N-Acetylcysteine and allopurinol up-regulated the Jak/STAT3 and PI3K/Akt pathways via adiponectin and attenuated myocardial postischemic injury in diabetes
Authors: Aimin Xu
Xiaowen Mao
Shigang Qiao
Paul M Vanhoutte
Junwen Wang
Kwok F J Ng
Shaoqing Lei
Haobo Li
Gordon T Wong
Zhengyuan Xia
Tingting Wang
Zipeng Liu
Michael G Irwin
Journal: Free Radical Biology and Medicine (2013)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: 15-F2t-IsoP; 15-F2t-isoprostane; ALP; APN; Adiponectin
Affiliations: Department of Anesthesiology, The University of Hong Kong, Hong Kong SAR, China.

Abstract:  N-Acetylcysteine (NAC) and allopurinol (ALP) synergistically reduce myocardial ischemia reperfusion (MI/R) injury in diabetes. However, the mechanism is unclear. We postulated that NAC and ALP attenuated diabetic MI/R injury by up-regulating phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathways subsequent to adiponectin (APN) activation. Control (C) or streptozotocin-induced diabetic rats (D) were untreated or treated with NAC and ALP followed by MI/R. D rats displayed larger infarct size accompanied by decreased phosphorylation of Akt, STAT3 and decreased cardiac nitric oxide (NO) and APN levels. NAC and ALP decreased MI/R injury in D rats, enhanced phosphorylation of Akt and STAT3, and increased NO and APN. High glucose and hypoxia/reoxygenation exposure induced cell death and Akt and STAT3 inactivation in cultured cardiomyocytes, which were prevented by NAC and ALP. The PI3K inhibitor wortmannin and Jak2 inhibitor AG490 abolished the protection of NAC and ALP. Similarly, APN restored posthypoxic Akt and STAT3 activation and decreased cell death in cardiomyocytes. Gene silencing with AdipoR2 siRNA or STAT3 siRNA but not AdipoR1 siRNA abolished the protection of NAC and ALP. In conclusion, NAC and ALP prevented diabetic MI/R injury through PI3K/Akt and Jak2/STAT3 and cardiac APN may serve as a mediator via AdipoR2 in this process. 

