发布日期:2025-04-01
通讯作者: 王恒, 李忠堂, 李中军

Synthesis and functional evaluation of low-molecular-weight dextran sulfate as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal transition in A549 cells

Guohao Song¹, Linbo Fu², Yidian Mo², Tongtong Geng², Ximin Lv², Xiangbao Meng², Heng Wang¹^,^*(), Zhongtang Li²^,^*(), Zhongjun Li²^,^*()

¹ School of Pharmacy/Key Laboratory of Xinjiang Phytomedicine Resource and Utilization Ministry of Education/Institute for Safflower Industry Research, Shihezi University, Shihezi 832003, Xinjiang, China
² State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

Received:2024-12-08 Revised:2025-01-28 Accepted:2025-02-16 Online:2025-03-31 Published:2025-04-01
Contact: Heng Wang, Zhongtang Li, Zhongjun Li
Supported by:
The National Natural Science Foundation of China (Grant Nos. 92478133, 81930097, and 82151223), and by the State Key Laboratory of Natural and Biomimetic Drugs (Grant No. K202430).

摘要/Abstract

摘要：

特发性肺纤维化(IPF)是一种慢性肺部疾病, 目前治疗方案有限。上皮间质转化(EMT)被认为是其核心病理机制。本研究旨在合成低分子量硫酸葡聚糖(LMW-DSS), 并评价其潜在的EMT抑制效应, 探索新型IPF治疗策略。以分子量2 kDa的葡聚糖为起始原料, 采用不同的硫酸化试剂制备了两种硫酸化产物: DSS-LS和DSS-HS。体外实验中, 我们以TGF-β1诱导的A549细胞作为肺纤维化模型, 通过CCK-8实验验证LMW-DSS无细胞毒性, Transwell实验显示LMW-DSS能显著抑制TGF-β1诱导的A549细胞迁移。通过qPCR和Western Blot实验验证LMW-DSS可以减少促纤维化细胞因子TGF-β1和VEGF-A的表达释放, 抑制EMT关键转录因子Snail及间充质标志物Vimentin的表达。因此, 在LMW-DSS治疗中观察到α-SMA的下调, 表现出对EMT发展进程的显着抑制。低分子量硫酸葡聚糖可以有效减缓肺纤维化进程, 展现出潜在的临床应用价值, 为开发新型抗肺纤维化药物提供了实验依据。

关键词: 特发性肺纤维化, 上皮间质转化, 硫酸葡聚糖, 转化生长因子-β1, A549细胞

Abstract:

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease characterized by excessive fibrotic remodeling, for which effective therapeutic options remain severely limited. Among the pathogenic mechanisms implicated in IPF, epithelial-to-mesenchymal transition (EMT) is recognized as a pivotal driver of fibroblast activation and extracellular matrix deposition. In this study, we aimed to develop low-molecular-weight dextran sulfate sodium (LMW-DSS) derivatives and assess their capacity to interfere with EMT, thereby offering novel therapeutic avenues for IPF management. Starting with dextran (2 kDa) as a precursor, we successfully synthesized two sulfated derivatives, DSS-LS and DSS-HS, via distinct sulfonation processes. Using a TGF-β1-stimulated A549 alveolar epithelial cell model, we demonstrated that LMW-DSS compounds exhibited no cytotoxicity, as validated by CCK-8 viability assays. Importantly, Transwell migration assays revealed that LMW-DSS markedly attenuated TGF-β1-induced A549 cell migration, indicating a potent anti-fibrotic effect. Moreover, qPCR and Western blotting analyses confirmed that LMW-DSS significantly suppressed the expression and secretion of key pro-fibrotic mediators, including TGF-β1 and VEGF-A, and downregulated critical EMT-associated markers such as Snail and vimentin. Notably, reduced α-SMA expression following LMW-DSS treatment further substantiated its role in hindering EMT progression. Collectively, these findings highlighted the capacity of LMW-DSS to effectively impede EMT and fibrotic processes, thereby delaying the progression of pulmonary fibrosis. This work not only underscored the therapeutic potential of LMW-DSS in IPF but also provided compelling experimental evidence to support its development as a promising anti-fibrotic agent for clinical application.

Key words: Idiopathic pulmonary fibrosis, Epithelial to mesenchymal transition, Dextran sulfate sodium, Transforming growth factor-β1, A549

Supporting:

宋国豪, 符林波, 莫伊点, 耿彤彤, 吕禧敏, 孟祥豹, 王恒, 李忠堂, 李中军. 低分子量硫酸葡聚糖的合成及其在抑制TGF-β1诱导的A549上皮-间充质转化中的作用[J]. 中国药学(英文版), 2025, 34(3): 210-222.

Guohao Song, Linbo Fu, Yidian Mo, Tongtong Geng, Ximin Lv, Xiangbao Meng, Heng Wang, Zhongtang Li, Zhongjun Li. Synthesis and functional evaluation of low-molecular-weight dextran sulfate as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal transition in A549 cells[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(3): 210-222.

图/表 7

Table 1. The sequences of primers employed in the study.

Scheme 1. Synthetic route of low-molecular-weight dextran sulfate sodium (LMW-DSS).

Figure 1. GPC analysis of the molecular weight of LMW-DSS.

Figure 2. In vitro effects of DSS-LS and DSS-HS on cell viability and migration in TGF-β1-induced A549 cells. Cell viability was assessed using the CCK-8 assay, and cell migration was evaluated via transwell assay to determine the effects of DSS-LS and DSS-HS on TGF-β1-stimulated A549 cells. Data are presented as mean ± SD from independent experiments and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Compared with the TGF-β1 group: *P < 0.01; compared with the Control group: ##P < 0.01; ns, no significant difference.

Figure 3. Effects of LMW-DSS on the expression of TGF-β1 and VEGF-A in TGF-β1-induced A549 cells. qPCR and WB analyses were performed to evaluate the regulatory effects of LMW-DSS on the mRNA and protein expression levels of TGF-β1 and VEGF-A in A549 cells following TGF-β1 stimulation. Data are presented as mean ± SD from independent experiments and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Compared with the TGF-β1 group: *P < 0.05, **P < 0.01, ***P < 0.0001; Compared with the Control group: ####P < 0.0001, ##P < 0.01, #P < 0.05; ns, no significant difference.

Figure 4. Effects of LMW-DSS on α-SMA expression in TGF-β1-induced A549 cells. WB analysis was conducted to assess the impact of LMW-DSS on α-SMA expression in A549 cells following TGF-β1 induction. Data are presented as mean ± SD from independent experiments and analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. Compared with the TGF-β1 group: **P < 0.01, ***P < 0.001, ****P < 0.0001; Compared with the control group: ####P < 0.0001, ##P < 0.01; ns, no significant difference.

Figure 5. Effects of LMW-DSS on the expression of snail and vimentin in TGF-β1-induced A549 cells. WB analysis was performed to evaluate the regulatory effects of LMW-DSS on the expression of Snail and vimentin in A549 cells stimulated with TGF-β1. Data are presented as mean ± SD from independent experiments and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Compared with the TGF-β1 group: ***P < 0.001, ****P < 0.0001; Compared with the Control group: ####P < 0.0001; ns, no significant difference.

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低分子量硫酸葡聚糖的合成及其在抑制TGF-β1诱导的A549上皮-间充质转化中的作用

Synthesis and functional evaluation of low-molecular-weight dextran sulfate as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal transition in A549 cells

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