I、紫鉚花素誘導神經母細胞瘤細胞凋亡之分子機制 II、靜脈精胺酸補充對亞急性腹膜炎大鼠免疫力及抗氧化能力的影響

I、Generation of reactive oxygen species mediates butein-induced apoptosis in neuroblastoma cells II、Effects of parenteral arginine on immunity and oxidant- antioxidant homeostasis in rats with subacute peritonitis

陳雅慧(Ya-Hui Chen)

指導教授 ： 許立松

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2013年)

https://doi.org/10.6834/CSMU.2013.00178

摘要

I、黃酮類物質(flavonoids)具有化學預防和化學治療的效用。紫鉚花素(Butein)是一種可從原生植物中分離，且具有生物活性的黃酮類化合物，已被證明會誘導人類癌細胞發生細胞凋亡。本篇研究動機主要是探究butein對神經母細胞瘤(neuroblastoma cells)細胞增殖(proliferation)和細胞凋亡(apoptosis)的分子機制為何。結果顯示，butein可抑制神經母細胞瘤Neuro-2A細胞的細胞存活，且隨著butein劑量和處理時間增加而更有效性抑制細胞存活(dependent manner)。Butein誘導Neuro-2A細胞細胞凋亡，其特徵在於使細胞週期停滯於sub-G1時期增加凋亡細胞分佈。Butein處理將顯著性增加細胞內活性氧ROS的含量和降低Bcl-2/Bax蛋白比值，誘導pro-caspase 3和PARP的裂解。若前處理抗氧化劑，N-乙酰半胱氨酸（NAC），能夠抑制butein誘導ROS的產生和細胞死亡。NAC也能恢復butein誘導的細胞凋亡相關蛋白的改變。總之，butein導致神經母細胞瘤細胞發生凋亡的分子機制可能是透過產生ROS，Bcl-2/Bax蛋白比值的改變，和pro-caspase 3和PARP的裂解。我們的研究結果顯示，butein可以作為一個治療神經母細胞瘤有療效的化療藥物。
II、背景：精胺酸缺乏和慢性炎症可能導致免疫功能低下。先前研究指出，亞急性腹膜炎大鼠於靜脈補充藥理劑量精胺酸後，將促進鳥胺酸生成及減少一氧化氮生成。雖已有報導提出精胺酸能減緩氧化壓力的發生，然而發炎時，精胺酸可能誘導過量一氧化氮生成，進而導致氮化壓力。因此，本研究探討給於靜脈不同劑量精胺酸補充對免疫細胞的亞群分佈和功能之影響，並同時探討其對全身和器官/組織脂質過氧化物和抗氧化酶活性的影響。此研究之結果將可提供臨床上亞急性腹膜炎患者，利用靜脈精胺酸補充劑，改善免疫力及抗氧化能力之資訊。材料：利用盲腸穿孔手術引起的亞急性腹膜炎雄性Wistar大鼠於靜脈輸入總熱量為1.61 (CP組)、2.85 (LA組)、4.08 ( MA組)或6.54% (HA組)的精胺酸補充營養液連續7天後，分析其周邊血淋巴細胞和脾臟細胞中T細胞、B細胞和單核細胞/巨噬細胞分佈及細胞激素分泌情形。此外，分析其全身及腹膜炎影響的靶器官/組織的脂質過氧化物、抗氧化劑、抗氧化酶的活性及硝基酪胺酸含量。本實驗亦包括一組未經手術，且經口餵養的健康大鼠作為參考，即為NC組。
結果：白血球細胞數量和血清中腫瘤壞死因子(TNF)-α、干擾素(IFN)-γ濃度在各組間皆無顯著差異。血清中硝酸鹽/亞硝酸鹽(NOx)和細胞間素(IL)-2含量皆隨精胺酸補充劑量的增加而顯著減少。靜脈補充精胺酸顯著降低亞急性腹膜炎大鼠周邊血淋巴細胞和脾臟細胞經刀豆素蛋白A(Con A)和脂多醣(LPS)刺激後TNF-α的分泌，且顯著降低周邊血淋巴細胞自發性IL-6和脂多醣刺激IFN-γ的分泌，而脾臟細胞經脂多醣刺激後IL-6的分泌也因精胺酸之補充而顯著降低。另外，LA組顯著增加周邊血淋巴細胞和脾臟細胞自發性IFN-γ的分泌；HA組顯著增加周邊血淋巴細胞自發性TNF-α和經Con A刺激後IL-4和IL-6的分泌。靜脈補充精胺酸顯著減少亞急性腹膜炎引起的增加肌肉TBARS和腎臟硫醇化合物含量，且顯著增加肝臟細胞色素P450酵素含量，並顯著減少腎臟的TBARS。MA組和HA組顯著逆轉腹膜炎引起的血漿NOx和TBARS含量升高及腎臟穀胱甘肽過氧化物酶活性下降的情形。當與CP組比較時，LA組顯著增加血漿穀胱甘肽過氧化物酶酵素活性，MA組顯著降低肺臟NOx和增加肌肉NOx含量。結論：本研究指出當靜脈補充精胺酸的劑量為總熱量4%時，可改變亞急性腹膜炎大鼠周邊血淋巴細胞和脾臟細胞的免疫力，但補充劑量超過總熱量的6%時，可能對亞急性腹膜炎大鼠產生不良影響。此外，靜脈補充精胺酸的劑量為總熱量4至6%時，可減少亞急性腹膜炎大鼠脂質過氧化物的產生和提高抗氧化酶的活性，但可能會造成亞硝化壓力的升高；這些結果顯示，靜脈精胺酸補充可調節氧化-抗氧化平衡，且其作用與補充劑量及組織反應有關。

