目的 研究綠原酸對(duì)大鼠嗜堿性白血病細(xì)胞 RBL-2H3 脫顆粒作用及可能機(jī)制。方法 將不同質(zhì)量濃度(3.125、6.250、12.500、25.000、50.000、100.000、200.000 μg·mL-1)的綠原酸作用于 RBL-2H3 細(xì)胞,通過實(shí)時(shí)細(xì)胞分析(RTCA)系統(tǒng)檢測(cè)綠原酸干預(yù)后引起的細(xì)胞指數(shù)(CI)值變化,以評(píng)價(jià) RBL-2H3 細(xì)胞脫顆粒情況;利用甲苯胺藍(lán)染色觀察細(xì)胞形態(tài)變化;化學(xué)熒光法測(cè)定組胺釋放率;底物顯色法測(cè)定β-氨基己糖苷酶釋放率、類胰蛋白酶釋放量;網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)綠原酸誘導(dǎo)RBL-2H3 細(xì)胞脫顆粒的潛在機(jī)制,并應(yīng)用實(shí)時(shí)熒光定量PCR(qRT-PCR)對(duì)預(yù)測(cè)通路的主要靶點(diǎn)細(xì)胞外調(diào)節(jié)蛋白激酶(ERK1)、c-Jun氨基末端激酶(JNK)、p38絲裂原活化蛋白激酶(MAPK)mRNA水平進(jìn)行檢測(cè)。結(jié)果 綠原酸質(zhì)量濃度 ≥ 6.25 μg·mL^-1時(shí),可使RBL-2H3細(xì)胞的CI值在給藥20min后呈現(xiàn)先快速上升后下降的趨勢(shì);綠原酸質(zhì)量濃度 ≥ 12.5 μg·mL^-1時(shí),RBL-2H3 細(xì)胞逐漸變圓甚至呈現(xiàn)多邊形;與對(duì)照組比較,β-氨基己糖苷酶、組胺和類胰蛋白酶的釋放量顯著增加(P<0.05、0.01、0.001)。網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)發(fā)現(xiàn),綠原酸致細(xì)胞脫顆粒作用可能與MAPK、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)等信號(hào)通路相關(guān);qRT-PCR 結(jié)果證實(shí),與對(duì)照組比較,綠原酸可顯著增加 ERK1、p38MAPK mRNA表達(dá)(P<0.05、0.01),但對(duì)JNK mRNA表達(dá)影響不顯著。結(jié)論 綠原酸可通過激活ERK1、p38 MAPK通路誘導(dǎo) RBL-2H3 細(xì)胞發(fā)生脫顆粒,具有潛在的誘發(fā)類過敏反應(yīng)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

國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2018YFC1706804)