GSK‐3β inhibition protects the rat heart from the lipopolysaccharide‐induced inflammation injury via suppressing FOXO3A activity

Abstract Sepsis‐induced cardiac dysfunction represents a main cause of death in intensive care units. Previous studies have indicated that GSK‐3β is involved in the modulation of sepsis. However, the signalling details of GSK‐3β regulation in endotoxin lipopolysaccharide (LPS)‐induced septic myocardial dysfunction are still unclear. Here, based on the rat septic myocardial injury model, we found that LPS could induce GSK‐3β phosphorylation at its active site (Y216) and up‐regulate FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK‐3β inhibitors and further reversed through β‐catenin knock‐down. This pharmacological inhibition of GSK‐3β attenuated the LPS‐induced cell injury via mediating β‐catenin signalling, which could be abolished by FOXO3A activation. In vivo, GSK‐3β suppression consistently improved cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines in LPS‐induced model was also blocked by inhibition of GSK‐3β, which curbed both ERK and NF‐κB pathways, and suppressed cardiomyocyte apoptosis via activating the AMP‐activated protein kinase (AMPK). Our results demonstrate that GSK‐3β inhibition attenuates myocardial injury induced by endotoxin that mediates the activation of FOXO3A, which suggests a potential target for the therapy of septic cardiac dysfunction.

myocardial and mitochondrial energy metabolism disorders and apoptosis. 5,6 GSK-3β is a multifunctional serine/threonine kinase involved in regulating cell fate and differentiation processes in a variety of organisms 7 and can exert a central role in diverse signalling pathways such as Wnt, Notch, Hedgehog, NF-κB and AMPK. 8,9 Recently, GSK-3β has been identified as a crucial regulator in modulation of inflammation, 10,11 in which the knock-down of GSK-3β diminishes LPS-associated pro-inflammatory cytokines and significantly reduces the mortality of human monocytes. 10 The best characterized upstream kinase that inactivates GSK-3β through phosphorylation at ser-9 is Akt, which is well established as a protective effector in sepsis. 12 Similar to GSK-3β, Akt also directly attenuates the functions of FOXO3A through phosphorylation modification, and its inhibition causes FOXO3A activation. 13 FOXO3A is a member of FOXO transcription factor family consisting of FOXO1, FOXO3, FOXO4 and FOXO6, and mainly participates in the transcriptional regulation of autophagy, inflammation, apoptosis and cell cycle arrest. [14][15][16] Increasing evidence indicates that FOXO3A modulates inflammation through regulating NF-κB, 17 but its role in endotoxin-induced myocardial injury still remains unclear. It has been showed that GSK-3β positively regulates hepatoma cell proliferation through the transactivation activity of FOXO3A, 18 how about the cross-talking between FOXO3A and GSK-3β in endotoxin-induced heart injury has not been explored yet. FOXO3A could trigger cell apoptosis through promoting the expression of pro-apoptotic transcription factors such as Bim, PUMA, 19 Tradd 20 and Mxi1-0. 21 Hereby, we hypothesize that GSK-3β inhibition may protect against endotoxin-induced myocardial injury via FOXO3Adependent mechanisms.
In the present work, we found that pharmacological inhibition of GSK-3β preserved cardiac ejection fraction (EF) and fractional shortening (FS) in LPS-treated rats. The inhibition of GSK-3β reduced LPSinduced apoptosis in cardiomyocytes. FOXO3A was identified to be positively regulated by GSK-3β through degrading β-catenin, and the knock-down of FOXO3A in cardiomyocytes with siRNAs further confirmed its positive role on sepsis-induced cardiomyocytes dysfunction. These data indicate that both GSK-3β and β-catenin participate in the pathogenesis of endotoxin-induced cardiac dysfunction via activation of FOXO3A.

| Animals
All the rats were purchased from Slaccas Company (Shanghai, China) and kept in the animal facility at Tongji University. All of the procedures were approved by Institutional Animal Care and Use Committee at Tongji University (Approval No: TJLAC-016-022).
All of animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.

