Wang Chengwei, Wu Chunjie, Song Linjiang
State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdisciplinary, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
Int J Mol Sci. 2025 Feb 27;26(5):2119. doi: 10.3390/ijms26052119.
Saiga antelope horn (SAH), a rare traditional Chinese medicine, exhibits activities of anti-feverish convulsions and anti-inflammation, whereas its underlying mechanism and specific pharmacological components are still unclear. In the present study, transient receptor potential ankyrin 1 (TRPA1), a major transient receptor potential cation channel was used as a target protein to identified TRPA1 high-affinity peptides (THPs) from SAH digests. Firstly, the SAH was digested under in vitro gastrointestinal conditions. With the method of affinity ultrafiltration and liquid chromatography-mass spectrometry (AUF-LC/MS), about 200 peptides that have a high-affinity interaction with the TRPA1 protein were screened from SAH digests. Subsequently, bioactivity databases and molecular docking were further exploited to identified three THPs, including RCWPDCR, FGFDGDF, and WFCEGSF. Furthermore, RIN-14B cells, characterized by the high expression of TRPA1 on cell surfaces, were used as the cell model to investigate the biological effect of THPs. Immunofluorescence and ELISA were conducted and showed that THPs can increase the intracellular Ca concentration and serotonin (5-HT) secretion in RIN-14B cells by activating TRPA1, which is evidenced by impaired upregulation of intracellular Ca levels and 5-HT secretion after pretreatment with the TRPA1 inhibitor (HC-030031). Moreover, an analysis of Western blots displayed that THPs up-regulated the expression levels of the 5-HT synthesis rate-limiting enzyme (TPH1) and 5-hydroxytryptophan decarboxylase (Ddc), while serotonin reuptake transporter (SERT) levels were down-regulated, suggesting that THPs enhance 5-HT secretion by regulating the 5-HT synthesis pathway. In summary, our findings demonstrate that THPs, which were identified from SAH digest via TRPA1-targeted affinity panning, exhibited the activation of the TRPA1 channel and enhanced 5-HT release in RIN-14B cells.
赛加羚羊角是一种珍稀的传统中药，具有解热惊厥和抗炎作用，但其潜在机制和具体药理成分仍不清楚。在本研究中，主要的瞬时受体电位阳离子通道——瞬时受体电位锚蛋白1（TRPA1）被用作靶蛋白，以从赛加羚羊角消化产物中鉴定TRPA1高亲和力肽（THP）。首先，在体外胃肠道条件下对赛加羚羊角进行消化。采用亲和超滤和液相色谱-质谱联用（AUF-LC/MS）方法，从赛加羚羊角消化产物中筛选出约200种与TRPA1蛋白具有高亲和力相互作用的肽。随后，进一步利用生物活性数据库和分子对接技术鉴定出三种THP，包括RCWPDCR、FGFDGDF和WFCEGSF。此外，以细胞表面高表达TRPA1为特征的RIN-14B细胞被用作细胞模型，以研究THP的生物学效应。进行了免疫荧光和酶联免疫吸附测定，结果表明THP可通过激活TRPA1增加RIN-14B细胞内的钙浓度和5-羟色胺（5-HT）分泌，TRPA1抑制剂（HC-030031）预处理后细胞内钙水平和5-HT分泌上调受损可证明这一点。此外，蛋白质印迹分析显示，THP上调了5-HT合成限速酶（TPH1）和5-羟色氨酸脱羧酶（Ddc）的表达水平，而5-羟色胺再摄取转运体（SERT）水平下调，这表明THP通过调节5-HT合成途径增强5-HT分泌。总之，我们的研究结果表明，通过以TRPA1为靶点的亲和淘选从赛加羚羊角消化产物中鉴定出的THP，在RIN-14B细胞中表现出对TRPA1通道的激活作用并增强了5-HT释放。
