RETRACTED ARTICLE: Amygdalin reduces lipopolysaccharide-induced chronic liver injury in rats by down-regulating PI3K/AKT, JAK2/STAT3 and NF-κB signalling pathways

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article:  Yang Yang, Jie Zhao, Xiaoqin Song, Lifeng Li, Fuqin Li, Jia Shang & Wei-Wei Wang (2019) Amygdalin reduces lipopolysaccharide-induced chronic liver injury in rats by down-regulating PI3K/AKT, JAK2/STAT3 and NF-κB signalling pathways. Artificial Cells, Nanomedicine, and Biotechnology, 47:1, 2688–2697, DOI: https://doi.org/10.1080/21691401.2019.1634084 It has come to our attention that the full authorship and affiliations for this manuscript were changed entirely since submission. We have contacted the authors for an explanation, and they responded to all of our queries on the matter, but they were unable to provide a satisfactory explanation. The authors apologise for this situation. As determining authorship is core to the integrity of published work, we are therefore retracting the article. The corresponding author listed in this publication has been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions.  The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’. 


Introduction
Chronic liver injury (CLI) is a severe complication commonly occurred in liver diseases, which causes approximately 80-90% dysfunction of liver cells [1,2]. Patients with CLI are prone to hepatic encephalopathy and suffer from impaired ability to produce protein. A wide range of symptoms may be manifested with CLI, from cerebral oedema and multiorgan dysfunction, which may lead to coma or even death [3]. About 60% of all CLI patients developed systemic inflammation regardless of their infection status [4], which are prone to end up with failure of multiple organs. Although the pathogenic aetiology remains elusive, such systemic multi-organ failure may result from a sequence of unfavourable conditions, such as inflammation induced by oxidative stress and apoptosis.
The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway are highly implicated in the mechanism underlying inflammatory response to tissue injury [5]. The SOCS3, also known as suppressor of cytokine signalling 3, was found to inhibit the activation of JAK2/STAT3 pathway during nephropathic lesion complicated with diabetes [6]. It is reasonable to infer that SOCS3 dysregulation and JAK2/STAT3 activation play an important role in diabetes-induced tissue injury. However, no specific experiments about hepatic JAK2/STAT3 signalling pathway and SOCS3 and tissue inhibitors of metalloproteinase-1 (TIMP-1), which is dysregulated in metabolic syndrome and associated with macro and microvascular complications in patients, expression are displayed in rat models of liver dysfunction [6,7]. Lipopolysaccharide (LPS), a product derived from the cell wall of Gram-negative bacteria, was demonstrated to elicit serious global problem of sepsis and activate response of immunological system [8,9]. The uncontrolled response of the immune system to LPS could result in septic shock and systemic inflammatory [10], by which, it has been used for establishing injury model in liver, lung and kidneys, etc. [11][12][13]. In addition, inflammatory cells, cytokines and chemical mediators are highly implicated in pathological mechanisms underlying liver injury directly induced by LPS [14]. For instance, Kupffer cells have an abundant expression of nuclear factor kappa B (NF-jB), a type of transcription factor that could dominate the transcription of a bunch of inflammatory genes, such as TNF-a [15]. Endotoxin could activate NF-jB, which prompt us to hypothesize that targeting NF-jB in Kupffer cells may relieve the systemic damages by alcohol lipid disease [16,17]. Therefore, we sought to identify the link between LPS-induced injury and NF-jB signalling pathway.
Amygdalin (AG), a natural product derived from the aromatic amino acid phenylalanine, has a lot of cell-protective functions in many cell lines [18][19][20]. Previous studies have suggested that AG has multiple pharmacological effects, including anticancer, antibiotic, and anti-inflammation activities [21,22]. Recent studies [23] have reported that AG markedly improved glucose tolerance, and ameliorates insulin action in obese and/or diabetic patients by activating a signalling pathway related to glucose metabolism. In addition, a large body of evidence demonstrates that AG plays an important role in enhancing insulin sensitivity and reducing hyperlipidaemia [24]. Therefore, it is reasonable to speculate that this mechanism has the potential to treat acute liver injury in patients. However, the underlying mechanism of AG on improving liver injury or dysfunction remains unclear. Therefore, further study is needed to elucidate the underlying metabolism.

