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標題: | 評估固態發酵發芽臺灣藜胜肽其延緩衰老之功效 Evaluation of Solid-state Fermentation of Chenopodium formosanum Sprouts Peptide with Anti-senescence Activity |
作者: | 黃筱筑 Hsiao-Chu Huang |
指導教授: | 鄭光成 Kuan-Chen Cheng |
關鍵字: | 延緩衰老,秀麗隱桿線蟲,臺灣藜,固態發酵,胜肽, anti-senescence,Caenorhabditis elegans,Chenopodium formosanum,solid-state fermentation,peptide, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 衰老是一種隨著時間的推移、年齡的增長所發生的自然現象,同時由於細胞和組織中各種有害變化的累積所引發的現象。當生物體暴露於某些外源性物質,可能導致內源性和外源性 ROS 之間不平衡,從而降低抗氧化防禦能力而引起衰老等現象發生。本研究目的為評估自發酵發芽臺灣藜中所分離出之 < 2 kDa 小分子胜肽產量於生物反應器發酵之最適發酵天數,並進一步鑑定其序列,運用 UVA 誘導細胞光衰老及動物模式生物秀麗隱桿線蟲評估胜肽延緩衰老之功效。將臺灣藜發芽四天並且接種 Rhizopus oligosporus 於生物反應器進行發酵,以發酵第四天為最適生產胜肽之天數,產量增加 5.89 倍。依據快速蛋白質液相層析圖譜得知同樣於發酵第 4 天有最高之 UV 280 nm 吸光值933.27,相比第 0 天增加 3.08 倍;透過蛋白質鑑定結果得知此胜肽之序列為 GGGGGKP (GRP)。評估 GRP 經 UVA 誘導 Hs68 細胞光衰老之保護效果,50 及 100 ug/mL GRP 分別回復細胞存活率至 85.52±14.96% 及 83.45±7.83%,增加 1.29 及 1.26 倍;並且分別減少 ROS 相對螢光強度 1.85 及 2.46 倍;於衰老指標酵素活性 SA-β‐gal 分別減少 8.13 及 7.00 倍。利用 RNA-seq 之基因富集結果推測預先添加 GRP 可能具有減緩經 UVA 所誘導之氧化壓力,因而回復細胞週期停滯,接著 mRNA 表現量顯示,50 及 100 ug/mL GRP 分別顯著增加 Nrf2 基因 3.96 與 10.69 倍的表現,此外其下游 HO-1 基因亦分別提升 6.67 與 9.30 倍,表示其可能透過 Nrf2 途徑減少 ROS 生成;另外於 50 及 100 ug/mL兩種 GRP 濃度可顯著降低 p53 基因表現達 4.09 及 2.69 倍 並且可延緩 p21 基因表現至 14.15 與 24.00 倍;接著利用 ChIP 分析,相比控制組,分別顯著上調各基因 GSR 61.94 與 28.79 倍、SOD 8.37 與 16.76 倍 及 NQO1 3.63 與 3.98 倍,證實 GRP 透過 Nrf2 轉錄因子調節途徑。研究顯示 GRP 有助於緩解 UVA 誘導之細胞週期停滯,達到延緩細胞衰老之功效。50 及 100 ug/mL GRP 相較於控制組,顯著延長秀麗隱桿線蟲之平均生命週期分別顯著提升 13.4% 及 11.0%,使生存曲線向右移;同時顯著增加咽部抽動速率 1.08 與 1.35 倍,以及改善 1.33 與 1.34 倍由促氧化劑誘導氧化壓力的生存率,接著 mRNA 表現量相較於控制組,50 ug/mL GRP 分別顯著提升 skn-1 下游基因 gcs-1、gst-4 與 gst-7 ( 3.3 倍、2.32 倍和 6.00 倍 )。本研究證實發酵發芽臺灣藜胜肽於體內及體外試驗皆具有延緩衰老之功效。 Population aging is an international concern. Taiwan has confronted an aging society since 1993. By the end of January 2021, the proportion of 65-year-olds has accounted for 16.2%, over 14% of the aging society defined by the United Nations. The purpose of this study is to evaluate the optimal fermentation days of < 2 kDa peptides isolated from fermented Chenopodium formosanum sprouts. Then evaluate the anti-aging effect of peptides through UVA-irradiation and the animal model Caenorhabditis elegans. The fourth day of fermentation was the optimum day for peptide production, and the yield increased by 5.89 times. The sequence is GGGGGKP (GRP), which is identified by protein identificaiton. To evaluate the protective effect of GRP on UVA-induced aging of Hs68 cells, 50 and 100 ug/mL GRP restored cell viability to 85.52±14.96% and 83.45±7.83%, respectively, increasing by 1.29 and 1.26 times; and reduced the ROS fluorescence intensity by 1.85 and 2.46 times; the aging indicator enzyme activity SA-β‐gal is reduced by 8.13 and 7.00 times respectively. Using the Gene Ontology results of RNA-seq, it is speculated that the pretreatment with GRP may relieve the oxidative stress induced by UVA irradiation, thereby restoring cell cycle arrest. Then mRNA expression showed that 50 and 100 ug/mL GRP significantly increased the overall gene expression of Nrf2 by 3.96, 10.69 times and its downstream HO-1 gene by 6.67, 9.30 times, respectively, indicating that the ROS generation may be reduced through the Nrf2 pathway. The gene expression of p53 was increased by 4.09, 2.69 times, p21 was improved by 14.15, 24.00 times, help to relieve UVA-induced cell cycle arrest and achieve the effect of delaying cell aging. Then, using ChIP analysis, compared with the control group, the genes GSR was significantly up-regulated by 61.94 and 28.79 times, SOD by 8.37 and 16.76 times, and NQO1 by 3.63 and 3.98 times, confirming that GRP regulates the pathway through the Nrf2 transcription factor. Compared with the control group, 50, 100 ug/mL GRP significantly prolong the average lifespan of C. elegans by 13.4±3.78 %, 11.0±2.77 % and increase the pharyngeal pumping rate by 1.08, 1.35 times. At the same time, it could improve survival of oxidative stress induced by juglone by 1.33, 1.34 times. Then, 50 ug/mL GRP increases gene expression of gcs-1, gst-4, gst-7 by 3.3, 2.32 and 6.00 times, respectively. This study confirmed that the fermented and germinated Chenopodium formosanum peptide has the effect of delaying aging both in vivo and in vitro. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90604 |
DOI: | 10.6342/NTU202302822 |
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顯示於系所單位: | 食品科技研究所 |
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