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本文旨在研究槲皮素(quercetin,QUE)预处理对内质网应激诱导剂衣霉素(tunicamycin,TM)所致RAW264.7巨噬细胞凋亡的抑制作用,并探讨可能的分子机制。体外培养RAW264.7巨噬细胞,给予20、40和80μmol/LQUE预处理30min,再加入5mg/LTM继续培养12h。分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况;免疫荧光细胞化学法和免疫印迹法检测转录激活因子6(activating transcription factor6,ATF6)核转位情况;分别采用免疫印迹法和实时定量聚合酶链反应(real-timePCR)技术检测促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)和抗凋亡蛋白Bcl-2蛋白及mRNA表达变化。结果显示,QUE(40和80μmol/L)预处理显著抑制TM所诱导的细胞活力降低和凋亡。与TM处理组比较,QUE预处理组内质网应激感受器ATF6由胞浆向核内转移明显减弱。TM在蛋白和转录水平均明显上调CHOP表达,并下调Bcl-2表达,而QUE则明显抑制上述变化,且呈浓度依赖性。以上结果表明,QUE可减轻TM所诱导的RAW264.7巨噬细胞凋亡,可能是部分通过抑制ATF6-CHOP信号途径实现的。
This study aimed to investigate the inhibitory effect of quercetin (QUE) pretreatment on the apoptosis of RAW264.7 macrophages induced by tunicamycin (TM), an endoplasmic reticulum stress inducer, and to explore the possible molecular mechanisms . RAW264.7 macrophages were cultured in vitro, pretreated with 20, 40 and 80μmol / LQUE for 30min, then added 5mg / LTM for 12h. The cell viability and apoptosis were detected by MTT assay and Annexin V-FITC double staining respectively. The nuclear translocation of activating transcription factor 6 (ATF6) was detected by immunofluorescence cytochemistry and immunoblotting. The mRNA and protein expressions of C / EBP homologous protein (CHOP) and anti-apoptotic protein Bcl-2 were detected by real-time PCR and real-time PCR. The results showed that pretreatment with QUE (40 and 80 μmol / L) significantly inhibited TM-induced cell viability and apoptosis. Compared with TM treatment group, QUE pretreatment group endoplasmic reticulum stress receptor ATF6 significantly decreased from the cytoplasm to the nucleus. TM significantly upregulated the expression of CHOP and down-regulated the expression of Bcl-2 at the protein and transcription levels, while QUE inhibited the above changes in a concentration-dependent manner. The above results showed that QUE could reduce TM induced apoptosis of RAW264.7 macrophages, which may be partly through the inhibition of ATF6-CHOP signaling pathway.