晚期糖基化白蛋白通过上调核苷酸结合寡聚结构域样受体蛋白3诱导巨噬细胞焦亡

Advanced glycated albumin induces macrophage pyroptosis via upregulating nucleotide-binding oligomerization domain-like receptor protein 3

本研究旨在探讨晚期糖基化白蛋白(advanced glycated albumin, AGE-alb)对巨噬细胞核苷酸结合寡聚化结构域样受体蛋白3 (nucleotide-binding oligomerization domain-like receptor protein 3, NLRP3)-caspase-1途径的影响,以阐明AGE-alb对巨噬细胞焦亡的影响及机制。体外培养RAW264.7巨噬细胞,分别给予AGE-alb (1、2、4和6 g/L)、对照白蛋白(C-alb,4 g/L)处理24h或以NLRP3抑制剂MCC950 (1μmol/L)预处理细胞,1h后以AGE-alb(4g/L)处理24h。MTT法检测细胞活力,试剂盒测定caspase-1和培养基乳酸脱氢酶(lactate dehydrogenase, LDH)活性以及白细胞介素-1β(interleukin-1β, IL-1β)、IL-18浓度,TUNEL法和Hoechst 33342/PI双染法检测细胞死亡情况,Westernblot分析NLRP3、procaspase-1和cleavedcaspase-1表达变化。结果显示: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、TUNEL阳性和PI阳性细胞率显著增加,且呈浓度依赖性,并可促进IL-1β和IL-18分泌。AGE-alb显著上调NLRP3表达和caspase-1活性,尤其在4和6 g/L浓度时更为明显。然而,MCC950预处理可抑制AGE-alb所诱导的RAW264.7巨噬细胞活力降低、LDH漏出、TUNEL阳性和PI阳性细胞率增加以及IL-1β和IL-18分泌,并可抑制AGE-alb所致的caspase-1活化。以上结果表明,AGE-alb可诱导RAW264.7巨噬细胞焦亡,其机制至少部分是通过激活NLRP3-caspase-1信号途径实现的。

The purpose of the present study was to investigate the effect of advanced glycated albumin(AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb(1, 2, 4 and 6 g/L) and control albumin(C-alb, 4 g/L) for 24 h, or preincubated with MCC950(1 μmol/L) for 1 h and then treated with AGE-alb(4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase(LDH) activity and the levels of interleukin-1β(IL-1β) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL-or PI-positive cells in a concentration-dependent manner.Additionally, AGE-alb promoted IL-1β and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950(an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL-or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1β and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.