關鍵字

細胞凋亡 ； 紫鉚花素 ； 神經母細胞瘤 ； 精胺酸 ； 腹膜炎 ； 免疫力 ； 抗氧化能力

並列摘要

I、Flavonoids exhibit chemopreventive and chemotherapeutic effects. Butein, a bioactive flavonoid isolated from numerous native plants, has been shown to induce apoptosis in human cancer cells. In the current study, the molecular mechanisms of butein action on cell proliferation and apoptosis of neuroblastoma cells were evaluated. Treatment with butein decreased the viability of Neuro-2A neuroblastoma cells in a dose- and time-dependent manner. The dose-dependent nature of butein-induced apoptosis was characterized by an increase in the sub-G1 phase population. Treatment with butein signifi¬cantly increased intracellular reactive oxygen species (ROS) levels and reduced the Bcl-2/Bax ratio, triggering the cleavage of pro-caspase 3 and poly-(ADP-ribose) polymerase (PARP). Pre-treatment with the antioxidant agent, N-acetyl cysteine (NAC), blocks butein-induced ROS generation and cell death. NAC also recovers butein-induced apoptosis-related protein alteration. In conclusion, butein-triggered neuroblastoma cells undergo apoptosis via generation of ROS, alteration of the Bcl 2/Bax ratio, and cleavage of pro-caspase 3 and PARP. Our results suggest that butein may serve as a potential therapeutic agent for the treatment of neuroblastoma.
II、Background: Arginine deficiency and chronic inflammation may cause immune dysfunction. The authors previously showed that a pharmacological dose of parenteral arginine facilitates ornithine rather than nitric oxide production in subacute peritonitis. Beneficial effects of arginine on oxidative stress have been reported; however, arginine-induced excess production of nitric oxide (NO) needs to be concerned in inflammation. Herein, they investigated the effects of different doses of parenteral arginine supplementation on immunocytic subpopulation distribution and function. In addition, dose effects of parenteral arginine on lipid peroxidation (i.e., thiobarbituric acid reactive substances, TBARS) and activities of antioxidant enzymes in the circulation and organs/tissues were investigated.Materials: Male Wistar rats with cecal puncture-induced sub-acute peritonitis were infused with parenteral nutrition solutions containing 1.61, 2.85, 4.08, and 6.54% of total calories as arginine for 7 days, i.e., the CP, LA, MA, and HA groups, respectively. Distributions of T cells, B cells, and monocytes/macrophages and cytokine productions of peripheral blood lymphocytes (PBLs) and splenocytes were analyzed. In addition, levels of lipid peroxidation, antioxidants, antioxidant enzyme activity, and nitrotyrosine in the circulation and peritonitis- affected target organs/tissues also were analyzed. Healthy, orally-fed rats were as references.Results: There were no significant differences in circulating white blood cell numbers and serum tumor necrosis factor (TNF)-α and interferon (IFN)-γconcentrations among groups. Serum nitrate/nitrite (NOx) and interleukin (IL)-2 levels were significantly decreased by arginine in a dose-dependent manner. Animals supplemented with parenteral arginine had significantly decreased productions of concanavalin (Con) A- and lipopolysaccharide (LPS)-stimulated TNF-α in PBLs and splenocytes, spontaneous IL-6 and LPS-stimulated IFN-γin PBLs, and LPS-stimulated IL-6 in splenocytes. In addition, the LA group had significantly increased production of spontaneous IFN-γin PBLs and splenocytes. The HAgroup had significantly increased spontaneous TNF-α, and Con A stimulated IL-4 and IL-6 in PBLs. Parenteral arginine significantly attenuated sub-acute peritonitis-induced increases in TBARS in the muscle and thiol group in the kidneys, significantly increased cytochrome
P450 in the liver, and significantly decreased TBARS in the kidneys. In addition, peritonitis- elevated NOx (i.e., nitrate and nitrite) and TBARS in the plasma and peritonitis-declined glutathione peroxidase (GPx) activity in the kidneys were significantly reversed in the MA and HA groups. However, the LA group had significantly increased plasma GPx activity and the MA group had significantly decreased lung NOx and increased muscle NOx compared to the CP group. Conclusion: Parenteral arginine administration at approximately 4% of total calories may alter PBLs and splenocytic immunity, and >6% of total calories might not be of benefit in rats with subacute peritonitis. Parenteral arginine at 4 to 6% of total calories may attenuate lipid peroxidation and increase activities of antioxidant enzymes; however, nitrosative stress might be elevated. These results suggest that parenteral arginine supplementation may modulate oxidant-antioxidant homeostasis in dose- and organ-dependent manners.

並列關鍵字

apoptosis ； butein ； neuroblastoma ； arginine ； peritonitis ； immune function ； antioxidant enzymes ； nitric oxide

參考文獻

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