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目的:研究大蒜素对巨噬源性泡沫细胞凋亡和内质网应激(endoplasmic reticulum stress,ERS)凋亡通路关键分子半胱天冬酶-12(caspase-12)的影响,并探讨可能的分子机制。方法:体外培养RAW264.7巨噬细胞,分别给予大蒜素(12.5、25和50 mg/L)和4-苯丁酸(4-phenylbutyric acid,PBA;4 mmol/L)预处理1 h后,加入氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL;100 mg/L)或衣霉素(tunicamycin,TM;4 mg/L)处理24 h。分别采用MTT法和Annexin V-FITC/PI双染法检测细胞活力和凋亡情况;采用相应的试剂盒测定细胞内caspase-3和培养液中乳酸脱氢酶(lactic dehydrogenase,LDH)的活性;采用Western blot技术检测caspase-12的表达变化;油红O染色检测细胞内脂质蓄积;酶比色法测定细胞内总胆固醇含量。结果:与ERS抑制剂PBA相似,大蒜素可减轻oxLDL所致的巨噬细胞损伤,表现为细胞活力增加、LDH漏出减少、细胞凋亡率和caspase-3活性降低(P<0.05);ERS诱导剂TM可导致巨噬细胞活力下降,LDH漏出增多及细胞凋亡率升高(P<0.05),大蒜素可明显阻断TM的上述作用;大蒜素明显抑制ox-LDL所致的caspase-12活化(P<0.05);与TM组相比,大蒜素也可显著抑制TM所诱导的caspase-12活化(P<0.05)。另外,大蒜素还可显著抑制ox-LDL所诱导的巨噬细胞内脂质蓄积和泡沫细胞形成(P<0.05)。结论:大蒜素可减少ox-LDL所致的巨噬源性泡沫细胞凋亡,其机制可能与抑制caspase-12活化有关。
Objective: To investigate the effect of allicin on the apoptosis of macrophage-derived foam cell and endoplasmic reticulum stress (ERS) pathway, a key molecule of caspase-12, and to explore the possible Molecular mechanism. Methods: RAW264.7 macrophages were cultured in vitro and pretreated with allicin (12.5, 25 and 50 mg / L) and 4-phenylbutyric acid (PBA) The cells were treated with oxidized low-density lipoprotein (ox-LDL 100 mg / L) or tunicamycin (TM 4 mg / L) for 24 h. Cell viability and apoptosis were detected by MTT assay and Annexin V-FITC / PI double staining respectively. The activities of caspase-3 and lactic dehydrogenase (LDH) Western blot was used to detect the expression of caspase-12. Oil red O staining was used to detect the intracellular lipid accumulation. The intracellular total cholesterol content was determined by enzyme colorimetry. Results: Garlicin could reduce oxLDL-induced macrophages injury by increasing the cell viability, decreasing the leakage of LDH and decreasing the apoptosis rate and the activity of caspase-3 (P <0.05). ERS induced Agent TM can cause macrophage activity decreased, LDH leakage increased and apoptosis rate increased (P <0.05), allicin can significantly block the TM effect; allicin significantly inhibited ox-LDL-induced caspase-12 (P <0.05). Compared with TM group, allicin also significantly inhibited TM-induced caspase-12 activation (P <0.05). In addition, allicin also significantly inhibited ox-LDL-induced macrophage lipid accumulation and foam cell formation (P <0.05). CONCLUSION: Allicin can reduce the apoptosis of macrophage-derived foam cells induced by ox-LDL, which may be related to the inhibition of the activation of caspase-12.