Guhong Injection Protects Against Apoptosis in Cerebral Ischemia by Maintaining Cerebral Microvasculature and Mitochondrial Integrity Through the PI3K/AKT Pathway

Abstract

Guhong injection (GHI) can be used for the treatment of ischemic stroke. We investigated the antiapoptotic activity of GHI, its ability to repair the cerebral microvessels and mitochondria, and the PI3K/AKT signaling pathway of GHI against cerebral ischemia. Western blot and immunohistochemical analyses were used to determine the expression of cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), cytochrome c (Cyt-c), basic fibroblast growth factor (BFGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and proteins in the PI3K/AKT signaling pathway. Transmission electron microscopy and scanning electron microscopy were used to evaluate the structures of the cerebral microvasculature and cells. Hoechst 33342 staining was used to evaluate the nuclear morphology. FITC-AV/PI double staining was used to measure the antiapoptotic effects. The fluorescent dye JC-1 was used to measure mitochondrial membrane potential. The enzyme-linked immunosorbent assay (ELISA) was used to detect the activities of matrix metalloproteinase-9 (MMP-9). Biochemical assay kits were used to detect the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA). Compared with the middle cerebral artery occlusion (MCAO) group, there was decreased infarct volume and significantly improved neurological deficits in the GHI group. In addition, the expression of Bcl-2 was significantly upregulated, while the expression of Cyt-c, Bax, and cleaved caspase-3 was notably downregulated. GHI administration attenuated the pathological change and morphology of the cerebral microvasculature, and immunohistochemical staining indicated that the expressions of BFGF, VEGF, and TGF-β1 were significantly increased. The cell morphology, cell viability, cell nuclei characteristics, and mitochondrial morphology normalized following GHI treatment, which decreased the release of Cyt-c and the mitochondrial membrane potential. The levels of LDH, MMP-9, and MDA decreased, while SOD increased. Moreover, GHI administration inhibited the activation of the PI3K/AKT signaling pathway in rat brain microvascular endothelial cells (rBMECs) following oxygen/glucose deprivation (OGD) injury. Therefore, our results show that GHI administration resulted in antiapoptosis of cerebral cells and repair of cerebral microvessels and mitochondria via the PI3K/AKT signaling pathway.

Keywords: Guhong injection; PI3K/Akt pathway; antiapoptosis; cerebral microvascular; ischemic stroke; mitochondria.

FIGURE 1

Effect of GHI on neurological deficit scores in focal cerebral I/R rats. Groups were as follows: Sham group (Sham), ischemia-reperfusion group (I/R group), GHI 2.5 ml/kg group, GHI 5 ml/kg group, and GHI 10 ml/kg group at 1 and 7 days, respectively. Values were expressed as mean ± SD (n = 8). ^## p < 0.01 vs. the Sham group, *p < 0.05, **p < 0.01 vs. the I/R group.

FIGURE 2

Effect of GHI on cerebral infarct volume in the focal cerebral I/R rats. Infarct volume was assessed by TTC staining at day 7 after tMCAO. (A) Representative TTC staining of the cerebral infarct of rat brain. (B) Infarct volumes assessed by TTC staining. Groups were as follows: Sham group (Sham), ischemia-reperfusion group (I/R group), GHI 2.5 ml/kg group, GHI 5 ml/kg group, and GHI 10 ml/kg group. The data were expressed as means ± SD (n = 8). ^## p < 0.01 vs. the Sham group, *p < 0.05, **p < 0.01 vs. the I/R group.

FIGURE 3

Western blot analysis of cleaved caspase-3, Bcl-2, Bax, and Cyt-c in the cerebral I/R region. Groups were as follows: Sham group (Sham), ischemia-reperfusion group (I/R group), GHI 2.5 ml/kg group, GHI 5 ml/kg group, and GHI 10 ml/kg group. ^## p < 0.01 vs. the Sham group, *p < 0.05, **p < 0.01 vs. the I/R group.

FIGURE 4

Ultrastructure of microvessels in the cerebral cortex. The transmission electron micrographs of the cerebral cortex are displayed in a1–a3; the scanning electron micrographs of the cerebral cortex are displayed in b1–b3. a1, b1: Sham group; a2, b2: I/R group; a3, b3: GHI 10 ml/kg + I/R group. E: perivascular edema; EC: endothelial cell; SA: swelling astroglial process; and V: venules (bar = 50 μm).