| Endotoxemia model
All experiments were operated on male Sprague Dawley rats that weighed between 220 g and 250 g. Rats were randomly divided into four groups: sham group, endotoxin group, negative group and positive group. Endotoxemia was induced by intraperitoneal (ip) injection with 4 mg/kg LPS (Sigma). Negative controls or positive groups were pre-treated with 100 mg/kg NaCl (Sigma) or with 100 mg/kg LiCl (Sigma), respectively, 3 days before or after LPS injection. 6 hours after stimulation with LPS, cardiac functions were evaluated and samples from each group were collected. All rats were killed under deep anaesthesia with isoflurane (Gene&I).

| Quantitative real-time PCR
The total RNA was isolated from tissues or cells with TRIzol reagent (Invitrogen), and cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa) followed by a gene expression assay applying TB Green™ Premix Ex Taq™ (TaKaRa) on a Bio-Rad CFX Connect™ real-time system. The related primers were listed in Table S1.

| Immunofluorescence staining
Cells were seeded on glass slides in 48-well plate. After treatment, CMs were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature (RT) and then permeabilized with 0.1% triton X-100 before blocked with 10% goat serum for 1 hour. Primary antibodies (1:200) were incubated at 4°C overnight, following by fluorescent secondary antibodies (1:250) for 1 hour and Hoechst for 12 minutes at RT. Images were taken using Leica confocal microscope after blocking with aqueous mounting medium.

| TUNEL assay
CMs apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. After fixed and permeated, each sample was treated with 80 μL of TUNEL reaction mixture for 60 minutes at 37°C in dark, and then, the samples were incubated with Hoechst for 12 minutes at RT, following by rinsing with PBS three times, and finally imaged using Leica confocal microscope.

| Separation of nuclear and cytoplasmic protein
Nuclear and cytoplasmic proteins were separated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) according to the manufacturer's protocol. Proteinase inhibitors were added to each buffer immediately before use.
Cells were harvested by scraping and rinsing with ice-cold PBS, and then, the cells were lysed in ice-cold CER I buffer for 15 minutes, following by adding ice-cold CER II buffer and incubating on ice for 1 minute. Cell suspensions were centrifuged at 16 000 g for 5 minutes, and the supernatant containing the cytoplasmic proteins was collected for next experiments. The precipitation was resuspended with ice-cold NER buffer and incubated on ice for 40 minutes. The samples were centrifuged at 16 000 g for 10 minutes, and the nuclear protein was collected and stored at −80°C for further use.

| Echocardiography
After 6 hours of the treatment with LPS or saline through ip injection, rats were anaesthetized with isoflurane (3.0% induction in room air, followed with 0.5% maintenance in room air) and subjected to echocardiography using Vevo770 (Visual Sonics Inc) as previously described. 22 The M-mode images of left ventricular (LV) dimensions were obtained.
Echocardiography data were recorded and analysed individually.

| Wheat germ agglutinin staining
Cardiomyocyte size was evaluated using wheat germ agglutinin staining. The rat heart was fixed in 4% paraformaldehyde, and then, the frozen tissues were sectioned into 20 μm slides, rinsed with PBS and stained for cardiomyocyte membrane with FITC-conjugated wheat germ agglutinin (Sigma). Finally, the heart cross section was imaged with Leica confocal microscope.

| Statistical analysis
ANOVA test was used to compare among three or more groups, followed by Bonferroni's post hoc test. Student's t test was applied to compare two groups, and the error bar represented the standard error of mean (SEM). A value of P < .05 was considered significant.
All data were analysed using Prism 5.0 (GraphPad Software, Inc).

| LPS induces activation of GSK-3β in rat cardiomyocytes
Uncontrolled inflammation and apoptosis are two main features of endotoxin-induced cardiac dysfunction. 10,25 Here, we examined the apoptosis rate of CMs exposed to LPS at different concentra- showed a peak in the presence of 500 ng/mL LPS for 12 hours ( Figure 1D-F), which might be related to the potential role GSK-3β at the early stage of inflammation injury.

