低氘氫水實驗室

中文摘要

氧化應激在炎症的發生髮展過程中發揮著重要作用，一方面它可以直接損傷機體，另一方面它可以通過分子間相互作用間接地對機體造成損害[1]。 在參與炎症過程的眾多複雜因子中，活性氧( ROS ) 和活性氮 ( RNS ) 是最重要的。它們包括羥自由基、超氧陰離子、過氧化氫、一氧化氮、亞硝酸陰離子 [2.3]。有大量的實驗證明清除羥自由基和亞硝酸陰離子能夠明顯減輕炎症的嚴重程度 [4-6] 。最近的研究表明氫氣能夠選擇性地清除羥自由基和亞硝酸陰離子[7]，因此氫氣很有可能通過這一途徑而具有抗炎的效應。但是通過呼吸道吸入氫氣的方法很不方便而且存在安全隱患，於是我們將氫氣通過高壓溶解於生理鹽水中，並檢查這種含有氫氣的生理鹽水是否具有抗炎效應。 

本課題以角叉菜膠誘導小鼠足腫脹為實驗模型，研究氫生理鹽水對於該炎症模型是否具有抗炎效應，並以巨噬細胞為例從細胞水平研究氫生理鹽水對炎性細胞的影響。 

本實驗通過酶聯免疫吸附試驗檢測了細胞培養上清中的 TNF-α 的含量；實時定量 PCR 檢測了RAW264.7 及小鼠原代腹腔巨噬細胞中 TNF-α 的 mRNA 水平；足爪容積測量儀檢測了小鼠足腫脹的程度；組織化學切片觀察了炎症局部的中性粒細胞浸潤程度。 

研究工作取得的主要結果如下： 

1. 氫生理鹽水能夠抑制被 LPS 激活的巨噬細胞分泌 TNF-α ：氫生理鹽水和 100ng/ml 的 LPS 共同處理 RAW264.7 細胞 4h ， 4h 後收集上清檢測 TNF-α ，發現氫生理鹽水能夠顯著抑制由 LPS引起的 TNF-α 產生，而且這種效應具有劑量依賴性。 

2. 氫生理鹽水能夠減少活化的巨噬細胞合成 TNF-α 的 mRNA 水平：氫生理鹽水和 100ng/ml 的LPS 共同處理巨噬細胞 1h ， 1h 後裂解細胞通過實時定量 PCR 檢測 TNF-α 的 mRNA 水平，發現氫生理鹽水能夠抑制巨噬細胞 TNF-α 的 mRNA 的表達。 

3. 氫生理鹽水能夠顯著抑制角叉菜膠誘導的足腫脹：以 2.5ml/kg 、 4ml/kg 、 5ml/kg 、 10ml/kg 的氫生理鹽水的量處理角叉菜膠誘導足腫脹的動物模型，發現處理組腳腫脹的程度明顯低於對照組，而且 5ml/kg 這個劑量效果最好。

4. 氫生理鹽水能夠顯著抑制炎症局部中性粒細胞的浸潤：將氫生理鹽水處理組、非處理組及對照組的足爪做成石蠟切片， HE 染色後觀察中性粒細胞浸潤程度，清水處理組的中性粒細胞浸潤明顯比非處理組少。

我們的研究發現氫生理鹽水不論在 LPS 激活的巨噬細胞上還是在角叉菜膠誘導的足腫脹動物模型上都具有明顯的抗炎效應，這一效應很可能是通過清除羥自由基、亞硝酸陰離子等自由基來實現的。因此我們可以預測，氫生理鹽水憑藉其有效性、安全性、方便性、廉價性，很可能成為我們以後治療某些炎症疾病的藥物。在今後的工作中，我們將深入研究氫生理鹽水俱有抗炎效應的機制，著重從炎症局部的自由基變化著手，驗證我們對於其抗炎機制的猜想，檢測炎症局部羥自由基和亞硝酸陰離子的變化。

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 (O 2 – ), hydrogen dioxide (H 2O 2), 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 H 2selectively reduced •OH and ONOO – [7] . So, as a free radical scavenger , H 2may have the effect of anti-inflammation. However, inhalation of hydrogen gas may be not convenient for therapeutic use，so we dissolved molecular hydrogen (H 2) 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.

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