大蒜素通过抑制caspase-12减轻巨噬源性泡沫细胞凋亡

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(P0.05);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(P0.05),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(P0.05);与TM组相比,大蒜素也可显著抑制TM所诱导的caspase-12活化(P0.05)。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(P0.05)。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。

Allicin attenuates macrophage-derived foam cell apoptosis by inhibiting caspase-12

AIM: To investigate the inhibitory effect of allicin on apoptosis and caspase-12 activation of macrophage-derived foam cells, and to elucidate the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with allicin (12.5, 25 and 50 mg/L) or 4-phenylbutyric acid (PBA, 4 mmol/L) for 1 h and then treated with oxidized low-density lipoprotein (ox-LDL, 100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were examined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of caspase-3 in the cells and lactic dehydrogenase (LDH) in the medium were measured. The protein levels of caspase-12 were determined by Western blot. The intracellular lipid accumulation was measured with oil red O staining and the content of intracellular total cholesterol was determined by enzymatic colorimetry. RESULTS: Similar to the endoplasmic reticulum stress (ERS) inhibitor PBA, allicin inhibited ox-LDL-induced injury of RAW264.7 macrophages in a concentration-dependent manner, as determined by the increased cell viability and the decreased LDH leakage, apoptosis and caspase-3 activity. The decrease in cell viability and increases in LDH leakage and apoptosis induced by TM (an ERS inducer) were also suppressed by allicin. Moreover, similar to PBA, allicin remarkably inhibited ox-LDL- or TM-induced activation of caspase-12. Furthermore, allicin remarkably attenuated ox-LDL-induced lipid accumulation in the RAW264.7 cells and foam cells formation in a concentration-dependent manner. CONCLUSION: Allicin may inhibit macrophage-derived foam cell apoptosis induced by ox-LDL, and the mechanism is partially related to suppressing the activation of caspase-12.