脂氧素A4对LPS诱导小胶质细胞活化的影响及SIRT1/NF-κB信号通路在其中的作用  

Effect of lipoxin A4 on lipopolysaccharide-induced activation of microglia and role of SIRT1/NF-κB signaling pathway

作　　者：蒋素芳[1] 万倩 王雪吉 曹天宇 康荣田[1] 黄立宁[1,2] Jiang Sufang;Wan Qian;Wang Xueji;Cao Tianyu;Kang Rongtian;Huang Lining(Department of Anesthesiology,The Second Hospital of Hebei Medical University,Shijiazhuang 050000,China;The Key Laboratory of Neurology,Ministry of Education,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院麻醉科,石家庄050000 [2]临床神经病学教育部重点实验室,石家庄050000

出　　处：《中华麻醉学杂志》2023年第10期1177-1182,共6页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(82071901);河北省自然科学基金(H2019206492)。

摘　　要：目的评价脂氧素A4(LXA4)对脂多糖(LPS)诱导小胶质细胞活化的影响及沉默信息调节因子1(SIRT1)/NF-κB信号通路在其中的作用。方法实验Ⅰ:取生长状态良好的小鼠BV2小胶质细胞,采用随机数字表法分为4组(n=30):对照组(C组)、LXA4组、LPS组和LPS+LXA4组(LL1组)。实验Ⅱ:采用随机数字表法将小胶质细胞分为2组(n=30):LPS+LXA4组(LL2组)、LPS+LXA4+SIRT1抑制剂EX527组(LLE组)。C组正常培养;LXA4组和LPS组分别加入LXA4(终浓度100 nmol/L)、LPS(终浓度100 ng/ml)孵育24 h;LL1组和LL2组于LPS处理前1 h加入LXA4(终浓度100 nmol/L),其余处理方法同LPS组;LLE组于LXA4处理前30 min加入EX527(终浓度为5μmol/L),其余处理方法同LL2组。采用qRT-PCR法检测诱导型一氧化氮合酶(iNOS)、CD32、精氨酸酶1(Arg-1)、CD206以及IL-1β、IL-6、TNF-α和IL-10的mRNA表达,ELISA法检测上清液IL-1β、IL-6、TNF-α和IL-10浓度,DCFH-DA荧光探针法检测活性氧(ROS)含量,WST-8法检测SOD活性,Western blot法检测NADPH氧化酶2(NOX2)、过氧化物歧化酶1(SOD1)、血红素加氧酶-1(HO-1)、SIRT1和乙酰化NF-κB p65的表达。结果与C组比较,LPS组iNOS mRNA、CD32 mRNA、IL-1βmRNA、IL-6 mRNA和TNF-αmRNA表达上调,上清液IL-1β、IL-6和TNF-α浓度升高(P<0.05),Arg-1 mRNA、CD206 mRNA、IL-10 mRNA表达和上清液IL-10浓度差异无统计学意义(P>0.05),NOX2和HO-1表达上调,SOD1表达下调,SOD活性降低,ROS含量升高,SIRT1表达下调,乙酰化NF-κB p65表达上调(P<0.05)。与LPS组比较,LL1组iNOS mRNA、CD32 mRNA、IL-1βmRNA、IL-6 mRNA和TNF-αmRNA表达下调,Arg-1 mRNA、CD206 mRNA和IL-10 mRNA表达上调,上清液IL-1β、IL-6、TNF-α浓度降低,IL-10浓度升高,NOX2表达下调,HO-1和SOD1表达上调,SOD活性升高,ROS含量降低,SIRT1表达上调,乙酰化NF-κB p65表达下调(P<0.05)。与LL2组比较,LLE组上清液IL-1β、IL-6、TNF-α浓度升高,SOD活性降低,ROS含量升高(P<0.05),上清液IL-10浓度差异无统计学意义(P>0.05)。�Objective To evaluate the effect of lipoxin A4 on lipopolysaccharide(LPS)-induced activation of microglia and role of silent information regulator sirtuin 1(SIRT1)/NF-κB signaling pathway.Methods This experiment was performed in two parts.PartⅠThe well-growth BV2 microglia were divided into 4 groups(n=30 each)using a random number table method:control group(group C),LXA4 group(group LXA4),LPS group(group LPS)and LPS+LXA4 group(group LLI).PartⅡThe well-growth BV2 microglia were divided into 2 groups(n=30 each)using a random number table method:LPS+LXA4 group(group LL2)and LPS+LXA4+SIRT1 inhibitor EX527 group(group LLE).Cells in group C were commonly cultured without any treatment.In LXA4 group and LPS group,LXA4(final concentration 100 nmol/L)and LPS(final concentration 100 ng/ml)were added respectively,and then the cells were incubated for 24 h.In LL1 group and LL2 group,LXA4(final concentration 100 nmol/L)was added at 1 h before treatment with LPS,and the other treatments were similar to those previously described in LPS group.EX527(final concentration of 5μmol/L)was added at 30 min before treatment with LXA4,and the other treatments were similar to those previously described in LL2 group.The expression of inducible nitricoxide synthase(iNOS),CD32,arginine synthase 1(Arg-1),CD206,interleuckin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α)and IL-10 mRNA was detected by real-time polymerase chain reaction.The concentrations of IL-1β,IL-6,TNF-αand IL-10 in the supernatant were measured using enzyme-linked immunosorbent assay.The content of ROS was detected by DCFH-DA.The activity of SOD was measured by WST-8 assay.The expression of NADPH oxidase 2(NOX2),superoxide dismutase 1(SOD1),heme oxygenase-1(HO-1),SIRT1 and acetylated NF-κB p65 was detected by Western blot.Results Compared with group C,the expression of iNOS,CD32,IL-1β,IL-6 and TNF-αmRNA was significantly up-regulated,the concentrations of IL-1β,IL-6 and TNF-αin the supernatant were increased(P<0.05),no significant change was found in the exp

关 键 词：小神经胶质细胞 脂氧素类 抗衰老酶1 NF-κB 

分 类 号：R614[医药卫生—麻醉学]