香椿萃取物對於敗血症中自噬反應之影響

Effects of Toona sinensis on the autophagic demand during sepsis

郭純恩(Chun-En Kuo)

指導教授 ： 褚佩瑜

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2017年)

摘要

研究背景：許多研究顯示香椿(Toona sinensis, TS)具有抗細胞凋亡、抗氧化及抗發炎之特質，而香椿在敗血症之動物模型中，也能減少肝細胞傷害、減緩器官衰竭。
研究目的：藉由細胞自噬反應(autophagy)的促進劑-雷帕黴素(rapamycin)之介入，觀察自噬反應在敗血症中的影響、香椿萃取液在RAW 264.7細胞株中對自噬反應的影響，及其是否能改善敗血症動物模式的存活率。
實驗設計與方法：細胞實驗中，以細菌內毒素之脂多糖(LPS)誘發RAW 264.7細胞株發炎狀態後，觀察細胞存活率、抗氧化能力、氧化還原狀態、細胞激素釋放情形、及自噬反應相關之蛋白質表現。動物實驗中，採用C57BL/6品系之小鼠，於盲腸結紮穿刺(cecal ligation and puncture, CLP)手術前四週內給予香椿 (100 mg/kg body weight (BW)/day)補充，評估手術後36小時內之變化，並記錄敗血症動物模式的存活狀況。
結果：細胞實驗中，香椿提升穀胱甘肽(glutathione, GSH)含量、還原態及氧化態穀胱甘肽之比例(GSH/GSSG ratio); 降低氧化態(oxidized glutathione, GSSG)、一氧化氮(nitric oxide, NO)總量、硝酸鹽(nitrate, NO3)、亞硝酸鹽(nitrite, NO2)、超氧陰離子、超氧化物歧化酶(superoxide dismutase, SOD)及穀胱甘肽過氧化酶(glutathione peroxidase, GPx)之表現，及細胞介素-6 (interlukine-6, IL-6)之釋放; 香椿不影響細胞存活率。單獨香椿和香椿及與雷帕黴素結合使用均顯著降低丙二醛(malondialdehyde, MDA)含量。香椿葉萃取物能在mTOR蛋白(mammalian target of rapamycin)的S2448位置上抑制其磷酸化作用，且在較高劑量下(50，75 μg/ml) 結合雷帕黴素治療時增加細胞自噬蛋白LC3 II的表現，進而提升細胞自噬反應。動物實驗中，香椿不論單用或與雷帕黴素併用都無法減緩CLP手術後導致敗血症之死亡率。
結論： 在RAW 264.7細胞株中，提前補充香椿葉萃取物能透過加強抗氧化系統、抑制發炎前期細胞激素之釋放、促進自噬反應，減緩LPS引起之細胞傷害。雷帕黴素亦能影響氧化還原狀態及抗氧化能力。香椿能促進自噬反應，且並不是只透過mTOR調控路徑，但在動物實驗中無法降低CLP手術後導致敗血症之死亡率。

關鍵字

敗血症 ； 香椿 ； 自噬作用 ； 雷帕黴素 ； 抗氧化

並列摘要

Background
Toona sinensis (TS) was reported to have anti-apoptotic, anti-oxidant, and anti-inflammatory properties. TS also ameliorated the liver cell damage and prevent the organ failure in the sepsis animal models.

Aim
By the intervention of rapamycin, as the autophagy enhancing agent, to explore the role of autophagy involved in sepsis. To examine if TS affect autophagy in RAW 264.7 cell lines, and if TS could improve the survival rate of sepsis animal models.

Methods
In the in vitro studies, the lipopolysaccharides (LPS) induced the inflammation status on the RAW 264.7 cell lines. The cell viabilities, the antioxidation abilities, the redox status, the release of cytokines, and the expression of autophagy-related proteins were observed. The C57BL/6 mice, as the animal model in this study, were supplemented with TS leaf extracts (100 mg/kg body weight (BW)/day) in feed for 4 weeks prior to cecal ligation and puncture (CLP) operations, and they were observed for the following 36 hours. The survival conditions of the sepsis animal models were recorded.

Results
In the RAW 264.7 cell lines, the pre-supplementation of TS leaf extracts elevated the total glutathione (GSH) levels and the GSH/GSSG ratio, decreased the oxidized glutathione (GSSG), total nitric oxide (NO), nitrate (NO3), nitrite (NO2), and superoxide anion levels, and attenuated the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and the release of interlukin-6 (IL-6). TS did not influence the cell viability. TS alone and the combined use with rapamycin both significantly decreased the levels of malondialdehyde (MDA). TS leaf extracts could inhibit the phosphorylation of mammalian target of rapamycin (mTOR) on site S2448. When applied with relatively higher dosage (50 and 75 μg/ml) and with the adjuvant use of rapamycin, TS elevated the expression of LC3 II, which further boosted autophagy. TS alone or the combination use with rapamycin did not improve the mortality rate in the CLP-induced-sepsis animal model.

Conclusion
Pre-supplementation of TS leaf aqueous extracts may prevent cell damage through strengthening the antioxidant system, suppressing the release of pro-inflammatory cytokines and promoting autophagy in the LPS-induced RAW 264.7 cells. Rapamycin also affected the redox status and anti-oxidation ability in the cell lines. TS restored autophagy, not only just through m-TOR pathway. However, TS did not improve the mortality rate in the CLP-induced-sepsis animal model.

並列關鍵字

Sepsis, Toona sinensis ； autophagy ； rapamycin ； anti-oxidation

參考文獻

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