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研究生:詹昕樺
研究生(外文):Chan, Hsin-Hua
論文名稱:14-脫氧11,12-雙脫氫穿心蓮內脂改善棕櫚酸誘導的AML-12肝細胞自噬通量受損以及凋亡
論文名稱(外文):14-Deoxy-11,12-didehydroandrographolide attenuates palmitic acid-induced impairment of autophagy flux and apoptosis in AML-12 hepatocytes
指導教授:陳暉雯
指導教授(外文):Chen, Haw-Wen
口試委員:李宗貴李健群
口試委員(外文):Lii, Chong-KueiLi, Chien-Chun
口試日期:2023-06-07
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:營養學系碩士班
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:中文
論文頁數:77
中文關鍵詞:非酒精性脂肪肝病脂毒性自噬凋亡14-脫氧11, 12-雙脫氫穿心蓮內脂
外文關鍵詞:ApoptosisAutophagy14-Deoxy-11,12-didehydroandrographolide (deAND)LipotoxicityNon-alcoholic fatty liver disease (NAFLD)
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  • 收藏至我的研究室書目清單書目收藏:0
非酒精性脂肪肝病 (NAFLD) 為目前常見的慢性肝臟疾病之一,定義為非攝取過量酒精情況下,肝細胞脂肪含量超過5%,脂質代謝異常使得肝臟脂肪變性,增加脂毒性的風險,進而造成粒線體受損、阻礙自噬通量,若未能及時改善,將導致細胞死亡,進一步加速非酒精性脂肪肝炎(NASH)的進程。14-脫氧11, 12-雙脫氫穿心蓮內脂 (deAND) 具有抗氧化、抗發炎、抗癌等生理功效,但其是否可改善NASH的相關研究仍不多見,因此本研究利用小鼠肝細胞AML-12,以250 M棕櫚酸(PA)誘導其產生脂毒性,探討deAND是否可改善AML-12細胞因PA所造成的脂毒性傷害,並改善自噬通量受損及減少細胞凋亡的可能作用機制。結果顯示,PA處理AML-12 細胞24小時後,自噬相關蛋白p-AMPK、Beclin1、Atg5、Atg7表現減少,p-p62、p62和LC3-II表現增加,粒線體自噬相關蛋白Parkin、PINK1表現減少,當PA處理細胞時間延長至36小時後,凋亡相關蛋白cleaved caspase 3表現顯著增加;之外,當給予自噬抑制劑CQ或3-MA時,會加劇PA所造成的細胞凋亡;以RFP-GFP-LC3B螢光分析,顯示PA抑制AML-12細胞的自噬流動、增加自噬體的堆積。預處理7.5或15 µM deAND,不但可促進自噬流動,也可有效改善PA誘發的細胞凋亡,而且PA所誘導的活性氧生成明顯被抑制。綜合以上結果,PA使粒線體受損、阻礙自噬通量、造成肝細胞凋亡,藉由deAND的抗氧化功能,有效減少PA導致的活性氧生成及改善自噬流動,有效保護肝細胞,減少細胞凋亡。
Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. It is defined as the presence of fat > 5% of the liver weight without alcohol use. Abnormal lipid metabolism causes hepatic steatosis and increases the risk of lipotoxicity, which results in mitochondrial damage and impaired autophagic flux. Cell apoptosis accelerates the progression to NASH. 14-Deoxy-11,12-didehydroandrographolide (deAND) has been shown to possess numerous physiological activities such as anti-oxidant, anti-inflammatory, and anti-cancer, but there are few studies regarding its effect on NASH development. Therefore, we investigated whether deAND (7.5 and 15 μM) could improve 250 μM palmitic acid -induced lipotoxicity including impaired autophagy flux and increased cell apoptosis in mouse hepatic AML-12 cells and the possible mechanisms involved in this study. The results revealed that after palmitic acid treatment for 24 hours, the expression of autophagy-related proteins such as p-AMPK, Beclin1, Atg5, and Atg7 decreased, the protein expression of p-p62, p62, and LC3-II increased, and the protein expression of mitophagy such as PINK1 and parkin decreased. When the palmitic acid treatment was extended to 36 hours, the expression of apoptosis-related protein cleaved caspase 3 increased dramatically. Furthermore, we found that when the autophagy inhibitors CQ or 3-MA were given, the apoptosis caused by palmitic acid treatment would aggravate. In the RFP-GFP-LC3B fluorescence analysis, it was shown that palmitic acid treatment inhibited the autophagic flux of AML-12 cells and increased the accumulation of autophagosomes. When given the pretreatment of 7.5 or 15 µM deAND, it improved autophagic flux, mitigated palmitic acid-induced apoptosis, and inhibited palmitic acid-induced reactive oxygen species generation. In conclusion, palmitic acid impaired mitochondrial function and autophagic flux, which results in apoptosis of hepatocytes. Due to the anti-oxidant and anti-inflammatory abilities of deAND, it effectively reduces the generation of reactive oxygen species caused by palmitic acid and reverses the autophagic flux, which reduces cell apoptosis and protects liver cells.
中文摘要 IV
Abstract V
目錄 VII
圖目錄 IX
表目錄 XI
第一章 前言 2
第二章 文獻探討 3
2.1 NAFLD 3
2.1.1 NAFLD定義及盛行率 3
2.1.2 NAFLD病程及發病機制 4
2.1.3 NAFLD及脂毒性 6
2.2 自噬(Autophagy) 9
2.2.1 自噬訊號途徑 10
2.2.2 粒線體自噬(Mitophagy)途徑 13
2.2.3 自噬通量(Autophagy Flux) 15
2.2.4 NAFLD與自噬 17
2.3 凋亡 17
2.3.1 NASH中的自噬與凋亡 18
2.4 穿心蓮(Andrographis paniculate) 20
2.4.1 14-脫氧11,12-雙脫氫穿心蓮內脂(14-Deoxy-11,12-didehydroandrographolide, deAND) 23
第三章 研究動機 24
第四章 實驗架構 25
第五章 參考文獻 26
1. Introduction 42
2. Materials and Methods 45
2.1 Chemicals 45
2.2 AML-12 cell culture 45
2.3 Palmitic acid preparation 46
2.4 Cell viability assay 46
2.5 Western blot assay 47
2.6 Autophagic flux analysis 49
2.7 Reactive oxygen species (ROS) measurement 50
2.8 Statistical analysis 50
3. Results 51
4. Discussion 67
5. Reference 71

