Inhibition of FOXO3a/BIM signaling pathway contributes to the protective effect of salvianolic acid A against cerebral ischemia/reperfusion injury

Abstract

Salvianolic acid A (SalA) is an effective compound extracted from traditional Chinese medicine Salvia miltiorrhiza Bunge. The Forkhead box O3a (FOXO3a) signaling pathway plays crucial roles in the modulation of ischemia-induced cell apoptosis. However, no information about the regulatory effect of SalA on FoxO3a is available. To explore the anti-cerebral ischemia effect and clarify the therapeutic mechanism of SalA, SH-SY5Y cells and Sprague-Dawley rats were applied, which were exposed to oxygen glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion/reperfusion (MCAO/R) injuries, respectively. The involved pathway was identified using the specific inhibitor LY294002. Results showed that SalA concentration-dependently inhibited OGD/R injury triggered cell viability loss. SalA reduced cerebral infarction, lowered brain edema, improved neurological function, and inhibited neuron apoptosis in MCAO/R rats, which were attenuated by the treatment of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) specific inhibitor LY294002. SalA time- and concentration-dependently upregulated the phosphorylation levels of protein kinase B (AKT) and its downstream protein FOXO3a. Moreover, the nuclear translocation of FOXO3a was inhibited by SalA both in vivo and in vitro, which was also reversed by LY294002. The above results indicated that SalA fought against ischemia/reperfusion damage at least partially via the AKT/FOXO3a/BIM pathway.

Keywords: BIM; FOXO3a; Ischemia reperfusion; Neuroprotection; Salvianolic acid A.

Graphical abstract

Figure 1

Chemical structure of SalA (A) and its protective effect against OGD/R-induced SH-SY5Y cells viability loss (B) and (C). Cell morphology was obtained by an inverted microscope. Data was expressed as mean±SD of 4 independent tests. ^###P<0.001 compared with the control group, ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with the OGD/R group.

Figure 2

SalA increased AKT and FOXO3a phosphorylation, inhibited BIM and cleaved caspase 3 expression in OGD/R-injured SH-SY5Y cells. (A) The p-FOXO3a/FOXO3a ratio. (B) The p-AKT/AKT ratio. (C) The p-mTOR/mTOR ratio. (D) The p-GSK3β/ GSK3β ratio. (E) The expression of BIM. (F) The expression of cleaved caspase 3. Proteins were analyzed using Western blot method normalized to GAPDH. Data was expressed as mean ± SD of 4 independent tests. ^#P<0.05 and ^###P<0.001 compared with control group, ^**P<0.01 and ^***P<0.001 compared with OGD/R group.

Figure 3

SalA increased the phosphorylation levels of AKT and FOXO3a in a time- and concentration-dependent manner. (A) The levels of p-FOXO3a, FOXO3a, p-AKT and AKT in SH-SY5Y cells treated without or with SalA (5 μmol/L) for 60, 120, 240 and 360 min. (B) The p-FOXO3a/FOXO3a ratio in SH-SY5Y cells treated without or with SalA (5 μmol/L) for 60, 120, 240 and 360 min. (C) The p-AKT/AKT ratio in SH-SY5Y cells treated without or with SalA (5 μmol/L) for 60, 120, 240 and 360 min. (D) The levels of p-FOXO3a, FOXO3a, p-AKT and AKT in SH-SY5Y cells treated without or with SalA (0.05, 0.5, 5 and 50 μmol/L) for 240 min. (E) The p-FOXO3a/FOXO3a ratio in SH-SY5Y cells treated without or with SalA (0.05, 0.5, 5 and 50 μmol/L) for 240 min. (F) The p-AKT/AKT ratio in SH-SY5Y cells treated without or with SalA (0.05, 0.5, 5 and 50 μmol/L) for 240 min. Proteins were analyzed using Western blot method normalized to GAPDH. Data was expressed as mean ± SD of 4 independent tests. ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with the control group.

