探討 Hepsin 在 acetaminophen 導致的肝毒性中扮演的角色

Investigate the role of Hepsin in acetaminophen-induced hepatotoxicity

洪嘉璐(Chia-Lu Hung)

指導教授 ： 林淑華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703929

摘要

APAP (acetaminophen) 濫用為已開發國家急性肝衰竭的常見原因，在高劑量下 APAP 會經由細胞色素 (cytochrome) P450 CYP2E1 及 CYP1A2 氧化為親電性高的活性物質 NAPQI (N-acetyl-p-benzoquinone imine)。NAPQI會與細胞內的許多蛋白質形成非特異性的共價鍵結而破壞其功能造成細胞損傷，肝臟藉由glutathione (GSH) 與 NAPQI 共價結合以降低其肝毒性，當 GSH被大量消耗之後，ROS (reactive oxygen species) 及 RNS (reactive nitrogen species) 含量上升。最終使細胞氧化壓力上升、粒線體失去功能和DNA損壞。
Hepsin 為第二型穿膜絲胺酸蛋白酶，主要表現於肝臟。本實驗室先前發現 Hepsin 基因剔除小鼠在注射 400 mg/kg APAP 後，會因嚴重的肝損傷而在8~10小時內死亡。本篇論文首先確認 Hepsin 基因剔除小鼠在投予400 mg/kg APAP 4~6小時後，血清AST、ALT 及 HMGB1 相較野生型小鼠皆大量上升，表示 Hepsin 基因剔除小鼠肝損傷較嚴重。由肝組織 H E 染色可見野生型及基因剔除小鼠於投予 APAP 2小時後皆發現肝細胞空泡化，而 Hepsin 基因剔除小鼠在投予 APAP 4小時後較野生型小鼠有較高的壞死比例。實驗進一步證明野生型小鼠在注射 400 mg/kg APAP 1小時後，肝細胞中 Hepsin 蛋白表現量顯著下降，顯示 Hepsin 在 APAP 造成急性肝衰竭的早期即扮演角色。Hepsin 基因剔除小鼠在投予400 mg/kg APAP 的0.5~6小時間點，CYP2E1、CYP1A2 的表現量及肝組織 GSH 的消耗量均與野生型小鼠無差異，但肝組織中反應氧化壓力的硝基酪氨酸 (nitrotyrosine) 在2小時即顯著上升。由西方墨點法分析受 APAP肝毒性而活化的 MAPK 訊息傳遞路徑，發現 JNK 和 AKT 磷酸化程度在 Hepsin 基因剔除小鼠及野生型小鼠間並無差異，而 Hepsin 基因剔除小鼠在投予 APAP 2小時後 P38 磷酸化的比例和4小時後 ERK 磷酸化的比例皆顯著上升，而投予 P38 抑制劑，在施打 APAP 4小時後血清AST、ALT濃度明顯下降。初步推測，Hepsin 可能藉由抑制 P38 的活性，進而緩解 APAP 造成的肝臟損傷。

關鍵字

Hepsin 基因剔除小鼠 ； 乙醯胺酚 ； 急性肝衰竭 ； 訊息傳遞路徑 ； P38 激?

並列摘要

APAP overdose is the leading cause of acute liver failure in developed country. At high doses, cytochrome P-450 enzymes (CYP2E1, CYP1A2) convert APAP into a reactive quinone form, N-acetyl-p-benzoquinone imine (NAPQI). Although NAPQI is inactivated by conjugation with glutathione (GSH), once the pool of GSH is exhausted, remaining NAPQI covalently binds cellular proteins, causing the elevation of reactive oxygen species and nitrogen species. Finally, it results in oxidative stress, mitochondrial dysfunction, and DNA damage.
Hepsin, a type II transmembrane serine protease, is mainly expressed in the liver. Previous study in our lab found that Hepsin knockout (KO) mice treated with 400 mg/kg APAP resulted in severe liver injury and death of mice within 8 to 10 hours after APAP injection. Our data confirmed that the concentration of serum AST, ALT, and HMGB1 rises greatly in Hepsin knockout mice within 4 to 6 hours after 400 mg/kg APAP injection, indicated that Hepsin knockout mice had more serious liver injury. H E stain of hepatocytes appeared vacuolization in both wild type and knockout mice 2 hour after APAP injection, and higher proportion of necrosis in Hepsin knockout mice compare to wild type mice 4 hour after APAP injection. Challenged wild type mice with 400 mg/kg APAP and found that the endogenous Hepsin in the liver of WT mice was significantly decreased within 1 hour after APAP injection, indicating that Hepsin played a role in APAP-induced acute liver failure at early time points.
The protein level of P450 CYP2E1 and CYP1A2, and the GSH depletion level in liver tissue showed no difference between Hepsin knockout and WT mice under 400 mg/kg APAP treatment. However, the expression level of nitrotyrosine, which represents oxidative stress in cells, were elevated 2 hours after APAP treatment. The kinase of signaling pathway, which is activated under APAP-induced toxicity, were analysis by Western blots. We found phosphorylated JNK and AKT showed no difference between Hepsin knockout and WT mice, but p-P38/P38 and p-ERK/ERK ratios were significantly upregulated at 2 and 4 hour after APAP injection, respectively. Besides, the concentration of serum AST, ALT were attenuated in knockout mice pre-injection with P38 inhibitor 1 hour before 4 hour 400 mg/kg APAP been challenged. We proposed that Hepsin may be involved in APAP-induced liver injury by regulating the activity of P38 kinase.

並列關鍵字

Hepsin knockout mice ； acetaminophen ； acute liver failure ； signaling pathway ； P38 kinase

參考文獻

1. Leytus, S.P., et al., A novel trypsin-like serine protease (hepsin) with a putative transmembrane domain expressed by human liver and hepatoma cells. Biochemistry, 1988. 27(3): p. 1067-74.

2. Tsuji, A., et al., Hepsin, a cell membrane-associated protease. Characterization, tissue distribution, and gene localization. J Biol Chem, 1991. 266(25): p. 16948-53.

3. Vu, T.K., et al., Identification and cloning of the membrane-associated serine protease, hepsin, from mouse preimplantation embryos. J Biol Chem, 1997. 272(50): p. 31315-20.

4. Somoza, J.R., et al., The structure of the extracellular region of human hepsin reveals a serine protease domain and a novel scavenger receptor cysteine-rich (SRCR) domain. Structure, 2003. 11(9): p. 1123-31.

5. Herter, S., et al., Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers. Biochem J, 2005. 390(Pt 1): p. 125-36.