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合欢皮作为常用中药,临床常用于治疗失眠、焦虑、肿瘤等。现代药理中针对合欢皮总皂苷(TSAJ)抗肿瘤活性多有报道,但由于单体成分分离困难,药理机制难以开展。该研究采用MTT法和Hoechst 33258染色法,确认了TSAJ对肝癌细胞的生长抑制作用,构建了表面修饰有TSAJ的固相亲合微球,利用该固相微球特异性地从人肝癌细胞裂解液中富集靶点蛋白,再利用高分辨质谱对富集到的靶点蛋白进行鉴定。采用Matabo Analyst 3.0处理数据,筛选并确定了Exportin-2,Beta-actin-like protein 2,Myosin-9,Protein transport protein Sec61 subunit beta,Cytochrome c oxidase copper chaperone等5个靶点蛋白。这些蛋白分别具有调控细胞凋亡及细胞生长、分化、运动等功能,提示TSAJ的抗肿瘤机制可能与诱导细胞凋亡及抑制肿瘤血管生成有关。综上所述,该研究利用TSAJ修饰的固相亲和微球,特异性富集并鉴定了TSAJ的靶点蛋白,从靶点源头上解释了TSAJ抗肿瘤作用机制,同时也为中药复杂体系的分子药理机制研究提供了一种全新的思路。
Acacia skin as a commonly used traditional Chinese medicine, clinically used in the treatment of insomnia, anxiety, cancer and so on. The anti-tumor activity of TSAJ in modern pharmacology has been reported. However, due to the difficulty of separation of monomer components, the pharmacological mechanism is difficult to develop. In this study, MTT assay and Hoechst 33258 staining were used to confirm the inhibitory effect of TSAJ on the growth of hepatocellular carcinoma cells. A TSAJ-modified solid-phase affinity microsphere was constructed. The solid-phase microspheres were specifically purified from human liver cancer cell lysate In the target protein enrichment, and then use high-resolution mass spectrometry to identify the target protein. The data of Matabo Analyst 3.0 were used to screen and identify five target proteins of Exportin-2, Beta-actin-like protein 2, Myosin-9, Protein transport protein Sec61 subunit beta and Cytochrome c oxidase copper chaperone. These proteins have the functions of regulating apoptosis and cell growth, differentiation, exercise and so on. These results suggest that the anti-tumor mechanism of TSAJ may be related to the induction of apoptosis and the inhibition of tumor angiogenesis. In summary, the TSAJ-modified solid-phase affinity microspheres were used to specifically enrich and identify the TSAJ target protein. From the source of the target, the anti-tumor mechanism of TSAJ was explained. At the same time, Molecular pharmacological mechanism provides a new way of thinking.