Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice

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• 作者：Hong-Min Ni ; Mitchell R. McGill ; Xiaojuan Chao ; Kuo Du ; Jessica A. Williams ; Yuchao Xie ; Hartmut Jaeschke ; Wen-Xing Ding ; ^{wxding@kumc.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：AP ; autophagosome ; APAP ; acetaminophen ; APAP-AD ; acetaminophen protein adducts ; AMAP ; 3&prime ; -hydroxyacetanilide ; ALT ; alanine aminotransferase ; CQ ; chloroquine ; CYP2E1 ; cytochome P450 2E1 ; GSH ; glutathione ; HPLC-ED ; high pressure liquid chomatography with electrochemical detection ; LC3 ; microtubule-associated protein light chain 3 ; Leu ; leupeptin ; Ly ; lysosome ; NAPQI ; N-acetyl-p-benzoquinone imine ; mTOR ; mechanistic target of rapamycin ; NAC ; N-acetylcysteine ; PI ; propidium iodide
• 刊名：Journal of Hepatology
• 出版年：2016
• 出版时间：August 2016
• 年：2016
• 卷：65
• 期：2
• 页码：354-362
• 全文大小：3414 K

文摘

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown.

Methods

Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and high pressure liquid chomatography with electrochemical detection analysis.

Results

We found that APAP-AD were detected at 1 h, peaked at approximately 2 h, declined at 6 h and almost full removed at 24 h post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were colocalized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes.

Conclusions

Our data indicate that APAP-AD are removed though selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

Lay summary

Acetaminophen overdose can form acetaminophen protein adducts and mitochondria damage in hepatocytes resulting in liver injury. Activation of autophagy-lysosomal degradation pathway can help to remove acetaminophen protein adducts. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.