Gut microbiota mediates diurnal variation of acetaminophen induced acute liver injury in mice
Authors: Tian Lan
Zhanguo Liu
Bernd Schnabl
Shuwen Liu
Neil Kaplowitz
Liyan Zeng
Rui Li
Na Li
Xiaojiao Chen
Peng Chen
Yong Jiang
Hongwei Zhou
Sanda Win
Xiaoyu Yang
Shenhai Gong
Haihua Luo
Journal: Journal of hepatology (2018)
Discipline: CLINICAL MEDICINE
Keywords: Acetaminophen; Acute liver injury; Gut microbiota; Metabolomics.
Affiliations: State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China; Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China; Department of Pathophysiology, Southern Medical University, Guangzhou, China., Guangdong Pharmaceutical University, Guangzhou, China., School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China., State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China., Department of Intensive Care Unit, Zhujiang Hospital, Southern Medical University, Guangzhou, China., University of Southern California Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, USA., State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China; School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China., Department of Medicine, University of California San Diego, La Jolla, USA., State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China; Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China; Department of Pathophysiology, Southern Medical University, Guangzhou, China. Electronic address: ., State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China; Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China; Department of Pathophysiology, Southern Medical University, Guangzhou, China; Department of Intensive Care Unit, Zhujiang Hospital, Southern Medical University, Guangzhou, China. Electronic address: .

Abstract:  Background &amp; aims: Acetaminophen (APAP) induced hepatotoxicity is a leading cause of acute liver failure worldwide. It is well established that the liver damage induced by acetaminophen exhibits diurnal variation. However, the detailed mechanism for the hepatotoxic variation is not clear. Herein, we aimed to determine the relative contributions of gut microbiota in modulating the diurnal variation of hepatotoxicity induced by APAP. Methods: Male Balb/C mice were treated with or without antibiotics and a single dose of orally administered APAP (300 mg/kg) at ZT0 (when the light is on-start of resting period) and ZT12 (when the light is off-start of active period). Results: In agreement with previous findings, hepatic injury was markedly enhanced at ZT12 compared with ZT0. Interestingly, upon antibiotic treatment, ZT12 displayed a protective effect against APAP hepatotoxicity similar to ZT0. Moreover, mice that received the cecal content from ZT12 showed more severe liver damage than mice that received the cecal content from ZT0. 16S sequencing data revealed significant differences in the cecal content between ZT0 and ZT12 in the compositional level. Furthermore, metabolomic analysis showed that the gut microbial metabolites were also different between ZT0 and ZT12. Specifically, the level of 1-phenyl-1,2-propanedione (PPD) was significantly higher at ZT12 than ZT0. Treatment with PPD alone did not cause obvious liver damage. However, PPD synergistically enhanced APAP-induced hepatic injury in vivo and in vitro. Finally, we found Saccharomyces cerevisiae, which could reduce intestinal PPD levels, was able to markedly alleviate APAP-induced liver damage at ZT12. Conclusions: The gut microbial metabolite PPD was responsible, at least in part, for the diurnal variation of hepatotoxicity induced by APAP by decreasing glutathione levels. Lay summary: Acetaminophen (APAP) induced acute liver failure because of over dose is a leading public health problem. APAP-induced liver injury exhibits diurnal variation, specifically APAP causes more severe liver damage when taken at night compared with in the morning. Herein, we showed that gut microbial metabolite, 1-phenyl-1,2-propanedione is involved in the rhythmic hepatotoxicity induced by APAP, by depleting hepatic glutathione (an important antioxidant) levels. Our data suggest gut microbiota may be a potential target for reducing APAP-induced acute liver injury. 

Retracted: No

PubPeer comments:
 I have a concern about Fig. 5F. The number of GAPDH bands of PPD, APAP and PPD+APAP groups are all three. However, the number of GAPDH bands in control group is two. Besides, from the contrast figure, the lanes of detecting proteins are 12, but the lanes of GAPDH are 11. What happened here? ![file] ![file] , 
 ![file] Please also check this, all samples are marked in the blot. , 
 ![file] Thank you for your comment on our paper. You will see the bands of the blot for Gapdh is not that well separated, part of them are connected with each other, the bands in control gapdh had 3, the second one is between the red stick. Beside, you will see the total length of control group is similar to other group, if there are only 2 bands in control, each one band would be much wider than other single band, how it happen? So there are no problem for the sample number in the figure. But we appreciate your concern, thank you. , 
 #1 Here we have strong evidence to demonstrate Gapdh has 12 lanes, see below: ![file] These images are exactly same as we published but had longer exposure time. We can see Gapdh and APAP adducts are from same membrane, check the red circles that indicate the membrane feature. Beside, you can carefully check Gapdh blot, the second lane are marked as well. So, undoubtedly, these strong evidence demonstrate Gapdh and APAP adducts are from same membrane and thus, they both had 12 lane! , 
 Dear Dr. Chen, Thank you for posting the whole blots. The  dip in lane 2 of the GAPDH blot and the gap between lanes 11 and 12 in both blots are still intriguing, but I find these two original blots pretty convincing. By posting these blots, you were able to show that the published images correspond to the original blots without alteration. I don t have any concerns and I hope that user Platycheirus Stegnus feels the same way. Thank you for being so responsive and clear. Elisabeth Bik ,