探討熱休克蛋白Dnajb4/HLJ1在調節乙醯胺酚引發肝損傷的作用

Abstract

乙醯胺酚(APAP)是一種常見的藥物，適應症為鎮熱、解痛。處方建議劑量的乙醯胺酚會藉由肝臟進行代謝，一般不會有嚴重的副作用。然而當乙醯胺酚服用過量會對肝臟造成損傷，嚴重的話會發展成急性的肝衰竭。過量乙醯胺酚在進入體內後，會被肝臟的phase I酵素CYP2E1氧化代謝成高反應性的中間產物N-乙醯基對苯醌亞胺(NAPQI)，形成許多NAPQI-蛋白加合物(NAPQI-protein adduct)，進而使細胞面臨較大的氧化壓力和內質網壓力，最終導致急性的肝毒性與肝細胞壞死。目前臨床上對於乙醯胺酚造成的急性肝損傷，通常是給予穀胱甘肽(GSH)、N-乙醯半胱氨酸(NAC)等還原性物質，來減緩肝細胞受到的氧化壓力。儘管能夠對已經發生的肝損傷進行補救性治療，然而在臨床上仍無有效降低因為過量乙醯胺酚引起肝臟毒性的方法。過去的研究經常將肝臟損傷與熱休克蛋白一同提及，而HLJ1作為伴侶蛋白(chaperone)的一員，屬於熱休克蛋白40家族。我們實驗室長期研究HLJ1蛋白的生理功能，發現HLJ1基因剔除小鼠會因為內質網壓力導致脂肪代謝異常，並且對致肝癌劑更敏感，表明HLJ1在維持肝臟恆定與對抗環境壓力的生理功能中有重要的作用。然而HLJ1在肝臟中是如何調控乙醯胺酚引起的肝臟損傷，機制仍不清楚。 在本研究中，我們給予小鼠過量的乙醯胺酚刺激，發現相較於野生型小鼠，HLJ1基因剔除小鼠的肝臟損傷更為嚴重。除此之外，我們使用流體動力注射，在小鼠的肝臟成功表現HLJ1蛋白並給予乙醯胺酚刺激，結果發現在外表現HLJ1蛋白後，HLJ1基因剔除小鼠的肝臟損傷有明顯減輕的情形。為了瞭解乙醯胺酚在小鼠體內的代謝情形，我們建立了質譜儀的分析平台來檢測乙醯胺酚相關的代謝物，結果發現在HLJ1基因剔除小鼠的肝臟中產生較多的phase I代謝物，表示NAPQI-蛋白加合物較多。接著利用RT-qPCR分析肝臟中phase I和phase II酵素相關基因的表現量，並測量了肝臟中穀胱甘肽的含量與氧化還原狀態，發現野生型小鼠和HLJ1基因剔除小鼠的酵素基因表現量與穀胱甘肽都沒有顯著差異，顯示代謝產物量的差異不是因為HLJ1調控了代謝酵素基因所導致。為了釐清HLJ1調控肝臟損傷的機制，使用兩個cDNA array的資料庫來比對找出可能與HLJ1有互動的蛋白質，透過PEPPI的蛋白質交互作用預測，鎖定了HSPa1b (HSP70/72)可能會與HLJ1結合，來調控並緩解肝臟損傷。我們進一步使用免疫沉澱法確認了HLJ1和HSP70的結合。透過西方墨點法確認小鼠肝臟中的內質網壓力(ER stress)，發現原本就會因為乙醯胺酚上升的內質網壓力，在HLJ1缺失、無法與HSP70結合的情況下，內質網壓力會升高的更多，進而使肝臟細胞較容易死亡，造成肝臟損傷。 總而言之，HLJ1可以與HSP70結合，協助NAPQI-蛋白加合物的重新摺疊或降解、調控細胞緩解內質網壓力，進而使肝臟緩解過量乙醯胺酚造成的毒性。本研究結果透過了解HLJ1在肝臟損傷中所扮演的角色，希望可以作為未來發展潛在保肝藥物的依據，本研究的最終目標希望可以應用在肝臟保護藥物的開發上，藉由調控HLJ1的表現量或是與HSP70結合的相關機制，來達到保護肝臟的效果。

Acetaminophen (APAP) is a commonly used pain and fever medication that is generally safe when taken at recommended doses. However, excessive intake can lead to severe liver toxicity. Following APAP intake, it is metabolized by the CYP2E1 enzyme into the toxic metabolite NAPQI. Excessive APAP consumption leads to an overproduction of NAPQI, and the NAPQI-protein adducts causing increased ER stress and oxidative stress in hepatocytes and consequently resulting in liver damage. Despite administering reducing agents such as glutathione (GSH) and N-acetylcysteine (NAC) is effective in therapeutic treatment for existing liver injury, there is no clinically available strategy to reduce the hepatotoxicity of overdosing APAP effectively. Previous studies reported the association of heat shock proteins (HSPs) with liver damage, such as hepatic ischemia-reperfusion injury or APAP-induced liver injury. However, the function of HLJ1, a molecular chaperone from the HSP40 family, in regulating APAP-induced acute liver injury remains uncertain. This study examines the effect of HLJ1 on APAP-induced liver injury using HLJ1 whole-body knockout (Hlj1-/-) mice. Following APAP administration, Hlj1-/- mice exhibited more severe liver damage. Additionally, we successfully re-expressed HLJ1 protein in the liver of mice through hydrodynamic injection, and this effectively mitigates liver damage caused by APAP in Hlj1-/- mice. We established a mass spectrometry platform to examine APAP-related metabolites, and the results revealed that Hlj1-/- mice generated more phase I metabolite of APAP, indicating more NAPQI-protein adducts were generated. We further analyzed APAP-related metabolic enzymes using RT-qPCR and measured liver glutathione levels. However, the results showed no significant differences in the mRNA expression levels of the enzymes and the glutathione redox state, suggesting that the difference in APAP metabolites is not due to HLJ1 regulating metabolic enzymes. To elucidate the mechanism of how HLJ1 regulates liver injury, we compared two cDNA array datasets to identify potential proteins interacting with HLJ1. HSPa1b (HSP70/72) was identified by the PEPPI online server as a potential HLJ1 binding partner. We further confirmed the HLJ1 and HSP70 interaction by immunoprecipitation. Western blot analysis of ER stress markers in mouse livers revealed that APAP-induced ER stress increased even more in the absence of HLJ1, which prevents its binding with HSP70, leading to higher ER stress and greater susceptibility of liver cells to death and damage. In summary, HLJ1 can bind with HSP70 to regulate cellular ER stress response, thereby mitigating APAP-induced liver toxicity. Understanding HLJ1's role in liver injury offers a basis for developing potential liver-protective drugs. The ultimate goal of our study is to develop a liver-protective drug that reduces liver damage by modulating HLJ1 expression or its interaction with HSP70, thereby achieving liver protection.