Animal model and treatment
A total of 60 male Sprague-Dawley rats (180-220 g) were purchased from the Experimental Animal Center of Nanjing Medical University. The establishment of animal model was conducted as previously described [25]. Rats were raised at room temperature under 55 ± 5% humidity and a normal 12h light/12-h dark cycle with the lights on, giving standard chow and water randomly. After one week of acclimatization, rats were subject to LPS or AG administration. All procedures were designed to minimize the suffering of the animal and decrease the number of animals used and strictly complied with the China Council on Animal Care. Rats were deprived of food overnight. LPS was purchased from Sigma-Aldrich, St. Louis, MO and prepared in PBS at a concentration of 10 g/ml. AG was purchased from National Drug Research Institute, Beijing, China and prepared in PBS at a concentration of 50 mg/Kg. Rats were randomly divided into five groups as follows: (1) Control group; (2) 2.5 mg/Kg LPS; (3) 2.5 mg/Kg LPS þ 0.5 mg/Kg AG; (4) 2.5 mg/Kg LPS þ 1.0 mg/Kg AG and (5) 2.5 mg/Kg LPS þ 1.5 mg/Kg AG. After 1 week of adaption, rats were orally treated with AG every other day for 60 d. From the 30th day, 2.5 mg/Kg LPS was administered by a single i.p. injection every 3 d for remaining 30 d. Citrate buffer was administered in normal control animal. Before blood collection, rats were deprived of food overnight. Blood was sampled from the tail of rats, which was stored in pre-ice tubes. Serum was derived by applying 4000Âg to centrifugation machine under 4 C for 6 min. Liver tissues were quickly resected from the rats and weighed for further calculation. All serum and tissue samples were stored at liquid nitrogen for subsequent experiments.

Biochemical assay
The serum content of metabolic enzymes such as alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT) and endotoxin (LPS) serves as an indicator of liver damage. Therefore, the above enzymes were determined to evaluate hepatic function. Quantification of enzymes was performed using common biochemical kits (Nanjing Jiancheng Biotech, Nanjing, China).

Histological assessment of the liver
After fixation in 10% neutral buffered formalin, the rat liver tissues were then embedded in paraffin. Liver histological assessment was performed as previously described [26]. A rotary microtome was employed to cut the samples into 7 lm thick sections, which were then mounted on APES coated glass slides. After deparaffinization in xylene and rehydration gradually in water containing decreasing concentrations of alcohol, the haematoxylin-eosin reagent was used to stain the specimens. Each slide was evaluated by histopathologist blinded to the functional status of the liver. The sections were investigated by light microscope.

Measurement of pro-inflammatory cytokines
After weighing of liver samples, a mixture of protease inhibitors was added to the samples. The mixture is composed of 0.002% sodium azide, 1.0 mg/ml BSA, 2 mM PMSF, 10 nM EDTA, and 0.1 mg/ml soybean trypsin inhibitor, dissolved in isotonic PBS (pH 7.0). Tissues were homogenized and then lysed to release the pro-inflammatory cytokines. The lysates were incubated at 4 C for 3 h. Centrifugation at 10,000Âg for 10 min was applied twice to extract the supernatant for protein quantification. ELISA kits (R&D Systems, Minneapolis, MN) was used to quantify IL-1b, IL-6, TNF-a, IL-1, IL-8 and IL-18 levels in the liver according to the manufacturer's manuals [27].

Real-time quantitative PCR (RT-qPCR) analysis
Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from each rat liver according to manufacturer's manual. mRNA expressions of IL-1b, IL-6, TNF-a, IL-1, IL-8 and IL-18, PI3K, AKT, m-TOR, SOCS3, TIMP-1, STAT3, IKKb, IjBa, and JAK2 were evaluated by RT-qPCR. Briefly, SuperScript First-Strand Synthesis kit (RT-PCR; Invitrogen, Carlsbad, CA) was used to reversely transcribe the cDNA. Primers were designed and synthesized by Biogenes Biotechnology, and carefully checked in GenBank to ensure low similarities in between. SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) was added to the PCR mixtures and then subject to programmed PCR cycles. The fluorescence produced during the PCR reaction was read by BioRad iCycler iQ Detection System. In parallel with RT-PCR of target genes, rGAPDH levels were also quantified as an internal control. Normalization and fold change for each of the genes were R E T R A C T E D calculated using the 2 ÀDDCt method. Primer sequences are displayed in Table 1.

