OK432的辐射防护作用及其分子机制研究

The development of novel radioprotection drug with low toxicity and high-efficiency is of great importance. Our previous data showed some hints: We found that LPS, the ligand of TLR4, induced radio-resistance, and TLR4 in vivo ligand are required for basal radio-resistance. However, the toxicity of LPS severely limited the application of LPS as a radioprotection drug. In this proposal, the applicant focused OK432, the other ligand of TLR4, tried to appraise the radioprotection application prospect of OK432 and elucidate the underlying molecular mechanisms involved in the radioprotection of OK432. We firstly compared the toxicity between OK432 and LPS in three dose: 500μg/mice, 1000μg/mice and 5000μg/mice, and found that in the three dose, the toxicity of OK432 was much lower than LPS. After twice or triple usage of OK432, the survival rate of mice was almost 100%. Then, we assessed the radioprotection of OK432 and found that OK432 showed a significant radioprotection effect in 7.5 Gy and 9Gy. So we have confirmed that the low toxicity and the anti-radio effect of OK432 in vivo, and other researchers showed that IL-6 and G-CSF were MyD88 dependent radio-protect factors. . OK432 has been clinical used for 30 years, is cheap and easily stored. Our data showed the very low toxicity and radioprotection effect of OK432. All the features of OK432 favor that OK432 could be stockpiled for a nuclear war, dirty bomb, or nuclear accident. So OK432 is a potential candidate radioprotection drug, which worth further development. For the underlying molecular mechanisms involved in the radioprotection of OK432, we proposed our hypothesis: OK432 could induce significant radioprotection effect, and the underlying molecular mechanism may be OK432 activated TLR4-MyD88-NF-κB signal pathways to up-level radio-protect factors IL-6 and G-CSF via binding TLR4. . To radioprotection drug development, we intend to evaluate the efficiency of OK432 in four levels: molecular, cellular, tissue and organ, animal. We try to ascertain the optimum dose and administration of OK432. We want to see what effect OK432 exert on the radiosensitivity tissues. . To investigate the underlying mechanism OK432 in radioprotection, we intend to used KO mice to confirm the role of TLR4-MyD88-NF-κB signal pathways, then show the detail of the signal pathway by western blot and ELISA etc.. Applicant has been focusing the role of TLR4 in radio-biology for many years. Our rich experience in radiobiology is a advantage. Our radiobiology lab functioned perfectly for many years. In this project, we try to convert our previous study into something applicative useful, and our previous data in this project showed the key hints and great hope. We believe that, if funded, with our sincere efforts and professional laboratory, this project would propel the development of novel radioprotection drug and elucidation of the underlying molecular mechanism of anti-radio effect via TLR4..

研发毒性小，效应强的新型抗辐射药物具有重要的现实意义。我们之前的研究证明TLR4的配体脂多糖具有一定的辐射保护作用。但是脂多糖较大的毒性限制其作为抗辐射药物的应用。申请人将TLR4的另一个配体OK432作为潜在的抗辐射药物进行评价和研究。前期研究发现OK432抗辐射效应显著，且无毒性。 而文献检索提示IL-6和G-CSF为 MyD88依赖的辐射保护因子。因此，我们提出“OK432能诱导显著的辐射防护效应，其机制为OK432刺激TLR4，通过TLR4-MyD88-NF-κB信号通路上调辐射保护因子IL-6和G-CSF。”的假说。本项目拟从不同水平，详细评价OK432作为抗辐射药物的可行性及效用。并以OK432—TLR4—MyD88—NF-κB—IL-6/G-CSF为主线，从基因敲除小鼠层面，分子，信号通路等水平反复验证此假说。本项目在新型抗辐射药物的研发及其机制的阐明方面具有重要意义。

核能与核技术已大量运用于国民经济生活及国防。但是核事故核泄漏带来电离辐射损伤相关的医学问题却一直没有很好的处理措施.核事故和核恐怖不仅能在短时间内对公共健康造成较严重的损害，而且容易导致大规模的群体恐慌，甚至影响一个地区的稳定。因此，研制针对电离辐射损伤的抗辐射药物是非常必要的。部队目前装备的仍是上世纪六七十年代的仿制品；其中效果较好的防护剂成份都是雌激素，不适合男性为主的核潜艇艇员，对女性副作用也很大，而防护效果差，非常有必要更新换代。OK432已经应用于临床达30余年，未见其临床安全性问题的报道。二是OK432价格便宜，制作工艺成熟，其药剂性能稳定，-20℃至5℃时可保存36个月，室温下可保存27个月，方便作为核事故核恐怖袭击的战略储备药物。我们发现OK432能诱导显著的辐射防护效应，且OK432的辐射防护效应与给药剂量和给药时间显著相关。我们发现随着 OK432 剂量的提高，小鼠的生存率也得到显著提高， OK432 的剂量100μg/小鼠时，小鼠的生存率达到最大。在时间上，及辐照前24小时给药所起的辐照保护作用最大；辐照后（24,72,120小时）三次给药的抗辐照作用最大。在机制研究中，我们发现OK432在有无辐射前提下均能上调WT小鼠骨髓细胞数并抑制照后WT小鼠骨髓细胞的凋亡，且OK432作用是完全MYD88依赖，大部分TLR4依赖和小部分TLR2依赖的。进一步实验发现OK432辐射防护因子IL6和G-CSF且重复应用OK432可以进一步上调辐射防护因子IL-6和G-CSF。我们发现此作用也是完全MYD88依赖，大部分TLR4依赖和小部分TLR2依赖的。其机制为OK432刺激TLR4，通过TLR4-MyD88-NF-κB信号通路上调辐射保护因子IL-6和G-CSF。.总之，本项目发现了一个新型辐射防护药物OK432。从体内体外两方面证实了OK432的辐射防护作用。且OK432毒性低，价格便宜，易储存，可作为良好的战略储备辐射防护药物。在分子机制方面，发现OK432作用是完全MYD88依赖，通过TLR4-MyD88-NF-κB信号通路上调辐射保护因子IL-6和G-CSF。.本项目研究目标完成情况良好，研究内容已经发表4篇SCI论文，其中第二标注两篇，第三标注两篇；已经成文并投递2篇，都是第一标注；并培养硕士研究生两名。

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