| GSK-3β inhibition attenuates LPS-induced cardiac inflammation injury
CHIR-99021 and LiCl were widely used as GSK-3β inhibitors in a variety of cell types. 10 We applied CHIR-99021 and LiCl to CMs to assess their potential inhibition on GSK-3β. β-catenin was used as an indicator of GSK-3β activity because β-catenin could be phos-

| GSK-3β inhibition improves cardiac function in vivo
To evaluate the in vivo role of GSK-3β, LPS (4 mg/kg) was intraperitoneally administrated into rats to induce septic myocardial dysfunction as previously described, 12 and LiCl was also applied  Figure 3A). The up-regulation of β-catenin in heart suggested an inhibition role on GSK-3β ( Figure 3B). Lipopolysaccharide significantly impaired heart function, including decrement ejection and shortening fraction, while GSK-3β inhibition could improve the EF by 13.85% and FS by 12.22% in the rats ( Figure 3C; Figure S1).
Myocardium damage assay demonstrated that GSK-3β inhibition not only attenuated the LPS-induced injury in hearts ( Figure 3D) but, meanwhile, could partially alleviate the change in the cell size in LPStreated CMs ( Figure 3E). However, no obvious effect on cardiac dimensions was observed in the condition ( Figure S2).
TUNEL staining confirmed that GSK-3β inhibition also reduced apoptotic cells in heart challenged with LPS ( Figure 3F). Consistent with experiments in vitro, LPS could increase the expression level of GSK-3β, FOXO3A and Bim, which could be reversed when exposed to LiCl ( Figure 3G,H). In addition, the GSK-3β inhibition suppressed

| Inhibition of β-catenin aggravates the LPSinduced cell apoptosis
β-catenin is the major downstream transcription factors and proteolyzed by the destruction complex containing GSK-3β. 10 To determine the role of β-catenin on the GSK-3β regulation in LPS-induced apoptosis of CMs, the specific siRNAs were transfected into CMs to inhibit β-catenin expression ( Figure 4A,B). We treated CMs with LPS, two siRNAs that targeted β-catenin, and Wnt/β-catenin inhibitors, XAV939 and ICG-001. The knock-down of β-catenin really attenuated the Bcl-2 and increased Bim expression after LPS exposure ( Figure 4D; Figure S4). Meanwhile, TUNEL-positive cells were also significantly increased under the condition ( Figure 4C; Figure   . Then, we exposed CMs to β-catenin siRNAs as treated with GSK-3β inhibitor, CHIR-99021. The results shown that the effect of GSK-3β inhibition on sepsis was prevented by the knock-down of βcatenin ( Figure 4E,F), indicating that β-catenin exerts an important role in GSK-3β related sepsis.

| GSK-3β inhibition decreases FOXO3A expression mediated by β-catenin
We and LiCl) in the presence and absence of the FOXO3A activator TIC10 and exposed them to LPS. We found that TIC10 really increased the FOXO3A level ( Figure 5D), with an up-regulation of inflammatory cytokines IL-1β, TNF-α and iNOs as well ( Figure 5F).
In addition, TUNEL staining and Western blot analysis for Bcl-2/ Bim showed that TIC10 significantly enhanced the apoptosis of the CMs ( Figure 5E,G), indicating that the FOXO3A as one of the target of the GSK-3β could regulate endotoxemia cardiac dysfunction which might attribute to its pro-apoptotic effect on the CMs.

| FOXO3A knock-down attenuates LPS-induced cardiac inflammation injury
To confirm the FOXO3A function in the LPS-induced sepsis in CMs, knock-down assay showed the expression level of FOXO3A was significantly decreased in the CMs transfected with the siRNA targeting FOXO3A for 48 hours ( Figure 6A,B), in which the LPS-induced up-regulation of IL-6, IL-1β, TNF-α and iNOs was suppressed ( Figure 6C). Meanwhile, FOXO3A knock-down also attenuated the activation of NF-κB ( Figure S6). These results suggest that the LPS-induced FOXO3A could regulate inflammation process in CMs. Moreover, the down-regulation of FOXO3A could decrease the LPS-induced apoptotic index ( Figure 6D), accompanying with an increase in Bcl-2 and a decrease in Bim. All of the data indicated that FOXO3A was associated with the endotoxin-induced myocardial apoptosis ( Figure 6E,F) and played a crucial role in the inflammation injury of the heart.