Retracted: No

PubPeer comments:
 Figure 1. * Red boxes: the left-most heart in panel A (4 weeks) looks unexpectedly similar to the left-most heart in panel B (9 weeks), if one of the two is rotated a bit.  Here is a close up of the two panels.  , 
 Dear Elizabeth. Yes I agree. I ll need to look into this and get back to you as it was some time ago. Thanks for bringing it to my attention. , 
 Dear Elizabeth: Thank you for pointing this out. This is paper was revised twice. And, I check out original revision 2 (FRBM-D-12-01208R2) submission figure file, and fond that the total set of the four heart tissue images for Fig. 1 A was totally different from that of the published. And the images for Fig. 1B were correct, same as our revision submission. We wanted to check on the journal s submission record, and fond that the manuscript title etc is there, and the content of the files were removed in accordance with the journal s Retention Policy. Another interesting thing about this paper is that: When the paper was finally in print, we noted that the email addresses for the two corresponding authors were mismatched. e.g., zyxia@hku.hk belongs to Z. Xia, but printed as: zyxia@hku.hk (M.G. Irwin), and mgirwin@hku.hk belongs to M.G Irwin, but printed as: mgirwin@hku.hk(Z. Xia). We assumed that this error will not affect much things, and thus we did not request for a correction etc. Likewise, the same (I think this way also) or the very similar images for the very left control group (i.e. no treatment, no ischemic intervention) basically shall not affect the findings conclusion of the study. Probably, we should also contact the journal for correction, in the issue, as it is different from the email mismatching issue? Your kind suggestion please. Thanks and best regards, Zhengyuan Xia , 
 Dear Michael, Yes, I had noticed the email name switch, but that was very minor. However, I am not sure about your explanation about Figure 1. Are you suggesting you sent the correct photo to the journal, but that the journal uploaded the wrong photo? It sure looks like your photo to me, so how did the journal make a mistake? Because the photo shows at least 1 heart tissue from your lab it seems you must have sent this photo to the journal. What exactly went wrong then? , 
 Dear Elisabeth, Thank you for pointing this out. We take this matter very seriously. We were able to retrieve the original files from the study and have determined how this mistake happened. A: After carefully reviewing the folder of our revised manuscript, there are two points we need to clarify: 1. The experimental procedures related to Fig 1A and 1B were completed by one of the co-authors (HL), who was responsible for the preparation of animal models in the laboratory, and the data statistics were jointly completed by HL and the first author TW to ensure the reliability of the results. As such, both of these authors have access to and storage of all the related images. 2. The original Fig 1A is shown below: However, the reviewer 1 raised a question: u201cIn figure 1A, the images of the heart are not very clear, which should be improved or enlargedu201d. So in the revised version, TW enlarged the images and gave the following response: u201cWe have enlarged the image of heart in the revised manuscriptu201d. After completion of the revision (including manuscript text, figures, and point-to-point response to reviewers) by the 1st author, corresponding authors (primarily the last author Z. Xia) checked and agreed the submission but advised the submitting author to have a double check before uploading files. However, the images for Fig 1A were actually not very clear. We guess this was why the reviewer suggested they should be improved or enlarged. In order to better improve the quality of the figure, the submitting author helped us to re-select the representative images with higher-quality images. Because they were both controls of non-diabetic rats, and the different ages (4-5 weeks vs. 9-10 weeks of age) of non-diabetic control rats were used to facilitate the comparison that corresponded to different duration (4 weeks vs. 9 weeks) of diabetic rats, we may conceptually considered that they were young untreated control rats anyway, and thus were not that alert. This might be the major reason why we mistakenly selected untreated control images and even used the same one as the representative image for two different groups, although we can not clearly recall why this happened. B: The error had no any impact on the results because 1.tThe images for 4 weeks or 9 weeks were separately stored in different file folders, and the data were analyzed by two different investigators. The error happened at the very last stage of choosing representative images. 2.tThe image we used twice belongs to the 9 week non-diabetic control rats, and all the images for Fig. 1B are correct. For Fig1A, we demonstrated that the STZ-diabetic model (4 weeks diabetes) subjected to 30 min coronary occlusion 2 h reperfusion didnu2019t result in a significant increase in IS when compared to that of control rats. This phenomenon was repeatedly confirmed by us. The result was consistent with our previous studies (Tingting Wang, et al. PLOS ONE 2011; PMID: PMID: 21912612; Xiaowen Mao, et al. PLOS ONE 2013; PMID: 23874823). Moreover, in Fig 2A of the paper concerned, we again proved that the myocardial IS between control (C group) and 4 week diabetic rats (D group) didnu2019t reach statistical significance. Thus, we are confident that the error of this picture does not affect the conclusion of the article. However, we shall write to the journal to clarify this and check if publication of a correction is needed. We will also acknowledge your assistance in pointing out this error. C: Why rotate the image in Fig. 1B? We seriously questioned and recalled why some (not just the very left one, but also the 2nd left one etc.) images were rotated in Fig. 1B. We figured out that we normally consider two things when we chose a representative heart infarct image. The major consideration is the representativity of the image. Another is that we wanted to chose the same or similar size of images to make the images line up well. And, rotation is actually not necessary or should be prevented in order to avoid the confusion of image manipulation. D: Why we initially assumed that it might be the publisher who made the error After we noticed the error as pointed out by PubPeer, we checked the revision file of the figures and found that the images of Fig. 1A were different from those published. We then went to the journal submission system and to determine whether or not we submitted the file with error or it was the publisher somehow made a mistake. We found that the submission files were removed due to journal retention policy (as least it is no longer available to authors). Later on, the submitting author was able to retrieve the actually uploaded figure files. Then, we realized it was our mistake due to an error at the very last stage. ,