槲皮素对毒胡萝卜素诱导的巨噬细胞内质网应激凋亡途径的抑制作用及机制

目的: 研究槲皮素(quercetin, Que)对毒胡萝卜素(thapsigargin, TG)诱导的巨噬细胞RAW264.7内质网应激凋亡途径的抑制作用及机制。方法: 1 μmol/L TG作用RAW264.7细胞24 h诱导内质网应激,不同浓度Que(80、120或160 μmol/L)与TG共同作用后,MTT法检测细胞存活率,流式细胞术检测凋亡率及Ca²⁺]_i,激光共聚焦显微镜观察细胞形态变化,Western blotting法检测糖调节蛋白78 (glucose-regulated protein 78,GRP78)及C/EBP同源蛋白(C/EBP homologous protein, CHOP)的表达;Western blotting法检测Que(160 μmol/L)和(或)磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)抑制剂LY294002(15 nmol/L)与TG共同作用时GRP78和CHOP的表达。结果: Que能够抑制TG诱导的RAW264.7细胞内质网应激损伤,与TG组相比,细胞存活率升高(P<0.05),凋亡率降低(P<0.05),Ca²⁺]_i降低(P<0.05),GRP78及CHOP表达减少(P<0.05);LY294002单独作用可抑制TG诱导的GRP78及CHOP表达上调(P<0.05),但与Que联合应用与二者单独使用时抑制作用无显著差异。结论: Que可以抑制TG诱导的RAW264.7细胞内质网应激凋亡途径,该作用可能与其抑制PI3K信号通路从而降低CHOP蛋白的表达有关。

Protective effect of quercetin on ER stress-related apoptosis induced by thapsigargin in macrophages

AIM:To investigate the effects and possible mechanisms of quercetin（Que） on endoplasmic reticulum stress（ERS） -related apoptosis induced by thapsigargin（TG） in RAW264.7 cells.METHODS;ER stress of RAW264.7 cells were induced by TG at concentration of 1μmol/L for 24 h.After treated with different concentrations of Que（80,120 and 160μmol/L）,the cell viability was determined by MTT assay.The apoptotic rate and the changes of intracellular Ca⁺ concentration(Ca²⁺]₁) were determined by flow cytometry,and the cell apoptotic morphology was observed under laser scanning confocal microscope.The protein levels of glucose-regulated protein 78（GRP78 ） and C/EBP homologous protein（CHOP） were detected by Western blotting.The effect of Que on GRP78 and CHOP induced by TG with phosphatidylinositol 3 -kinase（PI3K） inihibitor LY294002 at concentration of 15 nmol/L was measured by Western blotting.RESULTS:Que suppressed ER stress-related injury induced by TG in RAW264.7 cells.Compared with TG group,the cell viability increased（P<0.05）,apoptotic rate andCa²⁺]₁ decreased（P <0.05） and the changes of apoptotic morphology were alleviated.The increase in GRP78 and CHOP induced by TG as an ER stress marker was suppressed by Que（P < 0.05）.The suppressive effect of Que on GRP78 and CHOP was reproduced by LY294002（P < 0.05）,but they failed to exhibit additive suppression.CONCLUSION:Que suppresses the ER stress induced by TG in RAW264.7 cells.The protective effect may be related to its suppression on PI3K signaling pathway.