Nobiletin Ameliorates NLRP3 Inflammasome-Mediated Inflammation Through Promoting Autophagy via the AMPK Pathway
Authors: Pei Jiang
Yujin Guo
Yi Qiao
Haidong Wang
Jun Zhang
Journal: Molecular Neurobiology (2020)
Discipline: NEUROSCIENCE & BEHAVIOR
Keywords: AMPK; Autophagy; Depression; NLRP3; Nobiletin.
Affiliations: Department of Pharmacy, The Affiliated Lianyungang Hospital of Xuzhou Medical University/The First People's Hospital of Lianyungang, Lianyungang, 222000, People's Republic of China., Affiliated Jining First People's Hospital of Jining Medical University, Jining Medical University, No. 6, Jiankang Road, Jining, 272000, Shandong, People's Republic of China., Department of Public Health, Jining Medical University, Jining, 272000, People's Republic of China., Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People's Republic of China., Affiliated Jining First People's Hospital of Jining Medical University, Jining Medical University, No. 6, Jiankang Road, Jining, 272000, Shandong, People's Republic of China. .

Abstract:  Multiple lines of evidence have shown that neuroinflammation and autophagy are highly involved in the process of depression. Nobiletin (NOB) displays neuroprotective effects and anti-depressant-like effects. Given the evidence that NOB exerts anti-inflammatory effects and regulates autophagy, we investigate the anti-neuroinflammatory properties and the effect of regulating the autophagy of NOB and subsequently uncover the potential anti-depressant mechanisms of NOB. The behavioral changes of rats were observed after prolonged lipopolysaccharide (LPS) treatment and NOB administration. Rat hippocampus and BV2 cells treated by LPS and NOB were evaluated. The methods of real-time PCR analysis, Western blot, immunostaining, and adenovirus transfection were employed to determine neuroinflammation, autophagic markers, and nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) activation. Our study showed LPS enhanced the expression of pro-inflammatory cytokines and NLRP3 inflammasome activation but inhibited autophagy in both rat hippocampus and BV2 cells. NOB significantly improved the behavioral deficits and ameliorated the neuroinflammation induced by LPS in rats. Furthermore, NOB promoted autophagy and attenuated NLRP3 inflammasome activation induced by LPS, involving in the process the adenosine monophosphate-activated protein kinase (AMPK) pathway. Neuroprotective and anti-depressant actions of NOB relied on its effects of promoting autophagy and suppressing the activation of NLRP3, in which process of AMPK pathway may be involved. 

Retracted: No

PubPeer comments:
 The same two blots appear both in the 2014 PhD thesis and in the 2015 Nature Plant paper, but one band has been replaced in the Nature Plant article. ![file] , 
 Two tables presented in the PhD thesis and in the Nature Plant paper seem to display the result of a single experiment, if you consider the numerous identical mean values to the tenth of a mm. The legends, however, state different plant numbers (25 in the thesis, 10 in the paper). Further analysis show that in the thesis, data is shown as mean and standard error, whereas in the paper it is given as mean and standard deviation. The standard error of the mean is usually estimated as the sample standard deviation divided by the square root of the sample size. Thus, because we have both SE and SD, we can go back to sample size, which in several instances in this table are problematic. For example for the Col0 Col0 case, sample size for rosette radius and number of rosette leaves is in fact around 10, but for plant height, computed sample size is only 6, which is less than 10 and not equal to 25 either. The same computation reveals sample size incoherence in all cases marked in red. The red box indicates the only condition for which the means are different in the thesis and in the paper, likely indicating a separate experiment. It is concerning, in this case, that the average plant height has doubled for the same condition in the data presented in the paper. Also, there is a mismatch between radius (paper figure) and diameter (paper and thesis legend). ![file] , 
 Quantitative RT-PCR results shown in this paper in figure 3B are significantly different to the same experiment presented in the author s PhD thesis. Both claim that presented results are means of 3 biological samples, so probably present the same dataset. The presented levels are relative to Col0 Col0 grafts which are set to 1, but then the relative levels for kao1kao2 kao1kao2 are 8 (thesis) vs 3.5 (paper) for ga20ox1 3 (thesis) vs 5 (paper) for ga3ox1 1 (thesis) vs 1.3 (paper) for ga2ox1 Could the authors comment on these differences in qRT-PCR results? ![file] , 
 #1 I am Patrick Achard, the corresponding author of this article. Thomas Regnault and I checked the original blots and indeed the contrast of a band appears different. A handling mistake likely occurred during figure preparation. I will inform the journal about this issue for correction. The original conclusions are unaffected; results were confirmed in independent experiments. #2 There is an error in the legends of the table in the thesis: the sample size was n 7 and not n = 25 as indicated. Moreover, in reanalyzing the data, we noticed that some of the values reported in the table of the article are incorrect (the conclusions are unaffected). During the project, several series of grafts were performed to score the morphological parameters. Thus, several tables were generated containing intermediate (such as reported in the thesis) and complete dataset. The mistake has been made in mixing these tables. The journal will be informed about this error for correction. We apologize for not having detected these errors before. #3 Experiments and way of representation are different. In the thesis is shown the technical variability of a single experiment while in the article is shown the biological variability of three independent experiments. , 
 Please reconsider this reply and provide raw data points that account for both the single experiment described in the thesis and the replicates not shown but mentioned in the thesis and advertised in the paper. ![file] ,