Neuroprotective Effect of Danhong Injection on Cerebral Ischemia-Reperfusion Injury in Rats by Activation of the PI3K-Akt Pathway

Abstract

Many traditional Chinese medicines, including Danhong injection (DHI), can be used to treat cerebral ischemia-reperfusion injury and have neuroprotective effects on the brain; however, few studies have explored the mechanism by which this effect is generated. In this study, we investigated the neuroprotective effect of DHI against cerebral ischemia-reperfusion injury mediated via the PI3K-Akt signaling pathway. After establishing the model of middle cerebral artery occlusion (MCAO), 60 male Sprague-Dawley rats were allocated to six groups as follows: sham, MCAO, DHI (MCAO + DHI), LY294002 (MCAO + LY294002 [PI3K-Akt pathway specific inhibitor]), DHI + LY294002 (MCAO + DHI + LY294002), and NMDP + LY294002 (MCAO + NMDP [nimodipine] + LY294002). Hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to evaluate the pathological changes of brain tissue and the degree of neuronal apoptosis. Real-time quantitative polymerase chain reaction (qRT-PCR), western blot analysis and enzyme-linked immunosorbent assays were used to measure the expression of Bad, Bax, Bcl-2, Bim, P53, MDM2, Akt, PI3K, p-Akt, p-PI3K, and Cyt-C. Compared with the MCAO group, brain tissue cell apoptosis was significantly reduced in the DHI group, and the brain function score was significantly improved. In addition, the expression of pro-apoptotic factors (Bad, Bax, and Bim) was significantly downregulated in the DHI group, while expression of the anti-apoptotic factor Bcl-2 was significantly upregulated, and expression of the apoptotic gene p53 was also significantly attenuated. Moreover, this neuroprotective effect was attenuated by the PI3K-Akt signaling pathway inhibitor (LY294002). Thus, our results confirmed the neuroprotective effects of DHI in rats with ischemia-reperfusion injury and indicate that these effects on the brain are partly generated by activation of the PI3K-Akt signaling pathway.

Keywords: Danhong injection; PI3K-Akt pathway; apoptosis; ischemia-reperfusion; neuroprotection.

FIGURE 1

The chemical structures of five major components in DHI.

FIGURE 2

The neurological scores of rats in each group at 2, 24, 48, and 72 h after cerebral ischemia-reperfusion injury (n = 10). Compared with the Sham group, ^▲p < 0.05; compared with the MCAO group, *p < 0.05; compared with the LY294002 group, ^#p < 0.05. Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 3

The cerebral infarct volume of rats after cerebral ischemia-reperfusion injury by MCAO with TTC staining. (A) Results of TTC staining in each group. (B) Statistical results of cerebral infarct volume in each group (n = 3). Compared with the sham group, ^▲▲p < 0.01; compared with the MCAO group, **p < 0.01; compared with the LY294002 group, ^#p < 0.05. Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 4

Histopathological changes in hippocampus tissues of rats in each group were observed by HE staining. (A) The hippocampus tissues of each group rats at 200-fold magnification after HE staining, and necrotic cells indicated by black tips. (B) The hippocampus tissues of each group rats at 200-fold magnification after HE staining, and necrotic cells indicated by black tips (n = 3). Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 5

Apoptosis of brain tissue of rats in each group after TUNEL staining. (A) The apoptosis of rat brain tissue after TUNEL staining was observed under 200-fold magnification. (B) The apoptosis of rat brain tissue after TUNEL staining was observed under 400-fold magnification (n = 3). Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 6

The serum levels of rat Cyt-C and rat MDM2 of each group. (A) The expression level of cytochrome-c. (B) The expression level of MDM2 (n = 10). Compared with the sham operation group, ^▲p < 0.05; compared with the MCAO group, *p < 0.05, **p < 0.01; compared with the LY294002 group, ^#p < 0.05, ^##p < 0.01. Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 7

The protein strip diagram and protein expressions level of PI3K-Akt pathway related proteins in each group. (A) Band diagrams of p-Akt, p-PI3K, Akt, and PI3K in brain tissue of each group of rats determined by western blot. (B) The expression of p-Akt in brain tissue of each group rats determined by western blot. (C) The expression of p-PI3K in brain tissue of each group rats determined by western blot. (D) The expression of Akt in brain tissue of each group rats determined by western blot. (E) The expression of PI3K in brain tissue of each group rats determined by western blot (n = 4). Compared with the sham operation group, ^▲▲p < 0.01; compared with the MCAO group, *p < 0.05, **p < 0.01; compared with the LY294002 group, ^#p < 0.05, ^##p < 0.01. Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 8

The protein strip diagram and protein expressions level of apoptosis-related factors in each group. (A) Band diagrams of Bad, Bim, Bax, and Bcl-2 in brain tissue of each group of rats determined by western blot. (B) The expression of Bad in brain tissue of each group rats determined by western blot. (C) The expression of Bim in brain tissue of each group rats determined by western blot. (D) The expression of Bax in brain tissue of each group rats determined by western blot. (E) The expression of Bcl-2 in brain tissue of each group rats determined by western blot (n = 4). Compared with the sham operation group, ^▲▲p < 0.01; compared with the MCAO group, *p < 0.05, **p < 0.01; compared with the LY294002 group, ^##p < 0.01. Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.

FIGURE 9

The p53 mRNA expression level in each group determined by qRT-PCR (n = 4). Compared with the sham operation group, ^▲▲p < 0.01; compared with the MCAO group, *p < 0.05, **p < 0.01; compared with the LY294002 group, ^##p < 0.01. Sham, sham operation group; MCAO, middle cerebral artery occlusion; LY294002, 2-(4-morpholinyl)-8-phenyl-chromone; DHI, Danhong Injection; NMDP, nimodipine.