摘要
目的 探讨氢吗啡酮(HM)对脂多糖(LPS)诱导的小鼠肺泡巨噬细胞高尔基体应激的影响及其机制.方法 以10 mg/L LPS处理小鼠肺泡巨噬细胞系MH-S,构建肺泡巨噬细胞高尔基体应激模型.将细胞随机分为Control组(A组,正常培养)、LPS组(B组,给予10 mg/L的LPS)、LPS+HM组(C组,给予1 μmol/L HM和10 mg/L LPS)、Nrf2 siRNA+LPS+HM 组(D 组,Nrf2 siRNA 转染后给予 1 μmol/L HM 和 10 mg/L LPS)和 NC siRNA+LPS+HM组(E组,NC siRNA转染后给予1 μmol/L HM和10 mg/L LPS).采用ELISA法检测细胞上清液白细胞介素1β(IL-1β)和白细胞介素6(IL-6)含量;检测丙二醛(MDA)含量与超氧化物歧化酶(SOD)活性;采用Western blot法检测核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、高尔基体基质蛋白130(GM130)、高尔基体蛋白97(Golgin-97)、甘露糖苷酶Ⅱ(Mannosidase Ⅱ)、钙转运ATP酶2C型成员1(ATP2C1)蛋白表达水平;透射电镜下观察细胞高尔基体形态学改变.结果 与A组比较,B组IL-1β、IL-6、MDA含量显著升高及Nrf2、HO-1蛋白表达上调,SOD活性显著降低及GM130、Golgin-97、Mannosidase Ⅱ、ATP2C1蛋白的表达下调(F=2.33～47.61,P＜0.05);与B组比较,C组IL-lβ、IL-6、MDA含量显著降低,SOD活性显著升高及Nrf2、HO-1、GM130、Golgin-97、Mannosidase Ⅱ、ATP2C1 蛋白表达上调(P＜0.05);与 C 组比较,D 组 IL-1β、IL-6、MDA 含量显著升高,SOD 活性显著降低及 Nrf2、HO-1、GM130、Golgin-97、Mannosidase Ⅱ、ATP2C1 蛋白表达下调(P＜0.05).透射电镜显示,C组细胞高尔基体损伤、空泡化与碎片化较B组减轻;D组细胞高尔基体损伤、空泡化与碎片化较C组加重.结论 HM通过调控Nrf2/HO-1信号通路抑制LPS诱导的肺泡巨噬细胞高尔基体应激,减轻细胞炎症与氧化应激反应.
Abstract
Objective To investigate the effect of hydromorphone(HM)on Golgi stress in lipopolysaccharide(LPS)-at-tacked mouse alveolar macrophages and its mechanism.Methods The mouse alveolar macrophage cell line MH-S was attacked with 10 mg/L LPS to establish a model of Golgi stress,and then the cells were randomly divided into control group(group A,nor-mal culture),LPS group(group B treated with 10 mg/L LPS),LPS+HM group(group C treated with 1 μmol/L HM and 10 mg/L LPS),Nrf2 siRNA+LPS+HM group(group D treated with 1 μmol/L HM and 10 mg/L LPS after Nrf2 siRNA transfection),and NC siRNA+LPS+HM group(group E treated with 1 μmol/L HM and 10 mg/L LPS after NC siRNA transfection).ELISA was used to measure the content of interleukin 1β(IL-1β)and interleukin 6(IL-6)in cell supernatant;the content of malondialde-hyde(MDA)and the activity of superoxide dismutase(SOD)were measured;Western blot was used to measure the protein ex-pression levels of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),Golgi Matrix Protein of 130(GM130),Golgin-97,Mannosidase Ⅱ,and ATP2C1;transmission electron microscopy was used to observe the morphological changes of Golgi apparatus.Results Compared with group A,group B had significant increases in the content of IL-1β,IL-6,and MDA and the protein expression levels of Nrf2 and HO-1 and significant reductions in the activity of SOD and the protein ex-pression levels of GM130,Golgin-97,Mannosidase Ⅱ,and ATP2C1(F=2.33-47.61,P＜0.05).Compared with group B,group C had significant reductions in the content of IL-1β,IL-6,and MDA and significant increases in the activity of SOD and the protein expression levels of Nrf2,HO-1,GM130,Golgin-97,MannosidaseⅡ,and ATP2C1(P＜0.05).Compared with group C,group D had significant increases in the content of IL-1β,IL-6,and MDA and significant reductions in the activity of SOD and the protein expression levels of Nrf2,HO-1,GM130,Golgin-97,MannosidaseⅡ,and ATP2C1(P＜0.05).Transmission electron microscopy showed that compared with group B,group C had alleviation of the damage,vacuolization,and fragmentation of Golgi apparatus,and compared with group C,group D had aggravation of the da-mage,vacuolization,and fragmentation of Golgi apparatus.Conclusion HM inhibits Golgi stress in LPS-attacked alveolar mac-rophages by regulating the Nrf2/HO-1 signaling pathway,thereby alleviating cellular inflammation and oxidative stress response.
基金项目
山东省医药卫生技术发展计划项目(20210411-0960)
青岛大学附属医院青年科研基金(QDFYQN2023227)
国家科技期刊平台
NSTL
万方数据