劉承慈周妤芳Yu-Fang Chou營養學系2026-03-242023-10-2520212021-10-15https://handle.ncl.edu.tw/11296/w97ya5https://scholars.csmu.edu.tw/handle/123456789/7154近年研究指出，肥大細胞膜上具第四型組織胺受體(H4R)，且其活化會導致細胞脫顆粒作用和發炎反應。因此有學者推論當肥大細胞受到外源性刺激物活化時所分泌的histamine會惡化過敏反應。已知大豆異黃酮的主要活性成分genistein及daidzein對於多種先天和後天免疫系統的細胞活性具有調節的作用，但其中抗過敏效果之研究報導甚少，且其能否透過調節肥大細胞活性達到抗過敏之效果及其相關機制目前尚未釐清。本研究目的是探討在A23187、histamine及/或LPS誘導下，介入此兩種大豆異黃酮對於P815的脫顆粒作用、促發炎細胞激素釋放以及胞內發炎訊號關鍵蛋白質MyD88和NRLP3表現量之影響。本研究將P815細胞培養於熱失活DMEM (1×106 cells/ml)24小時，以serum-free DMEM wash out 6小時後，加入不同濃度大豆異黃酮genistein(0.01, 0.1, 1, 10µM)、daidzein(0.01, 0.1, 1, 10µM)或H4R專一性抑制劑–JNJ7777120(10µM)預培養1小時後，利用A23187(10 μM) 及/或以histamine(10 µg/ml)刺激1小時，並以A23187作為正對照組，進行β-hexosaminidase assay測定肥大細胞脫顆粒之活性；相同培養條件下，加入不同濃度大豆異黃酮genistein(0.01, 0.1, 1, 10 µM)、daidzein(0.01, 0.1, 1, 10 µM)、JNJ7777120(10 µM)預培養1小時，再利用histamine(10 µg/ml)及/或LPS(1 µg/ml)刺激6小時，收集細胞及其上清液分別測量細胞蛋白質MyD88和NLRP3表現量以及上清液中促發炎細胞激素含量。結果發現，雖然A23187刺激下則可顯著誘發細胞釋放顆粒酵素β-hexosaminidase之脫顆粒作用，但 histamine對於細胞脫顆粒作用之刺激效果並不顯著，且介入genistein(10 μM)和daidzein(10 μM)雖抑制A23187誘發之脫顆粒作用，但在histamine存在下其未影響細胞脫顆粒作用。在發炎反應方面，細胞受到histamine及/或LPS活化的情況下，促進肥大細胞產生促發炎細胞激素TNF-α及IL-6，且此作用顯著受到介入genistein (10 μM)或daidzein(10 μM)之抑制，其抑制程度與介入JNJ7777120相當。於胞內發炎訊號方面，透過histamine (10 µg/ml)或LPS (1µg/ml)刺激細胞，均可使胞內發炎訊號的關鍵蛋白MyD88和NRLP3之表現量上升，且介入genistein和daidzein都能顯著抑制此MyD88及NLRP3的表現量。綜合以上結果可知，genistein及daidzein這兩種大豆異黃酮能降低肥大細胞的脫顆粒作用及發炎反應，因而具有應用於抗過敏用途之潛力。Recent studies have shown that mast cells possess type IV histamine receptor (H4R). The activation of this receptor involves in degranulation and inflammatory responses of the cell. Consequently, it has been proposed that mast cells may response to extracellular histamine that secreted upon the induction of exogenous stimulus would worsen allergic condition. It is known that genistein and daidzein, two of the major soy isoflavones, are able to modulate the activity of several cell types of both innate and adaptive immune systems; however, studies on their anti-allergic activity are rare and their regulatory role on mast cells as well as the underlying mechanisms remained to be clarified. The aim of this study was to investigate the effects of these two soy isoflavones on the degranulation activity, the release of inflammatory cytokines, and the expression of intracellular inflammatory signals MyD88 and NRLP3 of P815 cells induced by A23187, histamine, and/or LPS. Cells were cultured in heat-inactivated DMEM (1×106 cells/ml) for 24 hours followed by washing out with serum-free DMEM for 6 hours, then different concentrations of genistein (0.01, 0.1, 1, 10 µM), daidzein (0.01, 0.1, 1, 10 µM), or H4R inhibitor JNJ7777120 (10 µM) were added to the culture medium and pretreated cells for 1 hour. Subsequently, cells were stimulated with A23187 (10 μM) and/or histamine (10 µg/ml) for 1 hour followed by the determination of degranulation activity of P815. A23187 was used as a positive control for degranulation response which was indicated by the secretion percentage of β-hexosaminidase activity. Alternatively, cells were pretreated with different concentrations of genistein (0.01, 0.1, 1, 10 µM), daidzein (0.01, 0.1, 1, 10 µM), or JNJ7777120 (10 µM) for 1 hour, followed by the stimulation of histamine (10 µM) and/or LPS (1 µg/ml) for 6 hours. Cell pellet and supernatant were then collected for analyzing the expression of MyD88 and NLRP3 proteins and for the pro-inflammatory cytokines, respectively. The results showed that despite that the degranulation of cells were significantly induced by A23187, reflected by significantly increased extracellular activity of β-hexosaminidase; however, this activity remained to be unaffected by the induction of histamine. In addition, both genistein (10 μM) and daidzein (10 μM) pretreatment significantly suppressed A23187-induced degranulation; however, neither of the soy isoflavons affected degranulation of cells in the presence of histamine. In the other hand, histamine and/or LPS significantly stimulated P815 to produce inflammatory cytokines TNF-α and IL-6, which was significantly reduced by genistein (10 μM) and daidzein (10 μM). Furthermore, western blotting analysis showed that histamine or LPS along significantly induced the expression of MyD88 and NLRP3 which were significantly suppressed by the pretreatment of cells with genistein and daidzein. In summary, both genistein and daidzein can reduce the degranulation and inflammatory activity of P815 cells. Consequently, genistein and daidzein have the potential to be used as anti-allergic agents.histamineA23187LPSmast cellsoy isoflavonegenisteindaidzeinallergythesis