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目的探讨中药活性成分雷公藤红素对TNF-α体外抗结肠癌活性的影响并研究其机制。方法用雷公藤红素联合TNF-α体外治疗结肠癌细胞系SW480,MTT法检测SW480细胞的细胞活力。SW480细胞用雷公藤红素联合TNF-α治疗后,Annexin V/PI染色法检测细胞的凋亡,Western blot法检测细胞caspase-8、caspase-9和caspase-3的活化和对CYLD蛋白表达的影响,并用免疫共沉淀法检测SW480细胞RIP1蛋白的泛素化水平。结果雷公藤红素联合TNF-α对结肠癌细胞系SW480的细胞活力抑制率和凋亡诱导活性均显著高于雷公藤红素及TNF-α单治疗组。雷公藤红素联合TNF-α对SW480细胞caspase-8、caspase-9和caspase-3的活化显著高于雷公藤红素及TNF-α单治疗组,且两者联合治疗后,caspase-8的活化时间显著早于caspase-9和caspase-3。SW480细胞用雷公藤红素联合TNF-α治疗后,其RIP1蛋白的泛素化水平显著低于雷公藤红素及TNF-α单治疗组。进一步研究发现,雷公藤红素能显著诱导SW480细胞CYLD蛋白的表达,而TNF-α对CYLD的表达水平无影响,当用小干扰RNA沉默CYLD的表达后,雷公藤红素对TNF-α的协同效应丧失。结论雷公藤红素通过促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性。
Objective To investigate the effect of Tripterine on the anti-colon cancer activity of TNF-α in vitro and its mechanism. Methods Tripterygium wilfordii combined with TNF-α in vitro treatment of colon cancer cell line SW480, MTT assay SW480 cell viability. SW480 cells were treated with tripterine and TNF-α, the apoptosis of SW480 cells was detected by Annexin V / PI staining, the activation of caspase-8, caspase-9 and caspase-3 and the expression of CYLD protein were detected by Western blot And the immunoprecipitation method was used to detect the ubiquitination level of RIP1 protein in SW480 cells. Results Tripterine combined with TNF-α inhibited cell viability and induced apoptosis of colon cancer cell line SW480 significantly compared with tripterine and TNF-α monotherapy groups. Tripterine combined with TNF-α on SW480 cells caspase-8, caspase-9 and caspase-3 activation was significantly higher than that of tripterine and TNF-α monotherapy group, and the combination of both, caspase-8 Activation time was significantly earlier than caspase-9 and caspase-3. The level of ubiquitination of RIP1 protein in SW480 cells treated with tripterine combined with TNF-α was significantly lower than that of tripterine and TNF-α monotherapy groups. Further study found that Tripterine significantly induced the expression of CYLD protein in SW480 cells, while TNF-α had no effect on the expression of CYLD. After silencing the expression of CYLD with small interfering RNA, Loss of synergy. Conclusion Tripterine enhances the apoptosis-inducing activity of TNF-α on colon cancer cells by promoting the de-ubiquitination of RIP1 protein.