Western blot
Frozen rat liver tissues were firstly homogenized in 1.5 ml RIPA buffer and the mixture was then centrifuged at 12,000 rpm for 15 min. Bradford method was applied to measure the protein level, before incubation in boiling water for 5 min. SDS-PAGE electrophoresis was performed on the total protein, which was then transferred electrophoretically to polyvinylidene difluoride (PVDF) membrane. The background protein was blocked by 5% skimmed milk for 3 h and primary antibodies were added to the membrane to incubate overnight at 4 C. HRP-conjugated goat anti-rabbit IgG (1:5000) was added to the immunoreactive bands. Immunoreactive bands were visualized using phototopehorseradish peroxidase Western blot detection system and quantified by densitometry using Molecular Analyst Software. Primary antibodies used were rabbit polyclonal antibodies against rPI3K (

Statistical analysis
Numerical data were presented as the mean ± standard error of the mean deviation (SEM). One-way analysis of variance (ANOVA) was performed to detect the difference of multi comparison, and Student-Newman-Keul's test was then employed to assess the difference between two treatments. A p-value <.05 was considered significant. GraphPad Prism 4 (GraphPad Software, Inc. San Diego, CA) was used to derive statistical graphs.

Results
Effects of amygdalin on body weight, relative liver weight and endotoxin levels of serum and liver in LPS-induced chronic liver injury rats Body weight, relative liver weight and endotoxin concentration in liver and serum are shown in Figure 1. In LPS-induced liver injury rats, body weights were decreased, while relative liver weight, endotoxin levels of liver and serum were significantly increased than the normal control group. Oral administration of AG demonstrated antagonizing effect against LPS, which significantly increased body weight and decreased liver weight, endotoxin levels in a dose-dependent manner in LPS-treated rats.

Effects of amygdalin on hepatic steatosis and alleviates the hepatic injury in LPS-induced rats
As summarized in Figures 1 and 2, the relative weight of the liver, steatosis and inflammation scores were significantly elevated in the LPS-treated rats compared to the normal control group ( Figure 2). Moreover, LPS-treated rats developed liver injury as indicated by increased serum and tissues levels of ALT, AST and ALP (Figure 3(A-F)), which were restored by 0.5, 1.0 and 1.5 mg/kg AG. The increased cell proliferation, a-SMA expression and ECM production were major events occur during phenotypic alterations in response to liver damage [28] and staining of a-SMA and collagen proteins were routinely measured to indicate liver injury [10,29]. Thus, a-SMA and COL1A1, a product of ECM, were measured to further confirm the successful establishment of liver injury rat model as well as the alleviation of AG on the hepatic injury. Quantification showed that COL1A1 and a-SMA expression were significantly elevated in LPS-induced rats, and immunostaining of them suggests fibrosis in LPS-induced rats (Figure 3(G-I)), which was restored by AG administration, indicating that liver injury was successfully established. These data suggest that pretreatment of AG effectively prevented these changes in LPSinduced liver injury rats.

R E T R A C T E D
Effects of amygdalin on the PI3K/AKT and JAK2/STAT3 pathway in LPS-induced liver injury The PI3K/AKT and JAK2/STAT3 pathway are suggested to be an essential pathogenic mechanism leading to liver dysfunction [30]. From the results of our study, the q-PCR ( Figure 5) and western blot (Figure 6(A-G)) analysis demonstrated that the expression levels of liver PI3K (p < .01), AKT (p < .01), m-TOR (p < .05), SOCS3 (p < .01), TIMP-1 (p < .01), STAT3 (p < .01) and JAK2 (p < .01) were elevated in LPS-treated rats. We found that this over-expression protein of liver PI3K, AKT, m-TOR, TIMP-1, STAT3 and JAK2 in LPS-treated rats was suppressed remarkably by the treatment of 1.5 mg/kg AG

Effects of amygdalin on the NF-jB pathway in LPS-induced liver injury
In LPS administrated inflammatory responses, activation of NF-LP pathway could be significantly observed in many animals model. Thus, in this part, IKKb, IjBa, p-NF-jB, IL-1b and IL-18 protein levels were analyzed to determine the role of NF-jB pathway in LPS-induced liver injury and protective effects of AG on inhibition of activation of NF-jB. mRNA quantification showed that LPS injection could enhance the up-regulation of NF-jB pathway related IKKb and IjBa in tissue, suggesting the NF-jB pathway was significantly activated under LPS administration in liver injury (Figure 7). Consistently, protein quantification further indicated that LPS could up-regulate the protein levels expression of the NF-jB pathway (Figure 8(A-E)). Of note, AG treatment has the ability to suppress activation of NF-jB, thereby inhibiting pro-inflammatory cytokines including IL-18 and IL-1b production. AG inhibits the NF-jB pathway in a doseindependent manner.

