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.

Keywords: FOXO3A; GSK-3β; NF-κB; cardiac dysfunction; inflammation injury; sepsis.

Figure 1

LPS induces inflammation injury and up‐regulates GSK‐3β in cardiomyocytes. A, B, CMs were treated with LPS (12 h) for different concentrations and stimulated with LPS (0.5 μg/mL) for different time. Western blot analysis for apoptosis‐related genes cleaved‐caspase3, Bim and Bcl‐2 expression (n = 3). C, qRT‐PCR analysis for the cytokines TNF‐α, IL‐1β, IL‐6 and iNOs (n = 3‐4). D, E, β‐catenin, GSK‐3β and p‐GSK‐3β (Y216) expression were measured by Western blot in CMs (n = 3). F, Immunofluorescence analysis for p‐GSK‐3β (Y216) and its location (n = 3). (Scale bar: 25 μm). *P < .05; **P < .01; or ***P < .001 and ****P < .0001 when compared with controls

Figure 2

Down‐regulation of GSK‐3β attenuates myocardial inflammatory injury in cardiomyocytes after LPS challenge. A, B, Relative β‐catenin expression was measured by Western blot in the CMs treated with GSK‐3β inhibitors LiCl (0‐50 mM) and CHIR‐99021 (10 μM) for 24 h (n = 3). C, qRT‐PCR analysis for pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and iNOs in CMs treated with LiCl (10 mM) and CHIR‐99021 (10 μM) in the presence of LPS (500 ng/mL) for 12 h (n = 3). D, E, TUNEL assay for apoptosis in CMs treated with LiCl (10 mM) and CHIR‐99021 (10 μM) in the presence of LPS (500 ng/mL) for 12 h. Nuclei were counterstained with the DNA‐intercalating dye Hoechst (blue). The lower panel was the percentage of TUNEL‐positive cells (n = 3). (Scale bar: 25 μm). F, Western blot for Bim and Bcl‐2 in CMs pre‐treated with LiCl (10 mM) for 12 h and followed by stimulation of LPS (500 ng/mL) for another 12 h. GAPDH was loaded as internal control (n = 3). *P < .05; **P < .01 and ****P < .0001 when compared with controls

Figure 3

Down‐regulation of GSK‐3β improves cardiac function and abrogates apoptosis in LPS‐treated rats. A, Schematic diagram for pharmacological inhibition of GSK‐3β. Rats were pre‐treated with LiCl (0‐200 mg/kg) for 3 d and then stimulated with LPS (4 mg/kg) for 6 h prior to analyse cardiac function and harvest heart tissue. B, The down‐regulation of GSK‐3β was measured by detecting the level of β‐catenin in CMs with Western blot. C, Echocardiography assays for left ventricular fractional shortening (%) and ejection fraction (%). D, Haematoxylin‐eosin (HE) staining for myocardial tissue (Scale bar: 50 μm). E, Wheat germ agglutinin staining (WGA) for myocardium (Scale bar: 25 μm; n = 5). F, TUNEL staining assay for apoptosis (n = 5; Scale bar: 75 μm). G, H, Western blot analysis for GSK‐3β, β‐catenin, FOXO3A, Bim and Bcl‐2. I, qRT‐PCR analysis for the cytokines TNF‐α, IL‐1β, IL‐6 and iNOs (n = 4). *P < .05; **P < .01; ***P < .001 and ****P < .0001 when compared with controls

Figure 4

β‐catenin mediates the effects of GSK‐3β on apoptosis of cardiomyocytes. A, B, qRT‐PCR and Western blot for expression level of β‐catenin (n = 3). C, Cell death in CMs was assessed by TUNEL staining (Scale bar: 25 μm). The right panel is the percentage of TUNEL‐positive cells (n = 3). D, E, Western blot analysis for Bcl‐2 and Bim under different conditions (n = 3). F, Cell death in CMs was assessed by TUNEL staining. (Scale bar: 25 μm). The right panel is the percentage of TUNEL‐positive cells (n = 3). *P < .05; **P < .01; ***P < .001 and ****P < .0001 when compared with controls

Figure 5

FOXO3A mediates the effects of GSK‐3β on the inflammation injury of cardiomyocytes. A, B, Immunoblots analysis for FOXO3A in CMs treated with LPS at different concentrations and time (n = 3). C, Western blot assay for FOXO3A in CMs transfected with β‐catenin for 24 h, followed by the treatment with LiCl (10 mM) or CHIR‐99021 (10 μM) for another 24 h (n = 3). D, Western blot for FOXO3A in CMs treated with TIC10 (10 μM) for 24 h (n = 3). E, Western blot for detecting the expression of Bcl‐2 and Bim. F, The mRNA expression level of pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and iNOs in CMs treated with LiCl (10 mM), CHIR‐99021 (10 μM) and TIC10 (10 μM) in the presence of LPS (500 ng/mL) for 24 h (n = 3), and apoptosis of the CMs was detected by (G) TUNEL staining. The right panel is the percentage of TUNEL‐positive cells (n = 3). (Scale bar: 25 μm). *P < .05; **P < .01; ***P < .001 and ****P < .0001 when compared with controls

Figure 6

FOXO3A knock‐down attenuates LPS‐induced myocardial inflammatory injury. A, B, qRT‐PCR and Western blot for FOXO3A in CMs transfected with siRNA for 48 h (n = 3). C, The mRNA levels of pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and iNOs were measured by qRT‐PCR in CMs transfected with FOXO3A siRNA and stimulated with LPS (500 ng/mL) for another 12 h (n = 3). D, TUNEL staining and E, F, qRT‐PCR and Western blot for Bcl‐2 and Bim of CMs pre‐treated with si‐FOXO3A for 24 h and challenged with or without LPS (500 ng/mL) for another 12 h (n = 3). (Scale bar: 25 μm). *P < .05; **P < .01; and ****P < .0001 when compared with controls

Figure 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