摘要
实验目的：
以传统中药雷公藤红素为先导化合物,对其进行结构修饰设计合成不同衍生物,研究雷公藤红素不同衍生物对不同肿瘤细胞的体外抗增殖能力以及对叔丁基过氧化氢(t-BHP)损伤的PC12细胞的保护作用,并且初步研究其抗氧化损伤作用的机制。以期获得药效更强,毒副作用更低的新化合物。初步探讨雷公藤红素的构效关系,为合理设计合成新的药物提供指导。
实验方法：
1.衍生物的化学合成：以雷公藤红素为先导化合物,对其不同部位进行结构修饰,引入有效药物基团,合成一系列雷公藤红素衍生物。
2.衍生物的结构鉴定：应用现代光谱学方法(1HNMR、ESIMS)对新合成的雷公藤红素衍生物进行结构鉴定。
3.雷公藤红素及其衍生物体外抗增殖能力的研究：培养C6、PC12细胞,用不同浓度的雷公藤红素及其衍生物作用于细胞,48 h后,采用MTT法测定细胞活性,计算各化合物的IC50,以顺铂为阳性对照,比较各化合物ICso。
4.雷公藤红素及其衍生物对t-BHP损伤的PC12细胞的保护作用研究：培养PC12细胞,首先用不同浓度的药物对细胞进行预处理,然后给t-BHP (200μm)建立氧化损伤模型,24 h后,采用MTT法测定细胞活性。以雷公藤红素为阳性对照药考察其衍生物的抗氧化活性。
5.雷公藤红素及其衍生物抗氧化作用机制研究：利用Western blot方法检测衍生物对热休克蛋白70(Hsp70)的诱导表达。以雷公藤红素为阳性对照比较Hsp70的表达量。
实验结果：
1.合成得到雷公藤红素衍生物Cel 1-13,并通过质谱、氢谱确认其结构。
2.体外抗增殖实验结果表明,雷公藤红素衍生物化合物Cel 1-7以及化合物Cel-13对PC12细胞以及C6细胞均有抑制作用,且这些雷公藤红素衍生物均比临床上常用的抗肿瘤药顺铂(DDP)的抗肿瘤活性强。其中,Cel-1和Cel-6的IC50值要小于天然产物雷公藤红素,说明其活性优于母核。
3.对t-BHP损伤的PC12细胞的保护作用研究结果表明,部分雷公藤红素衍生物在本实验的浓度范围内能够对t-BHP造成的细胞损伤起到保护作用。Celastrol和Cel-13能够诱导Hsp70的表达。
结论：
雷公藤红素衍生物化合物Cel 1-7以及化合物Cel-13对PC12细胞以及C6细胞均有抑制作用,Cel-、Cel-6、Cel-7、Cel-9和Cel-13在一定浓度下能够保护PC12细胞免受t-BHP引起的氧化损伤,Celastrol和Cel-13能够诱导Hsp70的表达,但这与它们对于t-BHP损伤的PC12细胞的保护作用并不完全一致。这说明雷公藤红素衍生物对于t-BHP损伤PC12细胞的保护作用并不完全是通过诱导Hsp70蛋白的表达来实现的,还可能与其他的机制有关。
Objectives
A series of Celastrol analogues were synthesized. The antiproliferative activity of these analogues was studied with C6 and PC 12 cells. Their protective effect against oxidative stress was investigated on neuronal PC12 cells. To understand the neuroprotective mechanism of action, we determined the expression of Hsp70 proteins by Western blotting. Our purpose of the present work was to obtain new compounds with higher activity and less toxicity. Analysis of structure-activity relationship of the new compounds provides new information for future study.
Methods
1. Synthesize a series of Celastrol analogues.
2. Characterize structures by 1H NMR and ESIMS.
3. Antiproliferative Assay:Cells (C6, PC12) were grown to confluence in a humidified atmosphere (37℃,5% CO2). Compounds were added and cells were returned to the incubator for 48 h. Cell viability was measured by the MTT method. IC50 values were calculated from three independent experiments using SPSS.
4. Cytoprotective effect against l-BHP-induced PC 12 cells damage:PC 12 cells were cultured. Drugs at different concentrations were added, and the cells were incubated at 37℃for 1 h. BHP (200μM) was then added, and the cells were incubated for another 24 h at 37℃under 5% CO2. Cell viability was measured by the MTT assay.
5. Neuroprotective mechanism of action:Western blotting for Hsp70 was performed following standard procedure. Expression of Hsp70 protein was compared with Celastrol.
Results
1. New analogues Cel 1-13 were synthesized, and the structures of all new compounds were identified by 1H NMR and ESIMS.
2. Analogues Cel 1-7 and Cel-13 showed better antitumor activity on PC 12 and C6 cells than DDP. Cel-1 and Cel-6 have considerable activity compare to the natural product.
3. Cel-6, Cel-7 and Cel-13 inhibit t-BHP-induced PC 12 cell damage. Cel-13 was more effective in protecting cells from t-BHP-induced cell damage than its parent Celastrol. Both Celastrol and Cel-13 up-regulated the chaperone protein Hsp70 at the tested concentrations.
Conclusions
Cel 1-7 and Cel-13 were active on PC 12 and C6 cells. Cel-6, Cel-7 and Cel-13 blocked t-BHP-induced PC 12 cell damage at the tested concentrations. Celastrol and Cel-13 up-regulated the chaperone protein Hsp70. These results suggest that the protective effect and the Hsp70 protein expression were not well-correlated for Celastrol and Cel-13. It remains to be uncovered if other mechanisms of action were in play for their neuroprotective effects.
引文
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