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本文旨在研究晚期糖基化白蛋白(advanced glycated albumin,AGE-alb)对巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键分子caspase-12的影响,以阐明AGE-alb对巨噬细胞凋亡的诱导作用及机制。体外培养RAW264.7巨噬细胞,分别给予AGE-alb(2、4和6 g/L)、正常对照白蛋白(control albumin,C-alb,4 g/L)、ERS诱导剂衣霉素(tunicamycin,TM,4 mg/L)处理,或以ERS抑制剂4-苯丁酸(4-phenylbutyric acid,PBA,5 mmol/L)预处理细胞1 h,然后与AGE-alb(4 g/L)共孵育24 h。采用MTT法检测细胞活力,TUNEL法检测细胞凋亡情况,试剂盒测定培养基乳酸脱氢酶(lactate dehydrogenase,LDH)活性,免疫印迹法检测caspase-12表达变化。结果显示:与TM相似,AGE-alb显著诱导RAW264.7巨噬细胞损伤,表现为细胞活力降低,LDH漏出和细胞凋亡率明显增加,且呈浓度依赖性。AGE-alb明显上调caspase-12活性,尤其在4和6 g/L浓度时更为显著(P<0.01)。然而,PBA可抑制AGE-alb所致的巨噬细胞活力降低以及LDH漏出和凋亡增加,且减轻AGE-alb诱导的caspase-12活化(P<0.05)。上述结果提示,AGE-alb可诱导RAW264.7巨噬细胞凋亡,其机制可能与激活caspase-12介导的ERS凋亡途径有关。
The purpose of this study was to investigate the effect of advanced glycated albumin (AGE-alb) on caspase-12, a key apoptotic pathway of macrophage endoplasmic reticulum stress (ERS) alb on macrophage apoptosis and its mechanism. RAW264.7 macrophages were cultured in vitro and treated with AGE-alb (2,4 and 6 g / L), control albumin (C-alb, 4 g / L) The cells were pretreated with 4-phenylbutyric acid (PBA, 5 mmol / L) for 1 h with tunicamycin, TM, 4 mg / L and AGE-alb ) For 24 h. Cell viability was measured by MTT assay. Cell apoptosis was detected by TUNEL assay. The activity of lactate dehydrogenase (LDH) in culture medium was assayed by kit. Caspase-12 expression was detected by Western blotting. The results showed that, similar to TM, AGE-alb significantly induced RAW264.7 macrophage injury, showing a decrease in cell viability, a significant increase in LDH leakage and apoptosis, and in a concentration-dependent manner. AGE-alb significantly upregulated caspase-12 activity, especially at 4 and 6 g / L concentrations (P <0.01). However, PBA inhibited the viability of macrophages induced by AGE-alb and the increase of LDH leakage and apoptosis, as well as AGE-alb-induced caspase-12 activation (P <0.05). These results suggest that AGE-alb can induce RAW264.7 macrophage apoptosis, which may be related to the activation of caspase-12 mediated ERS apoptosis pathway.