色胺酮抑制胸腺基质淋巴细胞生成素促肥大细胞增殖的药理机制

Pharmacological mechanism of tryptanthrin inhibition of mast cell proliferation promoted by thymic stromal lymphopoietin

摘要:

目的  探讨色胺酮抑制由胸腺基质淋巴细胞生成素（TSLP）促肥大细胞增殖的药理机制，为色胺酮治疗肥大细胞相关过敏性皮炎提供理论依据。方法  在TSLP刺激人源肥大细胞系-1（HMC-1）细胞增殖的前提下，同步采用色胺酮处理增殖的细胞，观察对细胞增殖的影响和相关蛋白水平变化。噻唑蓝（MTT）比色法检测不同浓度色胺酮处理对细胞活力的影响；溴脱氧尿苷渗入试验和Ki67 mRNA水平检测不同处理组细胞增殖情况；蛋白免疫印迹法（Western blot）检测鼠双微体基因2（MDM2）、P53、Caspase-3、多聚ADP-核糖聚合酶（PARP）剪切蛋白水平变化；实时荧光定量聚合酶链反应检测白细胞介素7受体α（IL-7Ra）和TSLPR mRNA的含量。结果  MTT比色法结果表明，色胺酮无细胞毒性；溴脱氧尿苷渗入试验和Ki67 mRNA水平的检测均显示色胺酮抑制TSLP刺激的肥大细胞增殖；色胺酮抑制TSLP刺激的HMC-1中MDM2蛋白表达水平，蛋白灰度分析比较差异有统计学意义；色胺酮处理后，HMC-1细胞中，P53蛋白和凋亡标志蛋白Caspase-3、PARP剪切蛋白水平升高，且蛋白灰度分析比较差异有统计学意义；TSLP上游调控蛋白IL-7Ra和TSLPR mRNA水平在色胺酮的作用下受到抑制。结论  色胺酮可抑制TSLP促肥大细胞增殖，对治疗肥大细胞相关过敏性皮炎有一定的潜力。

Abstract:

Objective To investigate the pharmacological mechanism of tryptanthrin (TR) in suppressing mast cell proliferation promoted by thymic stromal lymphopoietin (TSLP), so as to provide academic evidence for mast cell induced allergic dermatitis (AD) therapy through TR. Methods After HMC-1 cells were stimulated by TSLP, cell proliferation was observed with another treatment of TR, and the relative protein levels were detected as well. The cells were divided into four groups: blank control, TSLP stimulating, TR treating and TSLP combined with TR groups. Cell viability was tested by MTT assay with various concentrations of TR. Cell proliferation effect was verified through BrdU assay and Ki67 mRNA level detection. MDM2, p53, cleaved caspase-3 and cleaved poly-ADP-ribose polymerase (PARP) protein levels were measured by Western blot. IL-7Ra and TSLPR mRNA levels were detected by real-time fluorescent quantitative PCR. Results MTT assay showed TR had no cytotoxicity. BrdU and Ki67 assay indicated that TR evidently suppressed mast cell proliferation promoted by TSLP.  After TR treatment, MDM2 protein level decreased in HMC-1 cells and the difference had statistical significance; on the contrary, p53, cleaved caspase and cleaved PARP protein levels significantly elevated. The upstream regulatory protein IL-7Ra and TSLPR mRNA levels were inhibited obviously with TR treatment. Conclusions TR could suppress mast cell proliferation promoted by TSLP, and exhibits certain potential in AD therapy.