小窝蛋白-1脚手架区结构域多肽对脂多糖诱导肺泡巨噬细胞血红素加氧酶-1活性增加及M1/M2表型极化的影响

Effect of caveolin-1 scaffolding domain peptides on heme oxygenase-1 activity increasing and M/2 phenotype polarization in rat alveolar macrophages induced by lipopolysaccharide

目的探讨小窝蛋白-1脚手架区结构域（CSD）多肽对脂多糖（LPS）诱导肺泡巨噬细胞（AMs）血红素加氧酶-1（HO-1）活性增加和M/2表型极化的影响。方法应用生物信息学方法分析全长野生型CSD多肽和101位氨基酸缺失的截短型突变CSD多肽（Δ101CSD）与HO-1的结合情况。分离纯化培养大鼠原代AMs，细胞融合达到80%时用无血清培养基进行同步化处理，并分为5组：空白对照组不予任何处理；LPS组加入100 μ/的LPS处理16 h；LPS ＋血晶素组加入100 μ/的LPS和20 μmo/血晶素处理16 h；野生型CSD多肽＋ LPS ＋血晶素组在LPS处理前6 h加入10 μmo/野生型CSD多肽预处理；Δ101CSD ＋ LPS ＋血晶素组在LPS处理前6 h加入10 μmo/ Δ101CSD预处理。各组处理16 h后，激光共聚焦显微镜下观察AMs细胞膜小窝蛋白-1（Cav-1）与HO-1的结合情况；采用实时荧光定量反转录-聚合酶链反应（RT-qPCR）检测炎性因子白细胞介素-1β（IL-1β）、单核细胞趋化蛋白-1（MCP-1）以及AMs的M1型标志物肿瘤坏死因子-α（TNF-α）、诱导型一氧化氮合酶（iNOS）和M2型标志物白细胞分化抗原206（CD206）、IL-10的mRNA表达；采用分光光度计检测HO-1活性及一氧化氮（NO）含量。结果生物信息学分析结果显示：野生型CSD和Δ101CSD多肽均能与HO-1结合，且二者结合能力无明显差异；但101位Arg缺失导致Δ101CSD多肽与HO-1的部分结合区域消失。激光共聚焦显微镜下观察结果显示：空白对照组Cav-1和HO-1均低表达；LPS组和LPS ＋血晶素组Cav-1与HO-1大量结合；野生型CSD和Δ101CSD多肽均能明显减少LPS诱导的HO-1与Cav-1结合。HO-1活性分析结果显示：给予LPS刺激后，HO-1活性较空白对照组明显升高；血晶素可促进LPS诱导HO-1活性升高；给予两种CSD多肽预处理后，HO-1活性均进一步升高，以野生型CSD多肽作用更加显著，与LPS ＋血晶素组比较差异有统计学意义（pmol·mg-1·h-1：3？683±266比2？408±132，P〈0.05）。RT-qPCR检测结果显示：LPS可诱导细胞炎性因子及M1型标志物水平升高，M2型标志物水平下降；而血晶素可抑制LPS诱导的炎症反应及M/2表型极化。与LPS ＋血晶素组相比，给予野生型CSD多肽预处理后，AMs中炎性因子水平降低，同时可降低M1型标志物TNF-α和iNOS的mRNA表达水平〔TNF-α mRNA（2-？？Ct）：6.82±0.05比8.70±0.24，iNOS mRNA（2-？？Ct）：331.50±32.05比506.70±0.10，均P〈0.05〕，增加M1型标志物IL-10 mRNA的表达水平（2-？？Ct：269.09±6.54比119.05±3.30，P〈0.05）；而101位缺失部分削弱了CSD多肽对炎性因子的抑制作用，且只能降低iNOS mRNA的表达水平（2-？？Ct：429.11±8.92比506.70±0.10，P〈0.05），说明其促使AMs由M1表型向M2表型转化的能力较差。两种多肽对CD206的表达均无影响。 结论野生型CSD多肽可减少LPS诱导的Cav-1与HO-1结合、增加HO-1活性，促进AMs由M1表型向M2表型转化，抑制促炎因子表达。

