Retinoic acid alleviates the reduction of Akt and Bad phosphorylation and regulates Bcl-2 family protein interactions in animal models of ischemic stroke

Abstract

Ischemic stroke causes a lack of oxygen and glucose supply to brain, eventually leads to severe neurological disorders. Retinoic acid is a major metabolic product of vitamin A and has various biological effects. The PI3K-Akt signaling pathway is an important survival pathway in brain. Phosphorylated Akt is important in regulating survival and apoptosis. We examined whether retinoic acid has neuroprotective effects in stroke model by regulating Akt and its downstream protein, Bad. Moreover, we investigated the relationship between retinoic acid and Bcl-2 family protein interactions. Animals were intraperitoneally administered vehicle or retinoic acid (5 mg/kg) for four days before surgery and ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were performed 24 h after MCAO and cerebral cortical tissues were collected. Cresyl violet staining and TUNEL histochemistry were performed, Western blot and immunoprecipitation analysis were performed to elucidate the expression of various proteins. Retinoic acid reduced neurological deficits and histopathological changes, decreased the number of TUNEL-positive cells, and alleviated reduction of phospho-PDK1, phospho-Akt, and phospho-Bad expression caused by MCAO damage. Immunoprecipitation analysis showed that MCAO damage reduced the interaction between phospho-Bad and 14-3-3, which was attenuated by retinoic acid. Furthermore, retinoic acid mitigated the increase in Bcl-2/Bad and Bcl-xL/Bad binding levels and the reduction in Bcl-2/Bax and Bcl-xL/Bax binding levels caused by MCAO damage. Retinoic acid alleviated MCAO-induced increase of caspase-3 and cleaved caspase-3 expression. We demonstrate that retinoic acid prevented apoptosis against cerebral ischemia through phosphorylation of Akt and Bad, maintenance of phospho-Bad and 14-3-3 binding, and regulation of Bcl-2 family protein interactions. .

Fig 1. Retinoic acid improves neurobehavioral disorders caused by MCAO damage.

Neurological deficits scoring test (A), corner test (B), grip strength test (C), gross photographs (D), microscopic photographs of cresyl violet staining (F), and TUNEL staining (G) in vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. Retinoic acid improves neurological behavior deficits (A-C) and histopathological changes (D, F, G) in ischemic brain injury. Intact area were stained dark purple and ischemic area were not stained or stained light purple (D). Retinoic acid alleviated the increase in ischemic area due to MCAO damage. F represents microscopic photos of the square in photograph D. Arrows indicate damaged neurons with pyknotic nuclei, cytoplasmic vacuolation, and shrunken dendrites (F). Open arrows indicate the TUNEL-positive cells (G). Retinoic acid attenuated the increase in the number of damaged neurons and the number of TUNEL-positive cells caused by MCAO (E and G). Data (neurobehavioral test, n = 10; histopathological test, n = 5) are shown as the mean ± S.E.M. *p < 0.001, **p < 0.01 vs. vehicle + sham animals, #p < 0.001, ##p < 0.01 vs. vehicle + MCAO animals. Scale bar = 100 μm.

Fig 2. Retinoic acid alleviates decreases of phospho-Akt and phospho-Bad expression caused by MCAO damage.

Western blot analysis of phospho-PDK1, PDK1, phospho-Akt, Akt, phospho-Bad, and Bad in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. Phospho-PDK1, phospho-Akt, phospho-Bad expressions were decreased in vehicle + MCAO animals, retinoic acid treatment alleviated these decreases. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio, proteins intensity to β-actin intensity. Molecular weight (kDa) are depicted at right. Data (n = 5) are represented as mean ± S.E.M. *p < 0.001, **p < 0.01 vs. vehicle + sham animals, #p < 0.01 vs. vehicle + MCAO animals.

Fig 3. Retinoic acid attenuates reductions of phospho-Akt and phospho-Bad expression due to MCAO damage.

Immunohistochemical staining of phospho-Akt (A and C) and phospho-Bad (B and D) in cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. The positive cells of phospho-Akt and phospho-Bad were stained dark brown. Retinoic acid alleviated the decrease of phospho-Akt and phospho-Bad expression due to MCAO damage. The value of positive cells was expressed as a percentage of the number of positive cells to the number of total cells. Arrows indicate positive cells. Data (n = 5) are represented as mean ± S.E.M. *p < 0.001, **p < 0.01 vs. vehicle + sham animals, #p < 0.01 vs. vehicle + MCAO animals. Scale bar = 100 μm.

Fig 4. Retinoic acid alleviates decrease of phospho-Bad and 14-3-3 interaction caused by MCAO damage.

Western blot analysis of 14-3-3 (A and B) and immunoprecipitation analysis of phospho-Bad and 14-3-3 binding (C and D) in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. Retinoic acid attenuated the decrease of phospho-Bad and 14-3-3 interaction caused by MCAO damage. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio, proteins intensity to β-actin (B) or IgG (D) intensity. Molecular weights (kDa) are depicted at right. Data (n = 5) are represented as mean ± S.E.M. *p < 0.001, **p < 0.01, ***p < 0.05 vs. vehicle + sham animals, #p < 0.01, ##p < 0.05 vs. vehicle + MCAO animals.

Fig 5. Retinoic acid regulates Bcl-2 family protein interactions in MCAO animals.

Immunoprecipitation analysis of Bcl-2 and Bcl-xL to Bad (A and C) or Bax (B and D) in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. Retinoic acid alleviated the increase of Bcl-2/Bad and Bcl-xL/Bad binding and the decrease of Bcl/Bax and Bcl-xL and Bcl-xL/Bax caused by MCAO damage. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio of proteins intensity to IgG intensity. Molecular weights (kDa) are depicted at right. Data (n = 5) are represented as mean ± S.E.M. *p < 0.001, **p < 0.01 vs. vehicle + sham animals, #p < 0.01 vs. vehicle + MCAO animals.

Fig 6. Retinoic acid attenuates increases of caspase-3 and cleaved caspase-3 expression caused by MCAO damage.

Western blot analysis of caspase-3 and cleaved caspase-3 in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. Retinoic acid alleviated the increase of these proteins due to MCAO. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio of proteins intensity to β-actin intensity. Molecular weights (kDa) are depicted at right. Data (n = 5) are represented as mean ± S.E.M. *p < 0.001, **p < 0.01 vs. vehicle + sham animals, #p < 0.01 vs. vehicle + MCAO animals.

Fig 7. The neuroprotective mechanism of retinoic acid against MCAO damage.