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目的研究化合物1487B对肿瘤坏死因子-α(TNF-α)的作用,探讨其抗炎机制。方法利用荧光标记底物,检测在不同浓度化合物1487B作用下,THP-1细胞中肿瘤坏死因子-α转化酶(TACE)的活性;采用实时定量PCR法,分析肿瘤坏死因子-α的转录水平;利用酶联免疫吸附实验及免疫印迹,测定肿瘤坏死因子-α的分泌表达水平。结果在THP-1细胞中,1487B对胞内TACE酶活性有一定的抑制作用,实时定量PCR检测1487B可降低肿瘤坏死因子-α转录,且呈明显的剂量依赖性;免疫印迹及酶联免疫吸附实验检测终浓度为50、100、200μmol·L-1的1487B作用THP-1细胞后,肿瘤坏死因子-α前体的表达减少63%(P<0.01)、72%(P<0.01)、67%(P<0.01),肿瘤坏死因子-α的分泌水平降低24%(P<0.05)、31%(P<0.01)、70%(P<0.001)。结论 1487B通过抑制THP-1细胞胞内TACE酶活性和肿瘤坏死因子-α转录,减少肿瘤坏死因子-α生成,本实验初步阐明了化合物1487B的抗炎机制,为其进一步应用奠定了基础。
Objective To study the effect of compound 1487B on tumor necrosis factor-α (TNF-α) and its anti-inflammatory mechanism. Methods Tumor necrosis factor-α-converting enzyme (TACE) activity in THP-1 cells treated with different concentrations of compound 1487B was detected by fluorescent labeled substrate. The transcription level of tumor necrosis factor-α was analyzed by real-time quantitative PCR. The secretion of TNF-α was measured by enzyme-linked immunosorbent assay and Western blotting. Results In THP-1 cells, 1487B inhibited the activity of intracellular TACE enzyme. Real-time quantitative PCR assay 1487B could reduce the transcription of tumor necrosis factor-α, and showed a dose-dependent manner. Western blotting and enzyme-linked immunosorbent assay The expression of tumor necrosis factor-α (TNF-α) was decreased by 63% (P <0.01), 72% (P <0.01), and 67% after THP-1 cells were treated with 1487B at a concentration of 50,100 and 200μmol·L- (P <0.01). The secretion of tumor necrosis factor-α decreased by 24% (P <0.05), 31% (P <0.01), 70% (P <0.001). Conclusion 1487B can inhibit TNF-α production by inhibiting the intracellular TACE activity and TNF-α transcription of THP-1 cells. This experiment initially clarified the anti-inflammatory mechanism of compound 1487B and laid the foundation for its further application.