目的 研究绿原酸对大鼠嗜碱性白血病细胞 RBL-2H3 脱颗粒作用及可能机制。方法 将不同质量浓度(3.125、6.250、12.500、25.000、50.000、100.000、200.000 μg·mL-1)的绿原酸作用于 RBL-2H3 细胞,通过实时细胞分析(RTCA)系统检测绿原酸干预后引起的细胞指数(CI)值变化,以评价 RBL-2H3 细胞脱颗粒情况;利用甲苯胺蓝染色观察细胞形态变化;化学荧光法测定组胺释放率;底物显色法测定β-氨基己糖苷酶释放率、类胰蛋白酶释放量;网络药理学预测绿原酸诱导RBL-2H3 细胞脱颗粒的潜在机制,并应用实时荧光定量PCR(qRT-PCR)对预测通路的主要靶点细胞外调节蛋白激酶(ERK1)、c-Jun氨基末端激酶(JNK)、p38丝裂原活化蛋白激酶(MAPK)mRNA水平进行检测。结果 绿原酸质量浓度 ≥ 6.25 μg·mL^-1时,可使RBL-2H3细胞的CI值在给药20min后呈现先快速上升后下降的趋势;绿原酸质量浓度 ≥ 12.5 μg·mL^-1时,RBL-2H3 细胞逐渐变圆甚至呈现多边形;与对照组比较,β-氨基己糖苷酶、组胺和类胰蛋白酶的释放量显著增加(P<0.05、0.01、0.001)。网络药理学预测发现,绿原酸致细胞脱颗粒作用可能与MAPK、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)等信号通路相关;qRT-PCR 结果证实,与对照组比较,绿原酸可显著增加 ERK1、p38MAPK mRNA表达(P<0.05、0.01),但对JNK mRNA表达影响不显著。结论 绿原酸可通过激活ERK1、p38 MAPK通路诱导 RBL-2H3 细胞发生脱颗粒,具有潜在的诱发类过敏反应作用。

Objective To explore the degranulation response and mechanism of chlorogenic acid on RBL-2H3 cells. Methods After stimulation of RBL-2H3 cells with different concentration (3.125, 6.250, 12.500, 25.000, 50.000, 100.000, and 200.000 μg·mL^-1) of chlorogenic acid, the cell index (CI) was monitored by using real-time cell analyzer (RTCA) in order to evaluate the degranulation response of RBL-2H3. The cell morphology was observed by using toluidine blue. The fluorescence method were used to verify histamine release and beta-hexose glucosidase, tryptase release were used to verify the degranulation of RBL-2H3 cells by colorimetric method. Potential mechanisms of degranulation response of chlorogenic acid on RBL-2H3 cells was predicted by network pharmacology and the mRNA levels of extracellular regulatory protein kinase (ERK1), c-Jun amino terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK), which were the main targets of the predicted pathway, were detected by real-time quantitative PCR (qRT-PCR). Results Chlorogenic acid at concentrations greater than 6.25 μg·mL^-1 could significantly increase the CI value of RBL-2H3 cells and at concentrations greater than 12.5 μg·mL^-1 could make the cell morphology change obviously and increased levels of histamine, β-hexose glucosidase and tryptase released compared with control group (P<0.05, 0.01, and 0.001). A network pharmacology approach was find that the degranulation response of chlorogenic acid on RBL-2H3 cells was mainly associated with MAPK signaling pathways and PI3K/Akt signaling pathways. qRT-PCR assays confirmed that ERK1, p38 MAPK mRNA levels were significantly increased (P<0.05, 0.01) in chlorogenic acid group and chlorogenic acid didn't obviously affect the expression of JNK mRNA compared with control group. Conclusion Chlorogenic acid can induce degranulation response of RBL-2H3 cells via the ERK1, p38 MAPK pathway and maybe induce anaphylactoid reactions.

R285.5

国家重点研发计划项目(2018YFC1706804)