Figure 4

LY294002 reversed SalA-induced cell protective effect, and the AKT and FOXO3a phosphorylation effect in vitro. Cells were pre-treated with LY294002 (10 μmol/L) before OGD/R stimulation and SalA treatment. (A) Effects of LY294002 on the protective effects of SalA by MTT assay. (B) The p-AKT/AKT ratio in SH-SY5Y cells treated without or with LY294002 by Western blot assay. (C) The p-FOXO3a/FOXO3a ratio in SH-SY5Y cells treated without or with LY294002 by Western blot assay. (D) The expression and intracellular localization of FOXO3a (green fluorescence) were assessed by immunofluorescence assay. (E) and (F) The nuclear FOXO3a, cytosolic FOXO3a and BIM levels treated without or with FOXO3a siRNA by Western blot assay. Data was expressed as mean ± SD of 4 independent tests. ^##P<0.01 and ^###P<0.001 compared with control group, ^**P<0.01 and ^***P<0.001 compared with OGD/R group, ^{^^^}P<0.001 compared with OGD/R+SalA group. Scale bar=20 μm.

Figure 5

SalA inhibited MCAO/R injury induced FOXO3a nuclear translocation and upregulated AKT phosphorylation in rat brain cortex and hippocampus. (A) The levels of p-FOXO3a, FOXO3a, p-AKT and AKT in rat brain cortex treated by SalA without or with LY294002. (B) The p-FOXO3a/FOXO3a ratio in rat brain cortex treated by SalA without or with LY294002. (C) The p-AKT/AKT ratio in rat brain cortex treated by SalA without or with LY294002. (D) The levels of p-FOXO3a, FOXO3a, p-AKT and AKT in rat brain hippocampus treated by SalA without or with LY294002. (E) The p-FoxO3a/FoxO3a ratio in rat brain hippocampus treated by SalA without or with LY294002. (F) The p-AKT/AKT ratio in rat brain hippocampus treated by SalA without or with LY294002. (G) The expression and localization of FOXO3a (green fluorescence) and NeuN (red fluorescence) in rat brain cortex treated by SalA without or with LY294002 by immunofluorescence assay. (H) The expression and localization of FOXO3a (green fluorescence) and NeuN (red fluorescence) in rat brain hippocampus treated by SalA without or with LY294002 by immunofluorescence assay. Data was expressed as mean±SD of 4 independent tests. ^##P<0.01 and ^###P<0.001 compared with sham group, ^**P<0.01 and ^***P<0.001 compared with I/R group, ^{^^}P<0.01 compared with I/R+SalA group. Scale bar=50 μm.

Figure 6

SalA inhibited BIM and cleaved caspase 3 expression in rat brain cortex and hippocampus subjected to MCAO/R injury. (A) The expression of BIM in rat brain cortex treated by SalA. (B) The expression of cleaved caspase 3 in rat brain cortex treated by SalA. (C) The expression of BIM in rat brain hippocampus treated by SalA. (D) The expression of cleaved caspase 3 in rat brain hippocampus treated by SalA. Proteins were analyzed by Western blot assay normalized to GAPDH. Data was expressed as mean ± SD of 4 independent tests. ^###P<0.001 compared with sham group, ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with I/R group.

Figure 7

SalA reduced infarct volume, neurological deficit scores, and inhibited neuron and endothelial cells apoptosis in MCAO/R rats. (A) TUNEL and NeuN staining of cortex region. (B) The percent of TUNEL+NeuN positive cells of total neurons in cortex region. (C) TUNEL and NeuN staining of hippocampus region. (D) The percent of TUNEL+NeuN positive cells of total neurons in the hippocampus region. (E) TUNEL and GLUT1 staining of ischemic penumbra region. (F) The percent of TUNEL+GLUT1 positive cells of total GLUT1 positive cells in the ischemic penumbra region. (G) TTC staining of brain slices. (H) Percent of infarct volume calculated according to the thickness of brain slice and the area of infarct region by TTC staining (n=12). (I) Neurological deficit scores (n=18). ^###P<0.001 compared with sham group, ^***P<0.001 compared with I/R group, ^{^^}P<0.01 and ^{^^^}P<0.001 compared with I/R+SalA group. Scale bar=50 μm.