Fig 1.
Cerebral infarction (S1–S6) among the experimental groups after 30 min of MCAo followed by 7 d of reperfusion.
2,3,5-Triphenyltetrazolium chloride (TTC) staining shows normal brain tissue (deep red) and infarct tissue (white). Scale bar = 1 cm.
Fig 2.
Effects of P-FA, I-FA, and R-FA on cerebral ischemic areas and neurological behaviors 7 d after reperfusion.
(A) The percentage cerebral infarct areas in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups were evaluated 7 d after reperfusion (n = 6). (B) The neurological deficit scores among the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups were examined 1, 3, and 7 d after reperfusion. Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group.
Fig 3.
Effects of P-FA, I-FA, and R-FA on the cytosolic expression of p-JNK, JNK, p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-Akt, and Akt in the cortical penumbra.
(A) Representative Western blot images show cytosolic p-JNK, JNK, p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-Akt, and Akt expression in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups 7 d after reperfusion. The ratios of (B) p-JNK/JNK, (C) p-ERK/ERK, (D) p-p38 MAPK/p38 MAPK, and (E) p-Akt/Akt expression were calculated in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups (n = 5). cyto, cytosolic fraction. Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group.
Fig 4.
Effects of P-FA, I-FA, and R-FA on the cytosolic expression of HSP70, GFAP, p-p90RSK, and p-Bad in the cortical penumbra.
(A) Representative Western blot images show cytosolic HSP70, GFAP, p-p90RSK, and p-Bad expression in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups 7 d after reperfusion. Actin was used as a loading control in Western blot analysis. The ratios of (B) HSP70/actin, (C) GFAP/actin, (D) p-p90RSK/actin, and (E) p-Bad/actin expression were calculated in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups (n = 4–5). Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group.
Fig 5.
Effects of P-FA, I-FA, and R-FA on the cytosolic expression of p-CREB, CREB, Bcl-2, Bcl-xL, and Bax in the cortical penumbra.
(A) Representative Western blot images show cytosolic p-CREB, CREB, Bcl-2, Bcl-xL, and Bax expression in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups 7 d after reperfusion. The ratios of (B) p-CREB/CREB, (C) Bcl-2/actin, (D) Bcl-2/Bax, (E) Bcl-xL/actin, and (F) Bcl-xL/Bax expression were calculated in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups (n = 4–5). Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group.
Fig 6.
Effects of P-FA, I-FA, and R-FA on the mitochondrial expression of Bcl-2, Bcl-xL, AIF, and cytosolic expression of cleaved caspase-3 and AIF in the cortical penumbra.
(A) Representative Western blot images show mitochondrial Bcl-2, Bcl-xL, and AIF expression, and cytosolic cleaved caspase-3 and AIF expression in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups 7 d after reperfusion. HSP60 was used as a loading control in Western blot analysis. The ratios of (B) mitochondrial Bcl-2/Bax, (C) mitochondrial Bcl-xL/Bax, (D) mitochondrial Bax/HSP60, (E) mitochondrial AIF/HSP60, (F) cleaved caspase-3/actin, and (G) cytosolic AIF/actin expression were calculated in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups (n = 4–5). mito, mitochondrial fraction. Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group.
Fig 7.
Effects of P-FA, I-FA, and R-FA on the expression of cytochrome c and cleaved caspase-3 in the cortical penumbra.
Representative photographs show (A) cytochrome c and (B) cleaved caspase-3 expression in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups 7 d after reperfusion. (C) Representative photograph shows TTC-stained rat brain coronal section. The dotted line square indicates the region of evaluation of immunopositive cells. CP, cortical penumbra. Dotted line square = 1mm2. The bar graphs show the numbers of (D) cytochrome c- and (E) cleaved caspase-3-positive cells in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups (n = 3). Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group. Arrows in (A) and (B) point to cytochrome c- and cleaved caspase-3-positive cells, respectively. Scale bars equal 50 μm for (A) and (B).
Fig 8.
Effects of P-FA, I-FA, and R-FA on the expression of p-CREB/DAPI in the cortical penumbra.
Representative photographs show p-CREB (red) colocalizing with DAPI (blue) in the cortical penumbra in the Sham, Vehicle, B-FA, P-FA, I-FA, R-FA, and D-FA groups 7 d after reperfusion. Arrows point to p-CREB/DAPI double-labeled cells. Scale bars = 5 μm.
Fig 9.
Cerebral infarction among the D+Sham, D+Vehicle, D+I-FA, and SB+I-FA groups after 30 min of MCAo followed by 7 d of reperfusion.
(A) TTC staining shows normal brain tissues (S1–S6) (deep red) and infarct tissues (S1–S6) (white). Scale bar = 1 cm. (B) The percentage cerebral infarct areas in the D+Sham, D+Vehicle, D+I-FA, and SB+I-FA groups were evaluated 7 d after reperfusion (n = 3). Data are presented as mean ± SD. *P < 0.05 versus the Sham group; #P < 0.05 versus the Vehicle group.
Fig 10.
Effects of D+I-FA and SB+I-FA on the cytosolic expression of p-p38 MAPK, p38 MAPK, p-p90RSK, p-Bad, p-CREB, CREB, and Bcl-2/Bax.
(A) Representative images show cytosolic p-p38 MAPK, p38 MAPK, p-p90RSK, p-Bad, p-CREB, CREB, Bcl-2, and Bax expression in the cortical penumbra in the D+Sham, D+Vehicle, D+I-FA, and SB+I-FA groups 7 d after reperfusion. The ratios of (B) p-p38 MAPK/p38 MAPK, (C) p-p90RSK/actin, (D) p-Bad/actin, (E) p-CREB/CREB, and (F) Bcl-2/Bax were calculated in the cortical penumbra in the D+Sham, D+Vehicle, D+I-FA, and SB+I-FA groups (n = 5). Data are presented as mean ± SD. *P < 0.05 versus the D+Sham group; #P < 0.05 versus the D+Vehicle group.
Fig 11.
Effects of D+I-FA and SB+I-FA on the mitochondrial Bcl-2 and Bax, and cytosolic cleaved caspase-3 expression in the cortical penumbra.
(A) Representative images show mitochondrial Bcl-2 and Bax, and cytosolic cleaved caspase-3 expression in the cortical penumbra in the D+Sham, D+Vehicle, D+I-FA, and SB+I-FA groups 7 d after reperfusion. The ratios of (B) mitochondrial Bcl-2/Bax, (C) mitochondrial Bax/HSP60, and (D) cleaved caspase-3/actin were calculated in the cortical penumbra in the D+Sham, D+Vehicle, D+I-FA, and SB+I-FA groups (n = 5). Data are presented as mean ± SD. *P < 0.05 versus the D+Sham group; #P < 0.05 versus the D+Vehicle group.