Dual regulatory roles of HMGB1 in inflammatory reaction of chondrocyte cells and mice
Authors: Li Wenzhao
Wang Junjie
Huang Jun
Huang Xianzhe
Ding Muliang
Yan Mingming
Ni Jiangdong
Song Deye
Journal: Cell Cycle (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Col2a1; HMGB1; Osteoarthritis; apoptosis; autophagy
Affiliations: Orthopedics Department,  The Second Xiangya Hospital of Central South University , Changsha, China

Abstract:  Osteoarthritis (OA) is one of the most common bone diseasesas it is reported that the impact of knee osteoarthritis symptomatic form is estimated at 240/100,000 people per year. The inflammation of articular cartilageis thought to be the pathologic drive for development of this disease. HMGB1(high mobility group box-1), a regulatory factor for gene transcription, could stimulate inflammation response. However, theexact regulatory role of HMGB1 in the inflammation of articular cartilage still need to be elucidated. In the current study, we used Quantitative Real-Time PCR(Q-PCR) to detect them RNA levels of Collagen Type II Alpha 1(Col2a1), Aggrecan, MMP3(Matrix Metallopeptidase 3), MMP13, ADAMTs4 and ADAMTs5; Enzyme-Linked Immunosorbent Assay(ELISA) was used to detect the content of IL-1β and calpain protein; Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) assay and flow cytometryanalysis; Western blot and immunofluorescence assays were applied to assess the expression of HMGB1; Lastly autophagic activity was mainly verified by monodansylcadaverine (MDC) staining. Our data revealed that in the early stage of chondrocyte inflammation(3 and 6 h of LPS stimulation), cytosolic HMGB1 attenuated inflammation response by facilitating cell autophagy and preventing cell apoptosis. While in the late stage (24 and 48 h of LPS stimulation), the extracellular HMGB1 stimulated inflammation reaction and contributed to the cartilage destruction in OA. 

Retracted: No

PubPeer comments:
 Two of the images in Figure 2b are the same image, with only adjustments made to the contrast. Since the intra-cellular staining applied in the paper destroys the cells, these cannot have been the same cells observed at two different time points. Something has gone wrong here. I urge the authors to check their raw data. ![file] , 
 To whom it may concern. I want to apologize for the delaying response. Because of the bad situation caused by corona virus in China, I didnu2019t check the email very frequently. Right after I saw your comment, I contacted with Dr. Li (the first author of this paper) and checked the details with him. Reasons for the pictures you mentioned are as follows: Firstly, we did paste the wrong picture to Control group in figure 2. The red channel of the fluorescence microscope in our Lab has poor imaging effect (usually after the fluorescent interference of other channels), we usually use Photoshop to adjust the brightness of the image after collecting the image under the uniform exposure conditions. Because the primary chondrocytes were treated with LPS and observed for a long time, there are so many pictures. Additional, after the unified brightness adjustment, the number of pictures doubled, so we did not uniformly named them all, this directly led us in the later image layout to mistake the images without brightness adjustment in other groups for the Control group, which HMGB1 is lower expression there. We will actively contact the publishing house to correct mistakes, and thank you very much for your careful review. Fortunately, this error does not affect our overall conclusion, because immunofluorescence (IF) is used to indicate the location of target proteins in cells, which is consistent with the original purpose of LPS-induced HMGB1 expression displacement in this paper. We can also clearly see from the results that with the prolonging of LPS treatment time, the cell nucleus of primary chondrocytes gradually becomes larger and rounder, accompanied by the expression displacement of HMGB1, and finally we can detect the secretion of HMGB1 in the cell supernatant by ELISA. We are very positive about this test conclusion. Secondly, this study is a continuation of my previous study on the relationship between HMGB1 and autophagy in osteosarcoma, and also is a preliminary exploration of this relationship in OA. In a follow-up study of the project, we also found that HMGB1 may be a better OA inflammatory inducer than LPS, which can effectively promote macrophage pyroptosis induced by inflammatory microenvironment (not yet published). Finally, if youu2019re interested in the research of HMGB1 in osteosarcoma or in OA, you may want to read my article  PMID:22102692 and  PMID: 22301993 published on Cancer Research and Autophagy, respectively. Feel free to communicate with me if you have any questions, thanks again. We will show the original pictures at each time point as follows: ![file] ![file] (the Control group) The expression was still low after background correction , 
 #2 Dear Dr. Jun, Thank you for responding so quickly despite your bad situation. I understand how the wrong image might get uploaded. But I would urge you to scrutinize all of your raw data carefully, not just this one image. There is, for example, another image duplication in Figure 5 (shown below). Here the same image has again been uploaded twice but with slightly different cropping. To make matters more confusing however, some of the imaged cells seem to have changed color intensity (highlighted in lime) whereas others have not. I do not think a simple (green) channel adjustment can explain this one, however, as the green tinted cell in the middle of the image maintains its color across both images. ![file] , 
 Oh I ve never changed the original pictures. I exported all the original pictures from the lab, and listed them as follows. 2.3MB single pictureuff0cand 4.7MB merged Big file, couldn t uplowed here (should be smaller than 1MB). Maybe I could send you by email if required. ![file] ![file] , 
 Dear Dr. Jun, I am not sure I understand? What you are showing here are clearly the same set of cells. ![file] But according to the paper, they have been treated with LPS for 6 hours (upper panel), or for 24 hours (lower panel). Since the imaging process effectively destroys the cells (the TUNEL protcol normally requires PFA fixation and permeabilisation of the cell walls), it would be impossible for the *same cells* to be imaged after 6 hours *and* 24 hours. Something does not add up here. Are you maintaining that this is not an error of the wrong images having been uploaded? , 
 Sorry, I didn t I didn t notice the mistake before. Since it has been more than two years since the experiment was tested, and the master student in charge of the experiment also graduated last year, I have difficulty in verifying the details. However, according to my personal experience, this situation may be caused by the following operating errors: u2460u00a0The difference of spot quantity caused by focusing light oru00a0dark fluorescence spots. u2461u00a0random fluorescence quenching during the shooting process. u2462u00a0The culture plates at different time points under the same group were marked wrong,u00a0or picture recording error after taking (There were a large number of fluorescence staining experiments in this project, which may cause errors in recording). Our team has been actively contacting the publishing house to revise,u00a0Whatever the reason, I m sorry for the mistake and thanks for pointing this out. ,