泛素修饰酶A20在脂肪变肝细胞和单核细胞中表达及通路研究

Expression of ubiquitin editing enzyme A20 and its pathway in steatotic hepatocytes and monocytes

目的 探讨泛素修饰酶A20在游离脂肪酸（FFA）刺激下的表达变化及作用通路.方法 用0.5 mmol/L的混合FFA刺激HepG2细胞和U937细胞,采用Western印迹法检测A20蛋白表达水平、NF-κB通路蛋白表达水平（磷酸化p65,磷酸化IκBα）和MAPK通路蛋白表达水平（磷酸化Jun激酶,Jun激酶,磷酸化细胞外信号调节激酶（ERK）,ERK,磷酸化p38和p38）.流式细胞仪检测细胞上清液中IL-12p、IL-1β、TNF-α、IL-6、IL-10和IL-8的浓度.统计学处理采用t检验.结果 A20蛋白水平可随着FFA刺激时间不同而发生变化.NF-κB和MAPK通路在FFA刺激后被激活.FFA刺激HepG2后,IL-6与IL-8分泌均增加,且均在24 h分泌量最大,与对照组相比,IL-8为（423.8±8.9）pg/mL比（12.4±4.5）pg/mL,差异有统计学意义（t=41.28,P＜0.01）;IL-6为（4 082.0±423.6） pg/mL比（52.9±29.5） pg/mL,差异有统计学意义（t=9.49,P＜0.01）.FFA刺激U937细胞后,与对照组相比,IL-8分泌增加,在一定时间内成时间依赖关系,24 h分泌量最大,为（200.6±5.7）pg/mL比（5.0±3.9） pg/mL,差异有统计学意义（t=28.16,P＜0.01）.未检测到IL-10、IL-12p、IL-1β和TNF-α的分泌.结论 用FFA模拟的体外脂肪变模型可刺激A20蛋白的表达变化和炎性因子的分泌,NF-κB通路和MAPK通路均参与了U937和HepG2对FFA的应答.

Objective To investigate the changes of A20 expression stimulated by free fatty acids （FFA） and its pathway.Methods HepG2 cells and U937 cells were stimulated by 0.5 mmol/L mixed FFA.The expression of A20,phosphor-p65 and phosphor-IκBα of neclear factor （NF）-κB pathway and phosphor-c-Jun N-terminal kinase （JNK）,JNK,phosphor-extracellular signal-regulated kinase （ERK）,ERK,phosphor-p38 and p38 of mitogen-activated protein kinase （MAPK） pathway were detected by Western blotting.The level of interleukin （IL）-12p,IL-1β,tumor necrosis factor （TNF）-α,IL-6,IL-10 and IL-8 cytokines in the supernatant of cell culture were detected by flow cytometry.T-test was performed for statistical analysis.Results The level of A20 changed along with the stimulated time of FFA.NF-κB and MAPK pathways were activated after FFA stimulation.The secretion of IL-6 and IL-8 increased after HepG2 cells stimulated by FFA and both reached peak at 24 hour.Compared with control group,the difference in IL-8 was statistically significant （（423.8 ± 8.9） pg/mL vs （12.4 ± 4.5） pg/mL,t=41.28,P〈0.01）.The difference in IL-6 was also statistically significant （（4 082±423.6） pg/mL vs （52.9±29.5） pg/mL,t=9.49,P〈0.01）.After U937 cells were stimulated by FFA,the secretion of IL-8 increased compared with control group.And in a certain period of time the secretion was time dependence.The maximum secretion of 24 hours was （200.6±5.7） pg/mL vs （5.0±3.9） pg/mL,and the difference was statistically significant （t=28.16,P〈0.01）.IL-10,IL-12p,IL-1β and TNF-α were detected.Both NF-κB pathway and MAPK pathway were detected.Conclusions The in vitro FFA mediated steatotic cell model could induce the expression change of A20 and secretion of inflammatory cytokines.NF-κB and MAPK pathways involved in the response to FFA in HepG2 cells and U937 cells.