龙胆苦苷通过TGF-β1/Smad信号通路调控人瘢痕疙瘩成纤维细胞的增殖及胶原蛋白的产生

Gentiopicroside Regulates the Proliferation and Collagen Production of Human Keloid Fibroblasts via the TGF-β1/Smad Signaling Pathway

摘要: 目的：探讨龙胆苦苷(Gentiopicroside, GPS)对人瘢痕疙瘩成纤维细胞(Human keloid fibroblasts, HKF)增殖、迁移、细胞外基质(ECM)相关蛋白表达以及TGF-β1/Smad通路的影响。方法：利用成品HKF进行培养。将HKF分为空白对照组、DMSO组、龙胆苦苷浓度组(分别为2.5、5、7.5、10、12.5、15、17.5、20、22.5 μg/mL)，每组培养24 h后，采用MTT实验检测HKF增殖情况。利用细胞划痕实验检测20 μg/mL龙胆苦苷在不同时间段下(0 h、24 h、48 h)对HKF迁移能力的影响。根据MTT的结果，将实验分为空白对照组，20 μg/mL龙胆苦苷组，通过Western blot实验分别检测Collagen I、Collagen III、纤连蛋白(FN)及相关蛋白α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)的表达情况。而后再将其分为对照组(HKF)、模型组(HKF + TGF-β1)、实验组(HKF + TGF-β1 + GPS)。对照组不加任何处理因素，用完全培养基培养细胞；模型组：向完全培养基内加10 ng/ml TGF-β1培养细胞；实验组向GPS处理后的细胞加入10 ng/mlTGF-β1培养细胞，培养24 h，通过Western blot实验探索龙胆苦苷对TGF-β1/Smad信号通路蛋白的表达情况。结果：根据MTT实验法得出龙胆苦苷处理HKF 24 h后，随着浓度的增加，细胞存活率呈现下降趋势，在药物浓度为20 μg/mL作用24 h后，抑制HKF效果最为显著。细胞划痕实验显示，与空白对照组相比，20 μg/mL龙胆苦苷组HKF迁移能力受到显著抑制(P < 0.01)；Western blot显示龙胆苦苷可下调Collagen I、Collagen III、FN1、α-SMA蛋白表达，并且可降低TGF-β1/Smad通路中Smad2及Smad3磷酸化蛋白表达。结论：龙胆苦苷可能通过TGF-β1/Smad信号通路，抑制HKF增殖、迁移及细胞外基质相关蛋白的表达。

Abstract: Objective: To investigate the effects of gentiopicroside (GPS) on the proliferation, migration, expression of extracellular matrix (ECM)-related proteins, and TGF-β1/Smad signaling pathway in human keloid fibroblasts (HKF). Methods: Commercially available HKF were cultured and divided into blank control group, DMSO group, and GPS concentration groups (2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 μg/mL). After 24 h of culture, MTT assay was used to detect HKF proliferation. Cell scratch assay was performed to assess the effect of 20 μg/mL GPS on HKF migration ability at different time points (0 h, 24 h, 48 h). Based on the MTT results, the experiment was further divided into blank control group and 20 μg/mL GPS group; Western blot assay was used to detect the expression levels of Collagen I, Collagen III, fibronectin (FN), and α-smooth muscle actin (α-SMA). Subsequently, cells were divided into control group (HKF), model group (HKF + TGF-β1), and experimental group (HKF + TGF-β1 + GPS). The control group was cultured in complete medium without any treatment; the model group was cultured in complete medium with 10 ng/mL TGF-β1; the experimental group was pretreated with GPS followed by 10 ng/mL TGF-β1 for 24 h. Western blot assay was used to explore the effect of GPS on protein expression in the TGF-β1/Smad signaling pathway. Results: MTT assay showed that after 24 h of GPS treatment, HKF viability exhibited a decreasing trend with increasing GPS concentration, and the inhibitory effect on HKF was most significant at 20 μg/mL. Cell scratch assay revealed that compared with the blank control group, the migration ability of HKF in the 20 μg/mL GPS group was significantly inhibited (P < 0.01). Western blot demonstrated that GPS downregulated the protein expression of Collagen I, Collagen III, FN, and α-SMA, and reduced the phosphorylated protein expression of Smad2 and Smad3 in the TGF-β1/Smad pathway. Conclusion: GPS may inhibit HKF proliferation, migration, and the expression of ECM-related proteins through the TGF-β1/Smad signaling pathway.