中国药物警戒 ›› 2024, Vol. 21 ›› Issue (1): 65-73.
DOI: 10.19803/j.1672-8629.20230708

• 中药毒性物质和机制研究专栏 • 上一篇    下一篇

雷公藤红素抑制前体脂肪细胞成脂分化的作用及机制研究

岳兰昕1, 沈磐1, 周磊1,2, 黄从书1,2, 周维1#, 高月1,*   

  1. 1军事科学院军事医学研究院辐射医学研究所抗辐射药物研究室,北京 100850;
    2天津中医药大学,天津 301617
  • 收稿日期:2023-11-07 出版日期:2024-01-15 发布日期:2024-01-18
  • 通讯作者: *高月,女,研究员·博导,中药药理与毒理。E-mail: gaoyue@bmi.an.cn; #为共同通信作者。
  • 作者简介:岳兰昕,女,硕士,中药药理与毒理。
  • 基金资助:
    国家自然科学基金资助项目（82192911）; 国家中医药管理局中医药创新团队及人才支持计划项目（ZYYCXTD- D-202207）

The inhibitory effect and mechanism of Celastrol on adipogenic differentiation in 3T3-L1

YUE Lanxin1, SHEN Pan1, ZHOU Lei1,2, HUANG Congshu1,2, ZHOU Wei1#, GAO Yue1,*   

  1. 1Anti-radiation Drug Research Laboratary, Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China;
    2Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
  • Received:2023-11-07 Online:2024-01-15 Published:2024-01-18

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摘要： 目的 研究雷公藤红素（celastrol,Cela）对前体脂肪细胞（3T3-L1）成脂分化的抑制作用及其具体机制。方法 采用CCK-8法确定雷公藤红素处理3T3-L1细胞的最佳浓度;油红O染色及免疫荧光法（脂肪细胞标志蛋白Perilipin1,Adiponectin）检测雷公藤红素对3T3-L1成脂分化不同时期的抑制作用;Western Blot（WB）检测不同浓度雷公藤红素处理后3T3-L1细胞中调控脂代谢以及分化相关蛋白的表达;对未分化（Con）、分化第1天（MDI）、分化第1天同时给药250 nmol·L-1雷公藤红素（MCT）的3T3-L1细胞进行WB检测以及microRNA（miRNA）测序,qPCR验证筛选得到的miRNA,进行KEGG与GO富集分析;过表达mmu-miR-5121 mimics和inhibitor,WB检测相关蛋白的变化,油红O 染色检测分化结果。结果 Cela处理3T3-L1细胞的最佳浓度为250 nmol·L-1和500 nmol·L-1;油红O染色及免疫荧光结果表明250 nmol·L-1与500 nmol·L-1Cela对3T3-L1成脂分化均有显著抑制作用,250 nmol·L-1在3T3-L1成脂分化早期（第1天）便能显著抑制3T3-L1成脂分化;250 nmol·L-1Cela处理3T3-L1 24 h后,能显著抑制3T3-L1中DFCP1、FAS、ACC、PPARγ等蛋白的表达;分化第1天FAS与ACC表达升高,采用250 nmol·L-1Cela处理后,ACC、FAS蛋白表达显著降低;miRNA测序筛选后qPCR验证结果表明mmu-miR-5121在分化第1天减少,但Cela处理后含量增加。转染5121 inhibitor提高了FAS与ACC的含量,5121 mimics则呈现相反作用。单独转染5121 inhibitor可使得3T3-L1分化成熟,而分化同时添加5121 mimics可以有效抑制分化。并且250 nmol·L-1Cela可以抑制5121 inhibitor引起的分化。结论 250 nmol·L-1雷公藤红素作用于3T3-L1成脂分化早期可显著抑制3T3-L1分化,其可能通过上调mmu-miR-5121的含量,抑制FAS与ACC的表达发挥作用。

关键词: 雷公藤红素, 前体脂肪细胞, 成脂分化, 脂代谢, 油红O染色, mmu-miR-5121

Abstract: Objective To study the inhibitory effect and mechanism of Celastrol (Cela) on adipogenic differentiation in preadipocytes (3T3-L1). Methods CCK-8 was used to determine the optimal concentration of Cela for 3T3-L1 cells. Oil red O staining and immunofluorescence (Perilipin1, Adiponectin) were used to imply the inhibitory effect of Celastrol on 3T3-L1 adipogenic differentiation at different stages. Western Blot (WB) was used to detect proteins expression related to lipid metabolism and differentiation in 3T3-L1 cells treated with different concentrations of Cela; WB, microRNA (miRNA) sequencing and qPCR were used to analyze undifferentiated (Con), differentiation Day1 (MDI), 250 nmol·L-1Cela treatment on the first day of differentiation (MCT), and KEGG and GO enrichment analysis were performed; mmu-miR-5121 mimics and inhibitor were overexpressed, then changes in related proteins were detected by WB, and differentiation results were detected by oil red O staining. Results The optimal concentrations of celastrol for treatment of 3T3-L1 cells were 250 nmol·L-1and 500 nmol·L-1. Oil red O staining and immunofluorescence results showed that 250 nmol·L-1Cela significantly inhibited the adipogenic differentiation of 3T3-L1, 250 nmol·L-1Cela at the early stage (the first day) significantly inhibited the adipogenic differentiation of 3T3-L1. WB results showed that treatment with 250 nmol·L-1celastrol significantly inhibited the protein expression of DFCP1, FAS, ACC, and PPARγ in 3T3-L1 cells. On the first day of differentiation, the expression of FAS and ACC increased. After treatment with 250 nmol·L-1Cela, the expression of ACC and FAS protein significantly decreased; miRNA sequencing screening and validation showed that mmu-miR-5121 decreased on the first day of differentiation, but increased after Cela treatment. Transfecting 5121 inhibitor increased the content of FAS and ACC, while 5121 mimics showed the opposite effect. Transfecting 5121 inhibitor alone led to adipogenic differentiation, while 5121 mimics could effectively inhibit normal differentiation. And 250 nmol·L-1Cela can inhibit differentiation induced by 5121 inhibitor. Conclusion 250 nmol·L-1Cela significantly inhibited the differentiation at early state of 3T3-L1 adipogenic differentiation, possibly by upregulating the content of mmu-miR-5121 and inhibiting the expression of FAS and ACC.

Key words: celastrol, 3T3-L1, adipogenic differentiation, lipid metabolism, oil red O staining, mmu-miR-5121

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