Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin
Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin
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DOI：
10.1073/pnas.0510352103
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发表时间：
2006-03-21
影响因子：
11.1
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Nakai, K;Kadiiska, MB;Mason, RP
自由基的形成已经在LPS诱导的炎症的各种实验模型中进行了研究。本文利用电子自旋共振（ESR）和自旋阱α-（4-吡啶-1-氧化物）-N-叔丁基硝酮，检测了LPS处理小鼠皮肤6 h后脂质提取物中α-（4-吡啶-1-氧化物）-N-叔丁基硝酮自由基加合物的ESR谱。ESR谱与脂质衍生的自由基加合物的捕获一致。此外，使用二甲基亚砜（DMSO）的二次自由基捕获技术证明了甲基自由基的形成，揭示了羟基自由基的产生。自由基加合物的形成被氨基胍、N-（3-氨甲基）苄基乙脒（1400 W）或别嘌呤醇抑制，表明诱导型一氧化氮合酶（iNOS）和黄嘌呤氧化酶（XO）在自由基形成中的作用。在iNOS敲除（iNOS（-/-））小鼠中，自由基形成也受到抑制，证明了iNOS的参与。在这些自由基加合物的形成中不需要NADPH氧化酶，因为LPS处理在NADPH氧化酶敲除（gp 91（phox-/-））小鼠中增加的ESR信号强度与在野生型小鼠中一样多。一氧化氮（（NO）-N-中心点）终产物在LPS处理的皮肤中增加。正如预期的那样，（NO）-N-中心点终产物不被别嘌呤醇抑制，但被氨基胍抑制。有趣的是，LPS处理的皮肤中硝基酪氨酸的形成也被氨基胍和别嘌呤醇独立地抑制。用三价铁螯合剂Desferal预处理对自由基形成没有影响。我们的研究结果表明，iNOS和XO，但无论是NADPH氧化酶或三价铁，协同工作，形成脂质自由基和硝基酪氨酸在皮肤炎症早期由LPS引起的。
Free radical formation has been investigated in diverse experimental models of LPS-induced inflammation. Here, using electron spin resonance (ESR) and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, we have detected an ESR spectrum of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts in the lipid extract of mouse skin treated with LPS for 6 h. The ESR spectrum was consistent with the trapping of lipid-derived radical adducts. In addition, a secondary radical-trapping technique using dimethyl sulfoxide (DMSO) demonstrated methyl radical formation, revealing the production of hydroxyl radical. Radical adduct formation was suppressed by aminoguanidine, N-(3-aminomethyl)benzylacetamidine (1400W), or allopurinol, suggesting a role for both inducible nitric oxide synthase (iNOS) and xanthine oxidase (XO) in free radical formation. The radical formation was also suppressed in iNOS knockout (iNOS(-/-)) mice, demonstrating the involvement of iNOS. NADPH oxidase was not required in the formation of these radical adducts because the ESR signal intensity was increased by LPS treatment in NADPH oxidase knockout (gp91(phox-/-)) mice as much as it was in the wild-type mouse. Nitric oxide ((NO)-N-center dot) end products were increased in LPS-treated skin. As expected, the (NO)-N-center dot end products were not suppressed by allopurinol but were by aminoguanidine. Interestingly, nitrotyrosine formation in LPS-treated skin was also suppressed by aminoguanidine and allopurinol independently. Pretreatment with the ferric iron chelator Desferal had no effect on free radical formation. Our results imply that both iNOS and XO, but neither NADPH oxidase nor ferric iron, work synergistically to form lipid radical and nitrotyrosine early in the skin inflammation caused by LPS.