Discussion
AG is a natural compound which was originally extracted from herbs, which has been reported to possess pharmacological properties in reducing glucose levels and antimicrobial activities [31,32]. Given the antibiotic function of AG reported in previous studies, we postulated that it might alter whole body inflammation response to ameliorate the liver dysregulation and improve inflammatory response for the LPSinduced liver injury by direct and/or indirect regulation of NF-rec JAK2/STAT3 and PI3K/AKT and signalling pathway in tissues. In this study, we showed that AG relieved liver inflammation response by regulating key factors implicated in the onset of liver injury. Many types of research have indicated that PI3K/AKT signalling pathway is responsible for inflammatory responses in diabetic mice, regulating insulin sensitivity, obesity as well as diabetes [30,33]. In accordance with a previous study [2], our study demonstrated the elevation of protein expression of PI3K/AKT signalling pathway in LPS-induced rat liver, affecting NF-jB signalling pathway significantly and resulting in changes of cell transcription that finally develops hepatic dysfunction. These results were similar to elevation of hepatic AST, ALT and ALP, which further indicated liver injury in LPS-induced rats. Moreover, this study

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suggests that in rats with liver injury, phosphorylated JAK2 could activate the transcription factor STAT3 [34,35]. In parallel with JAK2/STAT3 activation, SOCS3 was overexpressed in LPS-induced rats. SOCS3 is an intracellular negative regulator of JAK2/STAT3 pathway. We found that JAK2, STAT3 and SOCS3 were up-regulated in the liver of LPS-induced liver injury rats, indicating JAK2/STAT3 plays an important role in liver injury during the progression of LPS administration. AG was shown to alter PI3K, AKT, JAK2, STAT3, TIMP-1 and SOCS3 protein levels as well as serum and hepatic ALP, ALT and AST levels in LPS-induced rat liver injury.

R E T R A C T E D
The molecular mechanisms underlying resistance to apoptosis have long been implicating PI3K/AKT signalling and related pathways [36]. The over-activation of PI3K/AKT signalling has been found in focus tissues in patients affected by various diseases and phosphorylated AKT could stimulate the expression of apoptosis-related proteins, such as Bax and Caspase-9, and necrosis factor NF-jB, together with leading to initiation of resistance to apoptosis [37,38]. Despite the role of LPS in inducing liver injury remains elusive, the interactions between LPS activated molecules and apoptosisrelated proteins amount to a postulation indicating the possible sequence of biological events. In this study, we demonstrated a reduced expression of PI3K and phosphorylated AKT in AG-treated cells, suggesting that AG exerted an inhibitory effect on AKT activation. Although further studies may be warranted to elucidate the detailed biological events, the inhibition of AG may be functional in promoting the restoration of LPS-induced liver injury.
Deep into the molecular level, LPS treatment significantly increased liver levels of IL-1b, TNF-a and IL-6 in the liver of rats. Considering the essential role of TNF-a in liver injury and IL-6, involving substantial inflammation responses, we supposed that promotion of cytokines production, such as IL-1b and IL-6, as a sign of overactive inflammation responses may result from LPS treatment, which preliminarily establishes the link between LPS and liver injury [39,40]. Furthermore, AG has been reported as an activator of NF-jB Figure 7. Amygdalin inhibits NF-jB signalling pathways indicators expression in LPS-induced liver injury. q-PCR analysis for the mRNA levels expression of IKKb and IjBa expression. These results are expressed as mean ± SEM, n ¼ 10-12. ÃÃ p < .01 compared to control group, #p < .05 and ##p < 0.01 compared to LPS group.

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translocation by binding to the promoter sites of NF-jB thereby triggering activation of TNF-a transcription [41]. Of note, these results were recovered by the administration of AG, which down-regulated the expression of IKKb, IjBa, p-NF-jB, IL-1b and IL-18, as well as serum IL-6 and TNF-a, suggesting that amygdalin serves as a protective role in liver injury via its anti-inflammation effects of regulating NF-jB signalling pathway.
The present results showed that AG consumption reversed most of the pathological changes of the LPS-induced liver injury of rats. The beneficial effects of AG may be due to its strong anti-inflammatory abilities and improvement of hepatic dysfunction through inhibiting PI3K/AKT, JAK2/STAT3 and NF-jB signalling pathways. Therefore, AG possessed a significantly protective effect for CLI.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was supported by the National Natural Science Foundation of China [81701536].