砷引起大鼠皮膚血管滲漏時一氧化氮所扮演的角色

Role of nitric oxide in increased vascular leakage in rat skin induced by arsenic

陳偉琪(Wei-Chi Chen)

指導教授 ： 陳世杰

高雄醫學大學/醫學院/醫學研究所/碩士(2006年)

摘要

依照流行病學研究指出，自由基以及一氧化氮在砷鹽所造成心血管疾病的因果關係中，扮演了一個重要的角色。本實驗經由大鼠靜脈注射染劑 Evans blue ( EB )，接著於鼠背皮膚皮內局部注射砷鹽、砷鹽加上不同濃度的一氧化氮合成酶抑制劑：N-nitro-L-arginine-methyl ester (L-NAME) 或者是Hemisulfate salt（Aminoguanidine）以及氫氧根自由基清除劑hydroxyl radical scavenger：1,3-Dimethyl-2-thiourea (DMTU)之後，粹取組織中的EB並測量血管滲透度，探討：1.砷鹽注射對大鼠皮膚血管破壞及滲漏程度多寡的影響；2.不同濃度的L-NAME、Aminoguanidine或是DMTU，是否可以降低砷鹽注射所導致的血管破壞及滲漏程度。3.另外，注射NOS inhibitor，再給予一氧化氮供應劑：S-nitro-N-acetylpenicillamine (SNAP)，是否又會提升砷鹽注射所導致的血管破壞及滲漏。4.以上實驗同時以H&E形態學方法，評估對大鼠皮膚皮內注射砷鹽溶液後，在不同的時間裡，對血管之傷害程度的影響。5.最後，以免疫形態學的方式，觀察與評估一氧化氮衍生物在砷所造成的血管滲漏中所扮演的角色為何。實驗結果顯示，砷鹽注射時間越長，血管滲漏的程度越高。當加入L-NAME或是DMTU在各種抑制濃度(5 μg/site、25 μg/site、125 μg/site)之下，均可以將砷鹽所造成血管滲漏濃度過高的情況壓抑下來；而Aminoguanidine只有濃度達到125 μg/site時，才可以將砷鹽所造成血管滲漏過高的情況壓抑下來。而saline注射（控制組），僅造成非常少量的血管滲漏。至於在膠體碳滲漏標記以及H&E形態學實驗方面，血管滲漏的實驗結果顯示：砷鹽注射的時間越長，膠體碳所呈現出的血管滲漏情形越高，且血管被破壞後；紅血球外滲的情形越嚴重，當加入單一濃度（125 μg/site）的L-NAME或是Aminoguanidine時，僅少量膠體碳外滲的結果表示L-NAME或是Aminoguanidine具有抑制血管滲漏的效果，而控制組，僅造成非常少量的血管破壞與紅血球外滲。當砷鹽加入L-NAME或是Aminoguanidine後，又加入NO donor（SNAP）時，實驗結果指出：血管滲漏（EB）和單獨注射砷鹽相同。實驗結果同樣顯示：即使同時在L-NAME或是Aminoguanidine存在下，使得組織中的NO無法產生，但是高濃度的SNAP仍可提供足量的NO，所以血管的滲漏情形和單獨注射砷鹽所造成結果相同。至於在免疫組織化學染色方面，以nitrotyrosine的存在當作指標。實驗結果顯示：砷鹽注射時間越長，nitrotyrosine的呈色就越深，當砷鹽混合高濃度的L-NAME或是Aminoguanidine注射至鼠背皮膚後，可以看到L-NAME或Aminoguanidine抑制了NO的生成。綜合以上結果顯示：暴露於砷鹽的時間越長，NO的產生就越多，血管被破壞滲漏的情形就越嚴重。本研究的結論是：NO的過量產生參與砷所造成的血管傷害及滲漏。

關鍵字

血管破壞及滲漏 ； 心血管疾病 ； 一氧化氮 ； 砷鹽

並列摘要

A correlation between arsenic and cardiac/peripheral vascular disease has been established through epidemiological studies. Present studies have shown that the increased vascular leakage is induced by arsenic injection. However, the underlying mechanism remains unclear. Previous studies indicated that increased vascular leakage by stimuli including endotoxin induced acute inflammation can be diminished by treatment with a nitric oxide syhthase (NOS) inhibitor, indicating that nitric oxide (NO) is required for stimuli to cause vascular leakage. Therefore, the goal of the present studies is to investigate whether NO plays a role in increased vascular leakage induced by arsenic. Vascular permeability changes were evaluated by means of EB assay and the India ink tracer techniques. Rats were intravenously injected with Evans blue (EB) or india ink followed by intradermal injections of sodium arsenite or sodium arsenite combined with various doses of NOS inhibitor : L-NAME, aminoguanidine or hydroxyl radical (OH-) scavenger DMTU or NO donor : S-nitro-N-acetylpenicillamine combined with both sodium arsenite and NOS inhibitors.
The results (rats, n=6/group) of EB permeability assay showed that different doses (5-125 μg/site) of both L-NAME and DMTU, as well as only one dose(125 μg/site) of aminoguanidine, attenuated the increased vascular leakage induced by arsenic injection. In time course experiments, 10 min after arsenic injection, all doses of L-NAME and aminoguanidine attenuated the vascular leakage induced by arsenic. During the intervals of 30 to 50 min, all doses of L-NAME and only one dose (125 μg/site) of aminoguanidine attenuated the increased vascular leakage induced by arsenic. The amount of vascular leakage induced by a solution of SNAP (1.25 μg/site) combined with NOS inhibitors (125 μg/site) was the same as arsenic injection alone. The India ink tracer technique also demonstrated a time-dependent increases of vascular labeling in the tissues examined. In addition, we also investigated the NO production after arsenic injected in rats with nitrotyrosine staining. The results showed that the NO production was significantly increased after arsenic injection and reached a maximum at 50 min after arsenic exposure. The increased NO production in skin was correlated with the increased leakage of blood vessels. In conclusion, the results indicated that endogenous NO may play important roles in tissue damage (vascular permeability) caused by arsenic in rat skin in vivo.

並列關鍵字

sodium arsenite ； vascular leakage ； cardiac/peripheral vascular disease

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