| GSK-3β inhibition negatively involved in the activations of ERK/NF-κB signalling and positively regulated AMPK pathway
IκBα is degraded upon phosphorylation which results in the consequent release and nuclear translocation of NF-κB. 27 When cells were stimulated with LPS, the degradation of IκBα induced an increase in NF-κB level in the nuclear fraction ( Figure 7A-C). However, GSK-3β inhibitors LiCl or CHIR-99021 increased the IκBα level and reduced nuclear NF-κB only under LPS condition ( Figure 7A-C). The results suggest that NF-κB is involved in the effect of GSK-3β on regulating inflammation injury in heart.
In addition, ERK and AMPK signalling are two important pathways activated by LPS in many cell types. ERK contributes to the induction of inflammation and apoptosis, whereas AMPK exerts reverse effects in the processes through cross-talking with NF-κB. 28 As shown in Figure 7D

| D ISCUSS I ON
In the current study, we showed that GSK-3β was associated with cardiac dysfunction during sepsis, and its inhibition attenuated the effects in ischaemia/reperfusion (I/R). 10,31 However, other studies show Wnt inhibitors Dickkopf-1, LGK974 and iCRT3 attenuate LPSinduced inflammatory response in different cells and lung injury. 32,33 These findings suggest that GSK-3β is an important regulator for the balance between pro-and anti-inflammatory cytokines. 26,34 In the work, inhibition of GSK-3β really reduced inflammatory mediators such as TNF-α, IL-1β, IL-6 and iNOs in CMs.
Apoptosis of CMs has been showed to contribute to inflammation injury in heart. 25 Using TUNEL assay, we demonstrated that LPS transcriptionally promote the expression of FOXO3A. 18 Here, we showed that GSK-3β inhibition decreased FOXO3A level, which was reversed by β-catenin knock-down, proving that GSK-3β could modulate FOXO3A through degrading β-catenin. This finding is consistent with previous report that β-catenin interacts with FOXO and enhances FOXO transcriptional activity in mammalian cells. 41 However, activation of Wnt/β-catenin represses murine liver oxidative stress-induced apoptosis by inhibiting FOXO3A through targeting SGK1. 42 In addition, FOXO3A could also regulate β-catenin transcriptional activity through competing with TCF. 43 These results mean that β-catenin modulation on FOXO3A shows cell-type specific with an animal model-dependent pattern. Furthermore, β-catenin has been showed to be also involved in down-regulating FOXO3A through promoting Akt activity, 44 which is consistent with our results. Actually, the roles of FOXO3A in heart injury so far still remain controversial. FOXO3A protects the heart from pathological hypertrophy and improves cardiac function in ischaemia-reperfusion and hypertensive cardiac injury models. 45 On the other hand, FOXO3A leads to organ injury via promoting cell apoptosis through transcriptional activation of pro-apoptosis genes, such as Bim, PUMA and Mxi1-0. 19 46 AMPK activation generally elaborates a potential anti-inflammatory effect in vitro and in vivo. 8 Like previous findings, 11 our study demonstrates that inhibition of GSK-3β activity via pharmacological inhibitors could reverse pro-inflammatory signalling through blocking NF-κB and ERK 1/2 pathways. In addition, GSK-3β inhibition could enhance AMPK signalling activity, resulting in a suppression on the LPS-induced heart injury.
In summary, our research shows that GSK-3β is excessively ac- F I G U R E 7 GSK-3β inhibition inversely correlates with activation of ERK and NF-κB pathways and positively related with activations of AMPK signalling. A, Western blot analysis for IKBα (n = 3). B, C, Western blot for nuclear proteins and immunofluorescence staining for CMs treated with GSK-3β inhibitors with or without LPS (n = 3). (Scale bar: 25 μm). Phosphorylation of GSK-3β, ERK1/2 and AMPK was measured by Western blot in CMs treated with GSK-3β inhibitors, D-F, LiCl (10 mM) or G-I, CHIR-99021 with or without LPS (n = 3). *P < .05; **P < .01; ***P < .001 and ****P < .0001 when compared with controls. J, Graphic summary. FOXO3A is transcriptionally activated by LPS in CMs through the activation of GSK-3β/β-catenin pathway and regulates LPS-induced heart damage by targeting Bim and NF-κB

CO N FLI C T O F I NTE R E S T
The authors confirm that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.