摘要
目的 探讨血红素氧合酶-1/一氧化碳（HO-1/CO）通路对脂多糖（LPS）诱导大鼠Ⅱ型肺泡上皮细胞（AECⅡ）线粒体融合的影响。方法 体外培养大鼠AECⅡ细胞株RLE-6TN，待细胞融合度达到85%时传代培养，并随机分为7组（n=5）：空白对照组细胞常规培养；LPS组加入10 mg/L的LPS制备内毒素攻击AECⅡ模型；外源性一氧化碳释放分子-2（CORM-2，体外CO释放剂）＋LPS组（CL组）和氯高铁血红素（Hemin，HO-1诱导剂）＋LPS组（HL组）分别加入100 μmol/L的CORM-2或20 μmol/L的Hemin预处理1 h，再加入10 mg/L LPS孵育；锌原卟啉-Ⅸ（ZnPP-Ⅸ，HO-1活性抑制剂）＋ LPS组（ZL组）加入10 μmol/L的ZnPP-Ⅸ预处理0.5 h，然后加入10 mg/L的LPS孵育；CORM-2＋ZnPP-Ⅸ＋LPS组（CZL组）和Hemin＋ZnPP-Ⅸ＋LPS组（HZL组）先分别加入100 μmol/L的CORM-2或20 μmol/L的Hemin预处理1 h，其余处理同ZL组。LPS孵育24 h后，采用酶联免疫吸附试验（ELISA）测定细胞上清液中白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的含量；用蛋白质免疫印迹试验（Western Blot）测定HO-1、线粒体融合蛋白1和蛋白2（Mfn1、Mfn2）以及视神经萎缩蛋白1（OPA1）的蛋白表达。结果 与空白对照组比较，各处理组细胞上清液中IL-6和TNF-α含量升高，HO-1蛋白表达上调，线粒体融合相关蛋白Mfn1、Mfn2和OPA1蛋白表达下调。与LPS组比较，给予CORM-2或Hemin预处理后，IL-6、TNF-α含量均明显降低〔IL-6（ng/L）：48.6±3.7、48.4±3.1比58.7±2.5，TNF-α（ng/L）：40.7±5.3、39.4±4.3比51.8±5.1〕，HO-1、Mfn1、Mfn2、OPA1蛋白表达均明显上调（HO-1蛋白：0.873±0.051、0.839±0.061比0.671±0.044，Mfn1蛋白：0.673±0.037、0.654±0.025比0.568±0.021，Mfn2蛋白：0.676±0.044、0.683±0.035比0.571±0.043，OPA1蛋白：0.648±0.031、0.632±0.031比0.554±0.032，均P〈0.05）；而给予ZnPP-Ⅸ预处理后，IL-6、TNF-α含量及HO-1、Mfn1、Mfn2、O
Objective To investigate the effects of heme oxygenase-1/carbon monoxide （HO-1/CO） pathway on mitochondrial fusion in rat alveolar epithelial type Ⅱ cells （AECⅡ） stimulated by lipopolysaccharide （LPS）.Methods Once the cultured in vitro rat AECⅡcells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups （n = 5 each）. RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECⅡ cells. The cells in carbon monoxide-releasing molecule-2 （CORM-2, in vitro CO release agent） ＋ LPS group （CL group） and Hemin （HO-1 inducer） ＋ LPS group （HL group） were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-Ⅸ （ZnPP-Ⅸ, HO-1 inhibitor） ＋ LPS group （ZL group） was pretreated with 10 μmol/L ZnPP-Ⅸ for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 ＋ ZnPP-Ⅸ ＋ LPS group （CZL group） and Hemin ＋ ZnPP-Ⅸ ＋ LPS group （HZL group） were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in the supernatant were determined by enzyme linked immunosorbent assay （ELISA）, the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 （Mfn1, Mfn2） and optic atrophy 1 （OPA1） were determined by Western Blot.Results Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 （ng/L）： 48.6±3.7, 48.4±3
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2018年第3期209-213,共5页
Chinese Critical Care Medicine
基金
国家自然科学基金（81372096）
天津市卫生计生委基金项目（2014KZ048）
天津“131”创新型人才团队基金（2016-1-22）
