Chow Jyh-Ming, Lin Hui-Yi, Shen Shing-Chuan, Wu Ming-Shun, Lin Cheng-Wei, Chiu Wen-Ta, Lin Chien-Huang, Chen Yen-Chou
Section of Hematology-Oncology, Department of Internal Medicine, Taipei Municipal Wan Fang Hospital, Taipei Medical University, Taipei 110, Taiwan.
Toxicol Appl Pharmacol. 2009 Jun 15;237(3):357-65. doi: 10.1016/j.taap.2009.04.009. Epub 2009 Apr 17.
In the present study, zinc protoporphyrin (ZnPP), but not ferric protoporphyrin (FePP), tin protoporphyrin (SnPP), or zinc chloride (ZnCl(2)), at the doses of 0.5, 1, and 2 microM, dose-dependently inhibited lipopolysaccharide- (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages in a serum-free condition. NO inhibition and HO-1 induction by ZnPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). A decrease in the iNOS/NO ratio and an increase in HO-1 protein by ZnPP were identified in three different conditions including ZnPP pretreatment, ZnPP co-treatment, and ZnPP post-treatment with LPS and LTA. Activation of c-Jun N-terminal kinases (JNKs) and extracellular regulated kinases (ERKs) were detected in LPS-, LTA-, and PGN-treated RAW264.7 cells, and iNOS/NO production was blocked by adding the JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059. However, ZnPP addition potentiated ERK and JNK protein phosphorylation stimulated by LPS, LTA, and PGN. Increases in total protein ubiquitination and ubiquitinated iNOS proteins were detected in ZnPP-treated macrophages elicited by LPS according to Western and immunoprecipitation/Western blotting assays, respectively. The decrease in LPS-induced iNOS protein by ZnPP was reversed by adding the proteasome inhibitors MG132 and lactacystin. The reduction in HO-1 protein induced by ZnPP via transfection of HO-1 small interfering RNA did not affect the inhibitory effect of ZnPP against LPS-induced iNOS/NO production and protein ubiquitination induced by ZnPP in macrophages. Data of the present study provide the first evidence to support ZnPP effectively inhibiting inflammatory iNOS/NO production through activation of protein ubiquitination in a HO-1-independent manner in macrophages.
在本研究中，在无血清条件下，0.5、1和2微摩尔剂量的锌原卟啉（ZnPP）而非铁原卟啉（FePP）、锡原卟啉（SnPP）或氯化锌（ZnCl₂）可剂量依赖性地抑制脂多糖（LPS）、脂磷壁酸（LTA）和肽聚糖（PGN）诱导的RAW264.7巨噬细胞中诱导型一氧化氮合酶（iNOS）和一氧化氮（NO）的产生，同时血红素加氧酶1（HO-1）蛋白增加。单独添加胎牛血清（FBS）和牛血清白蛋白（BSA）可阻断ZnPP对NO的抑制作用和对HO-1的诱导作用。在包括ZnPP预处理、ZnPP共处理以及ZnPP与LPS和LTA后处理的三种不同条件下，均发现ZnPP可降低iNOS/NO比值并增加HO-1蛋白。在LPS、LTA和PGN处理的RAW264.7细胞中检测到c-Jun氨基末端激酶（JNKs）和细胞外调节激酶（ERKs）的激活，添加JNK抑制剂SP600125可阻断iNOS/NO的产生，但添加ERK抑制剂PD98059则不能。然而，添加ZnPP可增强LPS、LTA和PGN刺激的ERK和JNK蛋白磷酸化。根据蛋白质免疫印迹和免疫沉淀/蛋白质免疫印迹分析，在ZnPP处理的、由LPS诱导的巨噬细胞中分别检测到总蛋白泛素化和泛素化iNOS蛋白增加。添加蛋白酶体抑制剂MG132和乳胞素可逆转ZnPP对LPS诱导的iNOS蛋白的降低作用。通过转染HO-1小干扰RNA诱导的ZnPP对HO-1蛋白的降低并未影响ZnPP对巨噬细胞中LPS诱导的iNOS/NO产生和ZnPP诱导的蛋白泛素化的抑制作用。本研究数据提供了首个证据，支持ZnPP通过以不依赖HO-1的方式激活蛋白泛素化有效抑制炎症性iNOS/NO的产生。
