雷公藤红素抑制SK-N-SH细胞增殖的作用机制:细胞周期阻滞与靶点网络分析

The mechanism of celastrol in inhibiting the proliferation of SK-N-SH cells:cell cycle arrest and target network analysis

摘要

目的 探讨雷公藤红素对神经母细胞瘤SK-N-SH细胞增殖的抑制作用及潜在分子机制。方法 采用CCK-8法检测5 μmol/L雷公藤红素作用24、48、72 h后SK-N-SH细胞的增殖能力,流式细胞术分析细胞周期分布变化,蛋白质印迹法(Western blot)检测细胞周期相关蛋白Cyclin D1、p53、p21的表达水平;结合网络药理学筛选雷公藤红素作用于神经母细胞瘤的潜在靶点,并进行GO功能富集及KEGG通路富集分析。结果 雷公藤红素可显著抑制SK-N-SH细胞增殖,且呈时间依赖性,24、48、72 h增殖抑制率分别为9.69%(P<0.05)、20.41%(P<0.01)、24.51%(P<0.001);该药物可诱导SK-N-SH细胞发生G0/G1期阻滞,72 h时G0/G1期细胞比例升至64.36%(P<0.01);Western blot结果显示,雷公藤红素可下调Cyclin D1蛋白表达,同时上调p53、p21蛋白表达,且变化均呈时间依赖性。网络药理学筛选得到雷公藤红素与神经母细胞瘤的交集靶点21个,Cyclin D1位列核心靶点前10;GO富集分析显示靶点主要富集于“细胞对环境刺激的反应”等生物学过程,KEGG富集分析提示靶点显著富集于AGE-RAGE信号通路等肿瘤相关通路。结论 雷公藤红素可能通过激活细胞应激反应,调控p53/p21-Cyclin D1信号轴,诱导SK-N-SH细胞发生G0/G1期周期阻滞,进而抑制细胞增殖,Cyclin D1是其关键作用靶点之一。

Abstract

Objective To investigate the inhibitory effect of celastrol on the proliferation of neuroblastoma SK-N-SH cells and its potential molecular mechanism. Methods CCK-8 assay was used to detect the proliferation of SK-N-SH cells treated with 5 μmol/L celastrol for 24, 48 and 72 h. Flow cytometry was applied to analyze the changes of cell cycle distribution. Western blot was used to detect the expression levels of cell cycle-related proteins Cyclin D1, p53 and p21. Network pharmacology was combined to screen the potential targets of celastrol acting on neuroblastoma, and GO functional enrichment and KEGG pathway enrichment analyses were performed. Results Celastrol could significantly inhibit the proliferation of SK-N-SH cells in a time-dependent manner,with the proliferation inhibition rates of 9.69% (P<0.05), 20.41%(P<0.01) and 24.51%(P<0.001) at 24, 48 and 72 h,respectively. The drug could induce G0/G1 phase arrest in SK-N-SH cells, and the proportion of G0/G1 phase cells increased to 64.36% at 72 h (P<0.01). Western blot results showed that celastrol could down-regulate the expression of Cyclin D1 protein,and up-regulate the expressions of p53 and p21 proteins in a time-dependent manner. Network pharmacology screening identified 21 intersection targets of celastrol and neuroblastoma,with Cyclin D1 ranking among the top ten core targets. GO enrichment analysis showed that the targets were mainly enriched in biological processes such as "cellular response to environmental stimulus", and KEGG enrichment analysis indicated that the targets were significantly enriched in tumor-related pathways such as AGE-RAGE signaling pathway. Conclusion Celastrol may inhibit the proliferation of SK-N-SH cells by activating cellular stress response,regulating the p53/p21-Cyclin D1 signaling axis and inducing G0/G1 phase cell cycle arrest,and Cyclin D1 is one of its key action targets.

关键词

雷公藤红素 / 神经母细胞瘤 / SK-N-SH细胞 / 细胞周期阻滞 / Cyclin D1 / 网络药理学

Key words

Celastrol / Neuroblastoma / SK-N-SH cells / Cell cycle arrest / Cyclin D1 / Network pharmacology

参考文献

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基金资助

山东省中医药科技项目(M-2022007)

济南市医疗卫生行业高层次人才专项经费资助(202412)