探討台灣香檬果皮萃取物對乙醯胺基苯酚誘導小鼠肝損傷之功效與機制

Effect and mechanism of Citrus depressa Hayata peel extract against acetaminophen-induced liver injury in mice

蔡博堯(Po-Yao Tsai)

指導教授 ： 蘇正元

中原大學/理學院/生物科技研究所/碩士(2021年)

https://doi.org/10.6840/cycu202100505

摘要

乙醯胺基苯酚 (acetaminophen，APAP) 為一種廣泛被使用的止痛退燒藥，然而長期或突然服用過量的APAP會引起慢性或急性肝損傷。台灣香檬 (Citrus depressa Hayata，CD) 含有豐富的多甲氧基黃酮類化合物，如川陳皮素 (nobiletin，Nob) 和橘紅素 (tangeretin，Tan)，被證實其具有抗氧化和抗發炎等功效。故本研究乃針對台灣香檬果皮經烘乾再製備成乙醇萃取物 (ethanolic extract，CD-EE) 後，探討台灣香檬果皮與可能活性成分 (Nob和Tan) 對APAP長期誘導BALB/c小鼠慢性肝損傷之保健功效與作用機轉。實驗動物隨機分成控制組、APAP組、APAP + N-acetylcysteine (NAC) 正控制組，以及APAP + CD-EE (250和500 mg/kg bw)、APAP + Nob (50 mg/kg bw) 和APAP + Tan (50 mg/kg bw) 等組別 (n=8)，於第1週至第9週分別每天管餵動物NAC、CD-EE、Nob或Tan；自第2週至第9週期間，除控制組外，各組以每週兩次的頻率進行APAP (400 mg/kg bw) 腹腔注射。試驗期結束後，分析結果顯示CD-EE (250和500 mg/kg bw)、Nob和Tan顯著降低APAP所誘導升高的小鼠血清ALT和AST活性，並減少APAP所造成的肝臟病理發炎現象。另與APAP相較下，CD-EE (250和500 mg/kg bw)、Nob和Tan可增加肝細胞內Nrf2與Nrf2下游抗氧化/解毒代謝酵素NAD(P)H：quinone oxidoreductase 1 (NQO1)、heme oxygenase-1 (HO-1) 和UDP-glucuronosyl transferase 1A (UGT1A) 等mRNA和蛋白質表現量，以及glutathione peroxidase (GPx)、glutathione S-transferase (GST) 和catalase等酵素活性，並降低肝臟中脂質過氧化作用(TBARS值)。此外，CD-EE (250和500 mg/kg bw)、Nob和Tan亦調節表觀遺傳學相關DNA 甲基轉移酶 (DNA methyltransferases, DNMTs) 1、3a和3b等蛋白質表現量。綜合以上結果，推測CD-EE、Nob和Tan可能透過表觀遺傳學調控機制而活化Nrf2 路徑，並提高肝細胞內抗氧化/解毒代謝酵素活性，進而達到減輕APAP所誘導肝臟損傷作用之功效。

關鍵字

乙醯胺基苯酚 ； 扁實檸檬 ； 表觀遺傳學 ； 肝臟 ； Nrf2 ； 川陳皮素 ； 橘紅素

並列摘要

Acetaminophen (APAP) is a common medicine for relieve pain and reduce the fever. But long time used and overdose of APAP will cause liver injury. In this research, we are study for the effect and mechanism of ethanolic extract of Citrus depressa Hayata (CD-EE), Nobelitin (Nob), and Tangeretin (Tan) against APAP-induced liver injury in Balb/c mice. In order to evaluate the possible beneficial effects of CD-EE, in the animal experiment, the mice were randomly divided into control, APAP, APAP+N-acetylcysteine (NAC) for positive control, APAP+CD-EE (250 mg/kg and 500 mg/kg), APAP+Nob (50 mg/kg), and APAP+Tan (50 mg/kg). At the first week mice were pretreated with NAC、CD-EE、Nob or Tan once daily for nine weeks, and then at the second week injected intraperitoneally (ip) with a single dose of APAP (400 mg/kg) twice a week for eight weeks. We found that CD-EE, Nob, and Tan significantly decreased APAP-induced serum ALT and AST activities, and decreased APAP-induced the inflammation of liver pathology. Compare with APAP, CD-EE, Nob, and Tan can upregulation the mRNA and protein expression of Nrf2, NAD(P)H：quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and UDP-glucuronosyl transferase 1A (UGT1A), and also restoration of the activities GPx, GST and catalase, and reduces lipid peroxidation in the liver. In addition, pretreatment of CD-EE, Nob, and Tan can also downregulated expression DNA methyltransferases (DNMTs), including DNMT 1, DNMT 3a, and DNMT 3b. These results suggest that CD-EE, Nob, and Tan might reduce APAP-induced hepatotoxicity through epigenetically regulating Nrf2-mediated BALB/c mice defense system.

並列關鍵字

acetaminophen ； Citrus depressa hayata ； epigenetic ； liver ； Nrf2 ； nobiletin ； tangeretin

參考文獻

Adeva-Andany, M. M., Perez-Felpete, N., Fernandez-Fernandez, C., Donapetry-Garcia, C., Pazos-Garcia, C. (2016). Liver glucose metabolism in humans. Biosci Rep, 36(6), : e00416.

Ahmed, O. M., Fahim, H. I., Ahmed, H. Y., Al-Muzafar, H. M., Ahmed, R. R., Amin, K. A., . . . Abdelazeem, W. H. (2019). The Preventive Effects and the Mechanisms of Action of Navel Orange Peel Hydroethanolic Extract, Naringin, and Naringenin in N-Acetyl-p-aminophenol-Induced Liver Injury in Wistar Rats. Oxid Med Cell Longev, 2019, 2745352.

Almazroo, O. A., Miah, M. K., Venkataramanan, R. (2017). Drug Metabolism in the Liver. Clin Liver Dis, 21(1), 1-20.

Arakawa, M., Ito, Y. (2007). N-acetylcysteine and neurodegenerative diseases: basic and clinical pharmacology. Cerebellum, 6(4), 308-314. Bannister, A. J., Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res, 21(3), 381-395.

Beers, R. F., Jr., Sizer, I. W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem, 195(1), 133-140.