青藤碱对大鼠肥大细胞RBL-2H3活化的影响及机制

Effect of sinomenine on activation of rat mast cells RBL-2H3 and its mechanism

目的探讨青藤碱对大鼠肥大细胞RBL-2H3活化的影响及其作用机制。方法将培养的大鼠RBL-2H3细胞随机分为对照Ⅰ组和青藤碱组。青藤碱组用青藤碱溶液处理,对照Ⅰ组则用PBS处理。利用生化法检测两组细胞培养上清中β-氨基己糖苷酶释放率,并用荧光定量PCR和Western blot方法检测细胞中信号调节蛋白α(SIRPα)mRNA和蛋白的表达水平。构建SIRPα过表达重组质粒或SIRPαsiRNA,将RBL-2H3细胞分为对照Ⅱ组、空载质粒组、过表达组、对照Ⅲ组、空白组、沉默组,对照Ⅱ组和对照Ⅲ组不进行转染,空载质粒组转染空白质粒,过表达组转染SIRPα过表达质粒,空白组转染非特异性siRNA,沉默组转染SIRPαsiRNA,转染结束后向对照Ⅲ组、空白组、沉默组添加青藤碱处理,并检测SIRPα的表达及炎症因子白细胞介素(IL)-4、IL-6和肿瘤坏死因子-α(TNF-α)的含量。结果与对照Ⅰ组比较,青藤碱组细胞中β-氨基己糖苷酶释放率显著降低(P=0.000),SIRPαmRNA及蛋白表达量均显著升高(P=0.000)。与对照Ⅱ组和空载质粒组比较,过表达组细胞中SIRPαmRNA表达量显著升高(P=0.000),IL-4、IL-6和TNF-α含量均明显降低(P=0.000)。与对照Ⅲ组和空白组比较,沉默组细胞SIRPαmRNA表达量显著降低(P=0.000),IL-4、IL-6和TNF-α含量均显著升高(P=0.000)。结论青藤碱处理能够显著抑制大鼠RBL-2H3细胞活化并上调细胞中SIRPα基因的表达,而SIRPα基因介导了青藤碱的这种抑制作用。

Objective To explore the effect of sinomenine on activation of rat mast cells RBL-2H3 and the underlying mechanism. Methods Cultured RBL- 2H3 cells were randomly assigned to the controlⅠ group and the sinomenine group. Cells in the sinomenine group were treated with sinomenine,while those in the controlⅠ group were treated with PBS. β- hexosaminidase release in the supernatants of these two groups were determined by biochemical methods.The mRNA and protein expression levels of signal regulatory protein α（ SIRPα） in these cells were analyzed by qRT-PCR and Western blot. Recombinant plasmids with SIRPα overexpression or SIRPα siRNAs were established. Furthermore,RBL- 2H3 cells were divided into the control Ⅱ group,blank vector group,overexpression group,control Ⅲgroup,mock group,and knockdown group. Transfection was not performed in cells of the controlⅡ and controlⅢ groups.Cells of the blank vector group were transfected with blank vectors,while cells of the overexpression group were transfected with SIRPα recombinant vectors. Cells of the mock group were transfected with non- specific siRNA,while cells of the knockdown group were transfected with SIRPα siRNA. After transfection,sinomenine was added into the control Ⅲ group,mock group,and knockdown group,and expression of SIRPα as well as the levels of interleukin（ IL）- 4,IL- 6,tumor necrosis factor- α（ TNF- α） were determined. Results Compared with the controlⅠ group,β- hexosaminidase release in cells of the sinomenine group was significantly decreased（ P = 0. 000） and SIRPα mRNA and protein expressions were upregulated（ P = 0. 000）. Compared with the controlⅡ group and blank vector group,SIRPα mRNA expression was significantly upregulated（ P = 0. 000） and levels of IL- 4,IL- 6 and TNF- α were significantly decreased（ P = 0. 000）in cells of the overexpression group. In contrast,compared with the control Ⅲ group and mock group,SIRPα mRNA expression was significantly downregulated（ P = 0. 000） and levels of IL- 4,IL- 6 and TNF- α were significantly higher（ P = 0. 000） in cells of the knockdown group. Conclusion Sinomenine treatment significantly inhibits activation of rat RBL- 2H3 cells and upregulates SIRPα expression,which mediates the inhibitory effect on RBL- 2H3 cells.