博文
氢气通过抑制巨噬细胞激活治疗炎性反应
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中文摘要
氧化应激在炎症的发生发展过程中发挥着重要作用，一方面它可以直接损伤机体，另一方面它可以通过分子间相互作用间接地对机体造成损害[1]。在参与炎症过程的众多复杂因子中，活性氧(ROS)和活性氮(RNS)是最重要的。
它们包括羟自由基、超氧阴离子、过氧化氢、一氧化氮、亚硝酸阴离子[2.3]。有大量的实验证明清除羟自由基和亚硝酸阴离子能够明显减轻炎症的严重程度[4-6]。最近的研究表明氢气能够选择性地清除羟自由基和亚硝酸阴离子[7]，因此氢气很有可能通过这一途径而具有抗炎的效应。但是通过呼吸道吸入氢气的方法很不方便而且存在安全隐患，于是我们将氢气通过高压溶解于生理盐水中，并检查这种含有氢气的生理盐水是否具有抗炎效应。
本课题以角叉菜胶诱导小鼠足肿胀为实验模型，研究氢生理盐水对于该炎症模型是否具有抗炎效应，并以巨噬细胞为例从细胞水平研究氢生理盐水对炎性细胞的影响。
本实验通过酶联免疫吸附试验检测了细胞培养上清中的TNF-α的含量；实时定量PCR检测了RAW264.7及小鼠原代腹腔巨噬细胞中TNF-α的mRNA水平；足爪容积测量仪检测了小鼠足肿胀的程度；组织化学切片观察了炎症局部的中性粒细胞浸润程度。
研究工作取得的主要结果如下：
1. 氢生理盐水能够抑制被LPS激活的巨噬细胞分泌TNF-α：氢生理盐水和100ng/ml的LPS共同处理RAW264.7细胞4h，4h后收集上清检测TNF-α，发现氢生理盐水能够显著抑制由LPS引起的TNF-α产生，而且这种效应具有剂量依赖性。
2. 氢生理盐水能够减少活化的巨噬细胞合成TNF-α的mRNA水平：氢生理盐水和100ng/ml的LPS共同处理巨噬细胞1h，1h后裂解细胞通过实时定量PCR检测TNF-α的mRNA水平，发现氢生理盐水能够抑制巨噬细胞TNF-α的mRNA的表达。
3. 氢生理盐水能够显著抑制角叉菜胶诱导的足肿胀：以2.5ml/kg、4ml/kg、5ml/kg、10ml/kg的氢生理盐水的量处理角叉菜胶诱导足肿胀的动物模型，发现处理组脚肿胀的程度明显低于对照组，而且5ml/kg这个剂量效果最好。
4. 氢生理盐水能够显著抑制炎症局部中性粒细胞的浸润：将氢生理盐水处理组、非处理组及对照组的足爪做成石蜡切片，HE染色后观察中性粒细胞浸润程度，清水处理组的中性粒细胞浸润明显比非处理组少。
我们的研究发现氢生理盐水不论在LPS激活的巨噬细胞上还是在角叉菜胶诱导的足肿胀动物模型上都具有明显的抗炎效应，这一效应很可能是通过清除羟自由基、亚硝酸阴离子等自由基来实现的。因此我们可以预测，氢生理盐水凭借其有效性、安全性、方便性、廉价性，很可能成为我们以后治疗某些炎症疾病的药物。在今后的工作中，我们将深入研究氢生理盐水具有抗炎效应的机制，着重从炎症局部的自由基变化着手，验证我们对于其抗炎机制的猜想，检测炎症局部羟自由基和亚硝酸阴离子的变化。
英文摘要
Back ground: Oxidative stress is thought to play an important role in the pathogenesis of inflammation not only through direct injurious effects, but also by involvement in the molecular mechanism[1]. Among the complex factors involved in the process of inflammation, reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as the hydroxyl radical (•OH), superoxide anion (O2 –), hydrogen dioxide (H2O2), nitric oxide (NO), peroxynitrite (ONOO –), appear to be critical elements[2. 3]. There is a large amount of evidence to show that Inhibitors of NOS activity reduce the severity of inflammation[4-6]. It has been reported recently that H2 selectively reduced •OH and ONOO– [7]. So, as a free radical scavenger, H2 may have the effect of anti-inflammation. However, inhalation of hydrogen gas may be not convenient for therapeutic use，so we dissolved molecular hydrogen (H2) in saline under high pressure (0.6MPa), and examine the effects of hydrogen saline on inflammation models.
Methods: TNF-α in supernatants was evaluated by ELISA. RT-PCR was used to characterize the mRNA expression of TNF-α in RAW264.7 macrophages. The severity of inflammation damage was evaluated by paw volume measurement and inflammatory cells infiltration.
Results: Supernantant TNF-α level from activated macrophages (collected 4h after LPS stimulation) treated with saline were significantly higher than that treated with hydrogen saline. This result was similar in both murine peritoneal macrophages and RAW 264.7 murine macrophages which indicated that hydrogen saline has the ability to inhibit TNF-α production of macrophages. Expression of activated macrophage mRNA was determined 1hr after the treatment of hydrogen saline or saline. Results were expressed as the ratio of mRNA cytokine/mRNA microglobin. TNF-α mRNA of activated macrophages treated with hydrogen saline was lower than that treated with saline.
Hydrogen saline showed dose dependently down-regulated carrageenan-induced paw swelling, compared with the vehicle control group, which received an equal volume of vehicle only (saline). The treatment of animals with hydrogen saline produced a significant decrease in the number of infiltration neutrophils in the inflammatory paw.
Conclusions: Our findings indicate that the hydrogen saline had the effect of anti-inflammation in both the LPS-activated macrophages and paw oedema models. The possible mechanism may work by reducing •OH and ONOO –. So we could conclude that hydrogen saline may be a potential candidate for the therapy of inflammatory diseases, which is more convenient to be administered than inhaling hydrogen.
KEY WORDS: hydrogen saline, macrophages, TNF-α, anti-inflammation, carrageenan.
全文等待
https://blog.sciencenet.cn/blog-41174-254854.html
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