廣東住血線蟲感染造成氧化傷害及用N-acetyl-L-cysteine治療的作用機轉

Oxidative damage caused by infection with Angiostrongylus cantonensis and the mechanism in the treatment with N-acetyl-L-cysteine

鍾麗玉(Li-Yu Chung)

指導教授 ： 顏全敏

高雄醫學大學/醫學院/醫學研究所/博士(2011年)

摘要

本研究目的：第一、為以C57BL/6鼷鼠感染廣東住血線蟲的模式，探討宿主感染後是否引起活性氧的產生，因而導致氧化傷害，第二、為探討腦中樞活性氧增加的來源以及何種因子刺激或支持活性氧的產生，最後以N-acetyl-L-cysteine在體外細胞培育的模式，探討抗氧化劑對清除活性氧的機轉，並實際治療感染BALB/c鼷鼠效果的評估。C57BL/6鼷鼠感染30隻廣東住血線蟲第三期幼蟲後，腦脊髓液內活性氧濃度逐漸增加，在感染後12至30天活性氧濃度比感染前顯著增加，在感染後24天達最高峰，而於感染後45天降至正常值，雖然抗氧化酵素包括超氧歧化酶(superoxide dismutase)、catalase、麩氨基硫還原酶(glutathione reductase)、麩氨基硫氧化酶(glutathione peroxidase)、麩氨基硫硫轉移酶(glutathione-s-transferase)的活性及麩氨基硫(glutathione)的濃度，隨感染而顯著增加，然後也回復至正常值，但脂質、蛋白質及DNA過氧化指標也都增加，顯示仍然對腦中樞造成氧化傷害。檢查感染鼷鼠腦脊髓液的嗜酸性白血球及腦組織研磨均質液，發現腦脊髓液內活性氧是由嗜酸性白血球所產生，而嗜酸性白血球的數量自感染後在腦脊髓液內的波動，與吸附因子包括E-、L-selectin，細胞內吸附因子(intercellular adhesion molecule-1)及血管細胞吸附因子(vascular cell adhesion molecule-1)等的波動較一致，末稍血因IL-5濃度的增加所產生大量的嗜酸性白血球受這些吸附因子的幫助而進到腦部，而這些嗜酸性白血球也只有在IL-5的存在下才能持續產生活性氧。感染後20天的鼷鼠，腦脊髓液內嗜酸性白血球於體外單獨與IL-5或同時與N-acetyl-L-cysteine的作用，發現前者可以使嗜酸性白血球保持產生活性氧的能力，而N-acetyl-L-cysteine可以清除細胞外活性氧，並抑制細胞內活性氧的產生，在後者的作用24小時後，嗜酸性白血球乳酸脫氫酶顯著滲漏於培養液，血球粒腺體膜電位顯著下降，凋亡指數顯著上升，caspase-3活性顯著增加，P-ERK濃度顯著下降，而cytochrome c的濃度、caspase-9的活性、 P-Akt、P-P38及P-JNK的濃度則無顯著變化。BALB/c鼷鼠感染後12天即給予灌食N-acetyl-L-cysteine12天，則腦脊髓液內嗜酸性白血球凋亡指數顯著增加，活性氧濃度顯著減少，而有50%的存活率，的確可以臨床應用於緩解因廣東住血線蟲感染，對腦部的氧化傷害。

關鍵字

廣東住血線蟲 ； 氧化傷害 ； 嗜伊紅性腦膜炎

並列摘要

This study included three aims. The first aim was to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to lipid, protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at day 12-30 post-infection compared to that before infection, and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione-s-transferase, and glutathione as that in ROS in the CSF. Those antioxidants in the CSF of infected mice were all significant higher than they were before infection. However, malondialdehyde, protein carbonyl content and 8-hydroxy-2’-deoxyguanosine, biomarkers of oxidative to lipid, protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis. The second aim was to know the source of ROS and the regulation factor in the CSF of infected mice. Eosinophils increased in mice after infection and E-, L-selectin, ICAM-1, and VCAM-1 promoted the adherence of eosinophils to vascular endothelial cells, then they transmigrated into the CSF of infected mice. The measurement of CSF eosinophils and brain tissue homogenates found that ROS were from CSF eosinophils. IL-5 was found that it could maintain CSF eosinophils producing ROS in incubation in vitro. The third aim was to estimate the effect of N-acetyl-L-cysteine in the treatment of infected mice and the mechanism in decrease the level of ROS in the CSF of infected mice. In the incubation of CSF eosinophils from infected mice with IL-5 and N-acetyl-L-cysteine, the extracellular ROS produced by eosinophils were scavenged and the intracellular ROS in eosinophils were inhibited by N-acetyl-L-cysteine. The increase leakage of lactic dehydrogenase of eosinophil in culture medium and lower mitochondrial membrane potential of eosinophil became significant 24 hours after incubation. Further more, significantly decrease of P-ERK, significantly higher activity of caspase-3, and lower cytochrome c levels as well as caspase-9 activity were also found in the same incubation. Thus, the ERK route and cytoplasmic caspase-3 were correlated with the apoptosis of CSF eosinophils incubation with N-acetyl-L- cysteine in IL-5 rich medium. The use of N-acetyl-L-cysteine in the treatment of A. cantonensis infection in hosts are helpful in promotion apoptosis of CSF eosinophils and reduction ROS production in CSF. Indeed, 50% of BALB/c mice survived after treatment with N-acetyl-L-cysteine for 12 days at day 12 after infection.

並列關鍵字

Angiostrongylus cantonensis ； Oxidative damage ； Eosinophilic meningitis

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