圖目錄
第一部分
圖 2.1 NAFLD病程 (Jackson et al., 2022) 5
圖 2.2 NAFLD多重平行打擊說(Byrne & Targher, 2015) 6
圖 2.3 脂毒性誘發NASH的機制(Huby & Gautier, 2022) 7
圖 2.4棕櫚酸誘導脂毒性(Song & Malhi, 2019) 8
圖 2.5 脂毒性誘發細胞損傷之機制(Marra & Svegliati-Baroni, 2018) 9
圖 2.6 自噬的種類(Hosaka et al., 2021) 10
圖 2.7 自噬訊號途徑(Riebisch et al., 2021) 11
圖 2.8 粒線體融合與裂變機制(Ren et al., 2020) 14
圖 2.9 粒線體自噬機制(Hirota et al., 2012) 15
圖 2.10 p62介導的泛素化蛋白調控自噬的機制(Matsumoto et al., 2011) 16
圖 2.11 p62及Keap1-Nrf2路徑之相關性(Ichimura et al., 2013) 16
圖 2.12 細胞凋亡途徑 (Ichimura et al., 2013) 18
圖 2.13 細胞調節自噬與凋亡(Messer, 2017) 20
圖 2.14 穿心蓮外觀:植株(A)、花瓣(B)、葉子(C)、種子(D) (Murthy & Dalawai, 2021) 21
圖 2.15 穿心蓮主要的植化素(Chao & Lin, 2010) 22







第二部分
Figure 1. Effects of palmitic acid and deAND on cell viability in AML-12 cells. 55
Figure 2. Palmitic acid induces apoptosis as well as inhibits autophagy and mitophagy in AML-12 cells. 59
Figure 3. deAND improves palmitic acid-induced apoptosis as well as palmitic acid-inhibited autophagy and mitophagy in AML-12 cells. 61
Figure 4. deAND promotes autophagic flux in AML-12 cells. 62
Figure 5. deAND decreases palmitic acid-induced autophagosome formation and cell apoptosis in AML-12 cells. 63
Figure 6. deAND inhibits palmitic acid-induced ROS generation and induces expression of antioxidant enzymes in AML-12 cells. 66
Figure 7. Scheme summarizing the signaling pathways involved in the mitigation of palmitic acid-induced lipotoxicity development by deAND in AML-12 cells. 67

表目錄
第一部分
表 2.1 110年十大死因排名(衛生福利部,2023) 4
表 2.2 110年十大癌症死因排名(衛生福利部,2023) 4

第二部分
Table 2.1 The number of cells used for different experiments 46
Table 2.2 Preparation of RIPA buffer 48
Table 2.3 Preparation of 4X sample buffer stock 48
Table 2.4 Preparation of SDS-Polyacrylamide gel 49



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