5-HT2 and 5-HT2B antagonists attenuate pro-fibrotic phenotype in human adult dermal fibroblasts by blocking TGF-β1 induced non-canonical signaling pathways including STAT3: implications for fibrotic diseases like scleroderma
5-HT2 and 5-HT2B antagonists attenuate pro-fibrotic phenotype in human adult dermal fibroblasts by blocking TGF-β1 induced non-canonical signaling pathways including STAT3: implications for fibrotic diseases like scleroderma
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DOI：
10.1111/1756-185x.13386
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发表时间：
2018-12-01
影响因子：
2.5
通讯作者：
Agarwal, Vikas
中科院分区：
文献类型：
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作者：
Chaturvedi, Saurabh;Misra, Durga Prasanna;Agarwal, Vikas
背景微血管损伤后激活的血小板释放5-羟色胺（5-HT; 5-羟色胺）导致组织纤维化。5-HT以转化生长因子β 1（TGF-β 1）依赖性方式强烈诱导真皮成纤维细胞的细胞外基质合成。目的评价5-HT 2和5-HT 2B抑制剂（特桂胺，SB 204741）对硬皮病患者成人真皮成纤维细胞（HADF）的抗纤维化作用。方法按照两种策略评价5-HT 2和5-HT 2B抑制剂的抗纤维化疗效：将HADF与5-HT（1 μ M）/TGF-β 1（10 ng/mL）处理1小时，然后用5-HT（1 μ M）/TGF-β 1（10 ng/mL）和特脲胺或SB 204741（1 μ M，每种）24小时（治疗后策略）和HADF用特古脲或SB 204741治疗（各1 μ M）处理1小时，随后5-HT（1 μ M）/TGF-β 1（10 ng/mL）处理24小时（预处理策略）。对促纤维化基因（TGF β 1、COL 1A 1、COL 1A 2、ACTA 2、CTGF和FN 1）和抗纤维化基因（MMP 2/TIMP 1）的表达进行真实的实时定量聚合酶链反应。通过免疫印迹检测I型胶原、α平滑肌肌动蛋白（α-SMA）、Smad 3、ERK 1/2和STAT 3的磷酸化的表达。结果用5-HT/TGF-β 1刺激HADF细胞导致促纤维化基因的表达增加，而Terguide和SB 204741均显著降低促纤维化基因的表达。与抑制剂孵育后，抗纤维化基因的表达不受影响。在5-HT刺激的HADF中，用terguride和SB 204741处理减少I型胶原和α-SMA。在5-HT/TGF-β 1刺激的HADF中，terguride和SB 204741处理降低ERK 1/2和STAT 3磷酸化，但不影响Smad 3磷酸化。结论Terguride和SB 204741可降低HADF细胞的促纤维化潜能，并抑制TGF-β 1介导的非经典途径ERK 1/2和STAT 3，这些途径与促纤维化基因的调控和纤维化的发生有关。总之，我们的数据表明，5-HT抑制剂可能通过抑制TGF-β 1介导的非经典信号通路来减少纤维化。这些观察结果对纤维化疾病如硬皮病具有重要的治疗意义。
Background Release of 5-hydroxytryptamine (5-HT; serotonin) from activated platelets following microvascular injury leads to tissue fibrosis. 5-HT strongly induces extracellular matrix synthesis in dermal fibroblasts in a transforming growth factor beta 1 (TGF-beta 1)-dependent manner. Aim To evaluate anti-fibrotic properties of inhibitors of 5-HT2 and 5-HT2B (terguride, SB204741) respectively in human adult dermal fibroblasts (HADF) derived from a patient with scleroderma. Methods Anti-fibrotic efficacy of 5-HT2 and 5-HT2B inhibitors was evaluated as per two strategies: HADF were incubated with 5-HT (1 mu M)/TGF-beta 1 (10 ng/mL) for 1 hour followed by 5-HT (1 mu M)/TGF-beta 1 (10 ng/mL) and terguride or SB204741 (1 mu M, each) for 24 hours (post-treatment strategy) and HADF were treated with terguride or SB204741 (1 mu M, each) for 1 hour followed by 5-HT (1 mu M)/TGF-beta 1 (10 ng/mL) for 24 hours (pre-treatment strategy). Real time quantitative polymerase chain reaction for expression of pro-fibrotic (TGF Beta 1, COL1A1, COL1A2, ACTA2, CTGF and FN1) and anti-fibrotic genes (MMP2/TIMP1) was performed. Expression of type I collagen, alpha smooth muscle actin (alpha-SMA), phosphorylation of Smad3, ERK1/2 and STAT3 was examined by immunoblotting. Results Stimulation of HADF cells with 5-HT/TGF-beta 1 led to the increased expression of pro-fibrotic genes which was significantly reduced by both terguride and SB204741. Expression of anti-fibrotic genes was not affected upon incubation with the inhibitors. In 5-HT-stimulated HADF, treatment with terguride and SB204741 decreased type I collagen and alpha-SMA. In 5-HT/TGF-beta 1 stimulated HADF, terguride and SB204741 treatment reduced ERK1/2 and STAT3 phosphorylation but did not influence Smad3 phosphorylation. Conclusion Terguride and SB204741 reduce pro-fibrotic potential of HADF cells and suppress TGF-beta 1-mediated non-canonical pathways, ERK1/2 and STAT3 which have been implicated in the regulation of pro-fibrotic genes and in the development of fibrosis. Taken together, our data suggest that 5-HT inhibitors might reduce fibrosis via suppression of TGF-beta1-mediated non-canonical signaling pathways. These observations have important therapeutic implications for fibrotic disorders like scleroderma.