Tripterin (Celastrol) is a natural product, a proteasome inhibitor that inhibits the pancreatic rennet-like activity of the 20S proteasome (IC50=2.5 μM). Tripterin has anti-inflammatory, anti-infectious, and immunomodulatory properties.

20S proteasome:2.5 μM

方法：人前列腺癌细胞 PC-3 用 Tripterin (0.5-5 µM) 处理 12 h，使用 Z-GGL-AMC 检测蛋白酶体糜蛋白酶样活性。
结果：Tripterin 以浓度依赖的方式显著抑制 PC-3 细胞中蛋白酶体糜蛋白酶的活性，在 2.5 µM 时，达到约 55% 的抑制作用。[1]
方法：人慢性髓白血病细胞 KBM-5 用 Tripterin (2.5 µM) 孵育 6 h，随后用 TNF (1 nM) 处理 6-24 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：TNF 以时间依赖的方式诱导抗凋亡蛋白 IAP1、IAP2、Bcl-2、Bcl-XL、c-FLIP 和 survivin 的表达，Tripterin 抑制了表达。[2]

方法：为检测体内抗肿瘤活性，将 Tripterin (1-3 mg/kg，10% DMSO+70% Cremophor/ethanol (3:1)+20% PBS) 腹腔注射给携带人前列腺癌肿瘤 PC-3 的 nude immunodeficient 小鼠，每天一次，持续十六天。
结果：Tripterin 治疗显著抑制前列腺癌症异种移植物的生长，并抑制体内蛋白酶体活性和诱导细胞凋亡。[1]
方法：为检测体内抗肿瘤活性，将 Tripterin (1.25 mg/kg) 腹腔注射给携带前庭神经鞘瘤 SC4 的 BALB/c (nu/nu) 小鼠，每三天一次，持续两周。
结果：Tripterin 显著抑制肿瘤生长而不显示毒性。[3]

Inhibition of purified 20S proteasome activity: A purified rabbit 20S proteasome (0.1 μg) is incubated with 40 μM of various fluorogenic peptide substrates in 100 μL assay buffer (20 mM Tris-HCl (pH 7.5)), in the presence of Celastrol at different concentrations or in the solvent DMSO for 2 hours at 37 ℃, followed by measurement of inhibition of each proteasomal activity.

The anti-proliferative effect of celastrol on various human tumor cell lines is determined by the MTT uptake method. Briefly, 5×103 cells are incubated with Celastrol in triplicate in a 96-well plate at 37 ℃. MTT solution is then added to each well. After a 2 hours incubation at 37 ℃, extraction buffer (20% SDS, 50% dimethylformamide) is added, cells are incubated overnight at 37 ℃, and the optical density is then measured at 570 nm using a Tecan plate reader.(Only for Reference)