FIGURE 5

Expressions of BFGF, VEGF, and TGF-β1 immunostained tissue of focal cerebral I/R rats (magnification 200×). Groups were as follows: Sham group (Sham), ischemia-reperfusion group (I/R group), GHI 2.5 ml/kg group, GHI 5 ml/kg group, and GHI 10 ml/kg group.

FIGURE 6

Immunohistochemical analysis of BFGF, VEGF, and TGF-β1 cerebral I/R hippocampal CA1 region. Groups were as follows: Sham group (Sham), ischemia-reperfusion group (I/R group), GHI 2.5 ml/kg group, GHI 5 ml/kg group, and GHI 10 ml/kg group. The data were expressed as means ± SD (n = 3 rats per group). ^## p < 0.01 vs. the Sham group, *p < 0.05, **p < 0.01 vs. the I/R group.

FIGURE 7

Characterization of rBMECs under a microscope. (A) rBMECs at 2 d, number of microvascular segments that appeared like a string of beads, and the wall of the tube was smooth and clear, (B) confluent subcultured cells formed cobblestoned morphology, (C) staining by factor VIII of cells, and (D) negative control.

FIGURE 8

Effects of GHI on OGD-induced changes in cell morphology and viability (n = 6). (A) Control group, (B) OGD group, (C) GHI 25 μL/ml, (E) GHI 100 μL/ml, and (F) cell viability. ^## P < 0.01 compared with the Control group; *p < 0.05, **p < 0.01 compared with the OGD group.

FIGURE 9

Effects of GHI on histochemical characterizations of rBMECs in OGD injury (n = 6). Arrowheads in the pictures indicate the nuclei of apoptosis cells. (A) Control group, (B) OGD group, (C) GHI 25 μL/ml, (D) GHI 50 μL/ml, (E) GHI 100 μL/ml, and (F) integrated optical density. ^## P < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the OGD group.

FIGURE 10

Effects of GHI on apoptosis rates of rBMECs in OGD injury. Values were expressed as the means ± SD (n = 3). (A–E) Flow cytometry images and (F) statistical analysis of the apoptosis rate. ^# p < 0.01 compared with the control group; *p < 0.01 compared with the OGD group.

FIGURE 11

GHI treatment decreases the proportion of the JC-1 monomer of rBMECs under OGD injury (n = 3). (A–E) Flow cytometry images and (F) statistical analysis of JC-1. ^# p < 0.01 compared with the control group; *p < 0.01 compared with the OGD group.

FIGURE 12

Evaluation of the mitochondrial ultrastructure by electron microscopy (n = 3).

FIGURE 13

Effects of GHI on the release of Cyt-c from mitochondria after OGD-induced injury. GHI decreases the release of Cyt-c from mitochondria. Cyt-c (A,E,I), mitochondria (B,F,J), and DAPI (C,G,K) staining revealed an apparent maintained within the mitochondria under OGD conditions when treated with 100 μl/ml of GHI (I–L) compared to the without GHI. Cyt-c and mitochondria double-positive cells are shown in panels: (D,H,L). Red: Cyt-c, blue: DAPI, and green: mitochondria (n = 3).

FIGURE 14

Effects of GHI on the levels of LDH, MMP-9, SOD, and MDA in rBMECs after OGD-induced injury. The data were expressed as means ± SD (n = 8). ^# p < 0.01 compared with the Control group; *p < 0.05, **p < 0.01 compared with the OGD group.

FIGURE 15

GHI attenuated OGD-induced injury by regulating the PI3K/Akt pathway. (A) Representative immunoblots of Akt, p-Akt, Bax, Bcl-2, cleaved caspase-3, and caspase-3 proteins. (B) The ratio of p-Akt/Akt. (C) The ratio of Bcl-2/Bax. (D) The levels of caspase-3. (E) The levels of cleaved caspase-3. Data are shown as the mean ± SD (n = 3). ^# p < 0.05, ^## p < 0.01 compared with the Control group; ^* p < 0.05, ^** p < 0.01 compared with the OGD group, ^Δ p < 0.05, ^ΔΔ p < 0.01 compared with the GHI 100 μL/ml group.

FIGURE 16

Schematic illustration showing that Guhong injection protected antiapoptosis and the integrity of the cerebral microvasculature and mitochondria through the PI3K/Akt signaling pathways under cerebral ischemia.