川椒方含药血清对RBL-2H3细胞IL-4、TNF-α的影响

Impact of drug-containing serum of Chuanjiao decoction on IL-4 and TNF-α in RBL-2H3 Cells

目的探讨川椒方治疗变态反应性结膜炎可能的作用机制。方法实验分为空白组、模型组、阴性对照组、阳性对照组(酪氨酸激酶抑制剂,PP2)、川椒方治疗高、中、低剂量组共7组,观察川椒方含药血清对大鼠嗜碱性白血病细胞(rat basophilic leukemia,RBL-2H3)白介素4(IL-4)、肿瘤坏死因子α(TNF-α)的影响。川椒方高、中、低剂量含药血清组按筛选所得浓度和作用时间每孔分别给予含药血清40μL;PP2组于加抗原前30 min给予终浓度为1μM的PP2 40μL;阴性对照组于加抗原前2h给予10%空白血清40μL,酶联免疫吸附(ELISA)法检测细胞上清液中IL-4、TNF-α的分泌量,Real-Time PCR检测IL-4、TNF-α mRNA的表达。结果与空白组相比,模型组和阴性对照组肥大细胞脱颗粒明显(P<0.01);RBL-2H3细胞分泌IL-4和TNF-α增多(P<0.01);IL-4和TNF-α mRNA表达增高(P<0.01)。与模型组和阴性对照组比较,川椒方各浓度组和PP2组肥大细胞脱颗粒现象明显抑制(P<0.01);RBL-2H3细胞分泌IL-4和TNF-α减少(P<0.01);IL-4和TNF-α mRNA表达下降(P<0.01),且川椒方抑制肥大细胞脱颗粒作用与浓度呈正相关。结论稳定肥大细胞,抑制其脱颗粒,减少炎症因子释放可能是川椒方治疗变应性结膜炎的作用机制。

OBJECTIVE To observe the effects of Chuanjiao decoction（CJD） on basophilic leukemia（RBL-2H3） rat with allergic conjunctivitis（AC）, and to clarify its mechanism. METHODS For this experiment,RBL-2H3 cells were divided into the normal group, the model group, the blank serum group, and the low, medium,and high dose CJD groups, to study the impact of drug-containing serum of CJD on IL-4,TNF-α of RBL-2H3 cells.We chose the 10% concentration of serum and incubation with cell 120 min, before adding antigen for experiment.After induced RBL-2H3 cells degranulation by antigen, the number and the ratio of degranulated RBL-2H3 were determinated using toluidine blue staining method; The levels of tumor necrosis factor-alpha（TNF-α） and interleukin4（IL-4） in the culture media were determined using commercial ELISA kits. The levels of TNF-α and IL-4 mRNA were determined using commercial real time PCR. RESULTS Compared with the normal group, the ratio of degranulated RBL-2H3 was increased in the model and blank serum group（P〈0.01）; the expression of IL-4 and TNF-α in media was significantly increased in the model and blank serum group（P 0.01）; the mRNA levels for IL-4 and TNF-α were significantly increased in the model and blank serum group（P 0.01）; Compared with the model and blank serum group, the ratio of degranulated RBL-2H3 was decreased in the CJD groups and the PP2 group（P〈0.01）;the expression of IL-4 and TNF-α in media significantly decreased in the CJD groups and the PP2 group（P〈0.01）;and the CJD groups and the PP2 group significantly decreased IL-4 and TNF-α mRNA levels in Ag-stimulated RBL-2H3 cells. The inhibitory effect of CJD on mast cell degranulation was in a concentration dependent manner.CONCLUSIONS The mechanism of CJD in treating allergic conjunctivitis may be related to the inhibition of degranulation in mast cells.