ObjectiveTo investigate the effect of caveolin-1 scaffolding domain （CSD） peptides on heme oxygenase-1 （HO-1） activity increasing and M/2 phenotype polarization in rat alveolar macrophages （AMs） induced by lipopolysaccharide （LPS）.MethodsBioinformatics was used to analyze the binding of full-length wild-type CSD polypeptide and 101 amino acid deleted truncated mutant CSD polypeptide （Δ101CSD） to HO-1. Primary AMs were isolated from rats, when cell fusion reached 80%, they were synchronized with serum-free medium and divided into five groups： no treatment was given to the blank control group; LPS group was treated with 100 μ/ LPS for 16 hours; LPS ＋ hemin group was treated with 100 μ/ LPS and 20 μmo/ hemin for 16 hours; wild-type CSD polypeptide ＋ LPS ＋ hemin group was pretreated with 10 μmo/ wild-type CSD polypeptide 6 hours before LPS treatment; Δ101CSD ＋ LPS ＋ hemin group was pretreated with 10 μmo/ Δ101CSD polypeptide 6 hours before LPS treatment. After treatment for 16 hours, the co-localization between caveolin-1 （Cav-1） and HO-1 was displayed by confocal microscope; the mRNA expressions of inflammatory cytokines interleukin-1β （IL-1β） and monocyte chemoattractant protein-1 （MCP-1） and M/2 polarization cytokines tumor necrosis factor-α （TNF-α）, inducible nitric oxide synthase （iNOS）, leukocyte differentiation antigen 206 （CD206） and IL-10 were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction （RT-qPCR）; the HO-1 activity and nitric oxide （NO） production were determined by spectrophotometry.ResultsBioinformatics analysis showed： both wild-type CSD and Δ101CSD peptides could bind to HO-1, and there was no significant difference in the binding ability between the two peptides, but the deletion of 101 Arg resulted in the disappearance of part of the binding region between Δ101CSD and HO-1. The results of laser confocal microscopy showed： the expressions of Cav-1 and HO-1 were lowed in the blank control group, and Cav-1 was bound to HO-1 in LPS group and LPS ＋ hemin group. Both wild-type CSD and Δ101CSD peptides pretreatment could significantly reduce the binding of HO-1 to Cav-1 induced by LPS. HO-1 activity analysis showed： after LPS stimulation, the activity of HO-1 was significantly higher than that of the blank control group; the activity of HO-1 induced by LPS was increased by hemin; after pretreatment with two kinds of CSD peptides, the activity of HO-1 was further increased, and the effect of wild-type CSD peptide was more significant, which showed a statistically significant difference as compared with that of LPS ＋ hemin group （pmol·mg-1·h-1： 3？683±266 vs. 2？408±132, P 〈 0.05）. RT-qPCR results showed： LPS could induce elevation of cytokines and M1 markers and decrease of M2 markers, while hemin could inhibit LPS-induced inflammatory response and M/2 phenotypic polarization. Compared with LPS ＋ hemin group, after pretreatment with wild-type CSD peptide, the levels of inflammatory factors in AMs were decreased, and the mRNA expression levels of TNF-α and iNOS, M1 markers, were decreased [TNF-α mRNA （2-？？Ct）： 6.82±0.05 vs. 8.70±0.24, iNOS mRNA （2-？？Ct）： 331.50±32.05 vs. 506.70±0.10, both P 〈 0.05], and IL-10 mRNA expression level was increased （2-？？Ct： 269.09±6.54 vs. 119.05±3.30, P 〈 0.05）. The deletion of 101 site partially weakened the inhibitory effect of CSD peptides on inflammatory factors and only reduced the expression of iNOS mRNA （2-？？Ct： 429.11±8.92 vs. 506.70±0.10, P 〈 0.05）, indicating that its ability to transform AMs from M1 phenotype to M2 phenotype was poor. The two peptides had no effect on the expression of CD206.ConclusionWild-type CSD had beneficial effects of anti-inflammation by reducing Cav-1 binding to HO-1 induced by LPS, restoring the HO-1 activity and driving M2 phenotype in alveolar macrophages.