基因合成精氨酸脫亞氨酶的神經保護作用於神經及小神經膠質細胞共同培養系統

Abstract

一氧化氮 (nitric oxide, NO●) 在人體中具有雙重功能，既是重要的訊息調控因子，也涉及許多病理機轉。特別是誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 過度表現所產生大量的NO● 與許多神經疾病有關，例如：帕金森氏症、阿茲海默症、和腦缺氧所導致的神經損害等。 精氨酸脫亞氨酶 (arginine deiminase, ADI) 是一種具有催化精氨酸 (L-arginine, L-arg) 分解為瓜氨酸 (L-citrulline, L-cit) 能力的蛋白質，此一蛋白僅表現於多種微生物內，而未見於哺乳類細胞中，且ADI已經被證實可以抑制iNOS所產生的NO●。因此，為了解ADI在iNOS所造成的神經毒性中所扮演的角色，我們利用BV2 (大鼠小神經膠質細胞株) 和SHSY5Y (人類神經母細胞瘤) 建立了小神經膠質細胞和神經細胞的共同培養系統，而在此共同培養系統中，加入2 μg/mL 的脂多醣 (lipopolysaccharide, LPS) 和1 ng/mL的γ-干擾素 (interferon-γ, IFN-γ) 兩天，成功地引起神經細胞死亡。利用此實驗模式，加入1 mU/mL的以基因合成的ADI (recombinant ADI, rADI) 於共同培養系統中持續二天到三天後，分別使用MTT assay和Griess method測定該培養系統中細胞存活率和NO● 產量。另外，進行神經細胞免疫染色和同位素標定多巴胺攝取實驗，分別作為專一地測定神經細胞存活率和神經功能。 結果發現在第二天時，和起始細胞的MTT數值相比，在只有投與LPS/IFN-γ的細胞中，細胞存活率僅剩21.1±4.1 %；而合併使用rADI後，細胞存活率則維持在89.2±2.2 %。 並且合併使用rADI也讓LPS/IFN-γ刺激所產生的NO● 從67.0±1.3 μM減少為19.5±5.5 μM。神經細胞免疫染色的結果清楚顯示，rADI確實具有神經細胞保護作用。在多巴胺的攝取功能上，與起始細胞相比，在只有投與LPS/IFN-γ的細胞中減少到35.7±11.4%，合併使用rADI的細胞則維持在103.0±12.6 %。另一方面，我們發現在第四天時，連續投與三天的rADI比只投與二天具有更好的保護效果，從MTT assay的數值上來看，投與三天的rADI可以保持76.8±11.7 %的細胞存活率，而投與二天的只有42.7±2.0 %；從多巴胺的攝取功能來看，連續投與三天與二天的rADI分別為75.1±10.8%和34.5±10.5 %。 為了解rADI的神經保護效果機轉，在rADI加入共同培養系統後，補充L-arg到培養液中；結果顯示L-arg的補充，可使rADI的神經保護效果完全消失。接著，我們將1400W (iNOS選擇性抑制劑) 和vinyl-L-NIO (nNOS選擇性抑制劑) 分別加入共同培養系統中，發現1400W可以有效的增加細胞存活率和抑制NO●產生，其EC50及IC50分別為2.3 μM 和5.7 μM，但是加入vinyl-L-NIO卻沒有這樣的現象。從以上結果來看，在此共同培養系統中，rADI對神經細胞之保護機轉可能是抑制過多由iNOS所產生的NO● 而非nNOS。 綜合上述結果，本研究之結論為：在此共培養系統中，rADI可以保護神經細胞免於LPS/IFN-γ所引起的神經毒性，不只是增加細胞的存活率，也維持神經細胞的功能。並且，rADI的保護機轉可能是透過消耗L-arg後，抑制iNOS產生NO●，達到神經保護的效果。為評估rADI在iNOS所導致的神經疾病是否具有臨床治療之可能性，未來可利用初始細胞 (primary cell) 及人工腦缺氧小鼠(ischemic mice)實驗，證實rADI是否在其他細胞及動物中具有相似的效果。

Nitric oxide (NO●) plays double-edged roles in human, not only important in physiological functions but also involved in many pathological pathways. Particularly, the overproduction of NO● generated by inducible nitric oxide synthase (iNOS) is associated with many neuronal disorders such as Parkinson’s disease, Alzheimer’s disease, and cerebral ischemic neuronal damage, etc. Arginine deiminase (rADI), expressed in some microorganisms but not in mammalian cells, can catalyze L-arginine (L-arg) to L-citrulline (L-cit). ADI has been reported the inhibitory activity of iNOS-mediated NO●production. A co-culture of BV2 (a murine microglial cell line) and SHSY5Y (a human neuroblastoma cell line) was established to understand the role of rADI in iNOS-mediated neurotoxicity. The co-culture was treated with the combination of 2 μg/mL of lipopolysaccharide (LPS) and 1 ng/mL of interferon-γ (IFN-γ) for 2 days, and successfully induced iNOS-mediated neuronal death. In the co-culture system, 1 mU/mL of purified recombinant ADI (rADI) was administrated for 2 and 3 days into the co-culture with LPS/IFN-γ treatment. The cell viability and NO● production were measured by MTT assay and Griess method, respectively. In addition, the specific neuronal viability and functionality were analyzed by neuron-specific immunostaining and 3H-dopamine uptake assay, respectively. The results showed that rADI treatment significantly preserved the cell viability (89.2±2.2 % of the initial cells) and decreased the NO● production (19.5±5.5 μM) on day 2, compared with the cells with LPS/IFN-γ treatment only (21.1±4.1 % and 67.0±1.3 μM, respectively) by MTT assay and Griess assay. Furthermore, the results of immunostaining showed that rADI treatment substantially preserved the neurons, and the dopamine uptake function was also maintained on day 2 (from 35.7±11.4% to 103.0±12.6 % of the initial cells) by rADI treatment. In addition, the neuroprotection of 3-day rADI treatment was observed better than 2-day treatment on day 4 by MTT assay (76.8±11.7 and 42.7±2.0 % of the initial cells, respectively), and dopamine uptake assay (75.1±10.8 and 34.5±10.5 % of the initial cells, respectively). To understand the possible neuroprotection mechanism of rADI treatment, replenishment of L-arg into the co-culture system with rADI treatment was conducted. Besides, the treatments of 1400W (a selective iNOS inhibitor) and vinyl-L-NIO (a selective neuronal NOS (nNOS) inhibitor) in the co-culture system were also performed. The results showed that the replenishment of L-arg abolished the neuroprotective and NO● suppressive effect of rADI in the co-culture system. In addition, the treatment 1400W preserved the cell viability (EC50=2.3 μM) and inhibited NO● production (IC50=5.7 μM), but vinyl-L-NIO did not. The results indicated that the neuroprotective mechanism of rADI is via L-arg depletion which inhibits the NO● production produced by iNOS, but not nNOS. According to the accumulative results, the conclusions are that rADI can protect the neurons from LPS/IFN-γ induced neurotoxicity in the co-culture system. rADI not only preserves the neuron viability but also maintains the functionality. The protection mechanism of rADI may be via depletion of L-arg and subsequently inhibits the iNOS mediated NO● production. To evaluate the therapeutic role of rADI in iNOS mediated neuronal disorders, further investigations in the neuroprotection of rADI in primary cell and ischemic mice model are needed in the future.