内毒素攻击大鼠肺泡巨噬细胞时HO-1对高尔基体应激的影响

Role of HO-1 on Golgi stress in LPS-stimulated rat alveolar macrophages

目的:探讨内毒素诱导大鼠肺泡巨噬细胞损伤时血红素加氧酶1(HO-1)对高尔基体应激的影响。方法:体外培养大鼠肺泡巨噬细胞,采用脂多糖(LPS)诱导大鼠肺泡巨噬细胞建立细胞损伤模型。使用CCK-8法检测细胞活力;使用DCFH-DA探针检测细胞内活性氧簇(ROS)的生成;使用生物化学方法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平;使用TUNEL染色和凋亡相关蛋白caspase-3/7活性检测试剂盒检测细胞凋亡;使用RT-qPCR和Western blot法检测HO-1和高尔基体磷蛋白3(GOLPH3)的表达;使用Western blot法检测高尔基体结构相关蛋白GM130、golgin-97和mannosidase II的表达。使用小干扰RNA(siRNA)沉默HO-1后,重复以上检测。结果:LPS刺激肺泡巨噬细胞下调细胞活力、SOD活性及GM130、golgin-97和mannosidase II表达水平,上调ROS和MDA含量及HO-1和GOLPH3表达水平,并导致TUNEL标记阳性细胞数增多,caspase-3/7活性增强(P<0.05);HO-1基因沉默后,细胞活力、SOD活性及GM130、golgin-97和mannosidase II表达显著下降,ROS和MDA含量及GOLPH3表达显著上升,TUNEL标记阳性细胞数增多,caspase-3/-7活性显著增强(P<0.05)。结论:内毒素诱导大鼠肺泡巨噬细胞损伤时,HO-1可减轻氧化应激和高尔基体应激反应,减少细胞凋亡。

AIM:To evaluate the role of heme oxygenase-1(HO-1)on Golgi stress in lipopolysaccharide(LPS)-stimulated rat alveolar macrophages.METHODS:The injury model of rat alveolar macrophages was established by LPS stimulation in vitro.CCK-8 assay was applied to measure cell viability,and the DCFH-DA probing was used to detect the generation of reactive oxygen species(ROS).In addition,the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were assayed by SOD and MDA kits,and apoptosis was analyzed by TUNEL assay and caspase-3/7 activity determination.The mRNA expression levels of HO-1 and Golgi phosphoprotein 3(GOLPH3)were detected by RTqPCR,and the protein expression levels of HO-1,GOLPH3 and Golgi structure-related proteins(GM130,golgin-97 and mannosidase II)were detected by Western blot.HO-1 small interfering RNA(siRNA)was applied to reduce the expression of HO-1 in rat alveolar macrophages.RESULTS:Stimulation with LPS reduced cell viability and SOD activity,increased the levels of ROS and MDA,up-regulated the expression of HO-1 and GOLPH3,impaired the expression of GM130,golgin-97 and mannosidase II,and further increased TUNEL positive rate and caspase-3/7 activityin rat alveolar macrophages(P<0.05).Knockdown of HO-1 significantly inhibited cell viability and SOD activity,increased ROS and MDA levels,decreased the expression of HO-1,GM130,golgin-97 and mannosidase II,increased the expression of GOLPH3,and further increased TUNEL positive rate and caspase-3/7 activity(P<0.05).CONCLUSION:The HO-1 attenuates oxidative stress and Golgi stress response,and prevents apoptosis in LPS-stimulated alveolar macrophages.