Combined Use of Emodin and Ginsenoside Rb1 Exerts Synergistic Neuroprotection in Cerebral Ischemia/Reperfusion Rats

Abstract

Acute ischemic stroke (AIS) generally causes neurological dysfunction and poses a serious threat to public health. Here, we aimed to assess the independent and combined effects of ginsenoside Rb1 (GRb1) and Emodin on neuroprotection through regulating Connexin 43 (Cx43) and Aquaporin 4 (AQP4) expression in cerebral ischemia/reperfusion (I/R) model rats. Adult male Sprague-Dawley (SD) rats were randomly divided into five groups: sham group, I/R group, Emodin group, GRb1 group and Emodin+GRb1 group. They were further allocated to four subgroups according to the 6h, 1d, 3d, and 7d time points except the sham group. Based on the modified Longa suture method, the focal cerebral I/R model was established by middle cerebral artery occlusion (MCAO). The neurological deficit scores (NDS), blood brain barrier (BBB) permeability and cerebral infarction area were assessed at each corresponding time point. Cx43 and AQP4 levels were assessed by Real-time PCR and Immunofluorescence. Compared with I/R group, both the independent and combined use of GRb1 and Emodin could alleviate NDS, reduce the BBB permeability, reduce the infarction area and down-regulate Cx43 and AQP4 expression at 6h, 1d, 3d, and 7d after I/R (P < 0.05). The Emodin+GRb1 group had more significant effects than Emodin group and GRb1 group (P < 0.05). In conclusion, the combination of Emodin and GRb1 exerts synergistically neuroprotective functions through regulating AQP4 and Cx43 after I/R.

Keywords: Aquaporin 4; Connexin 43; cerebral ischemia/reperfusion; emodin; ginsenoside Rb1.

FIGURE 1

The neurological deficits in different groups after I/R in rats (mean ± SD, n = 20). NDS in Sham group, I/R group, GRb1 group, Emodin group, and Emodin+GRb1 group at 6h, 1d, 3d, and 7d. Kruskal–Wallis test followed by the Mann–Whitney U test. ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^#P < 0.05, ^●p < 0.01, compared with the Emodin+GRb1 group.

FIGURE 2

The EB content in different groups after I/R in rats (mean ± SD, n = 5). Evans blue leakage experiments for determining BBB permeability after I/R in rats in four sub-groups at 6 h, 1d, 3d, and 7d. One-way analysis of variance (ANOVA) was used for multiple group experiments comparisons and followed by Dunnett test for comparison of two groups within the multiple groups. ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^●p < 0.01, compared with the Emodin+GRb1 group.

FIGURE 3

The percentage of infarct area in different groups after I/R in rats (mean ± SD, n = 5). TTC staining was used to evaluate cerebral infarction area after I/R in rats in four sub-groups at (A) 6h, (B) 1d, (C) 3d, and (D) 7d. (E) Quantitative analysis for the results of (A–D). One-way analysis of variance (ANOVA) was used for multiple group experiments comparisons and followed by Dunnett test for comparison of two groups within the multiple groups. ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^●p < 0.01, compared with the Emodin+GRb1 group.

FIGURE 4

Immunofluorescence staining of AQP4 in I/R rats (mean ± SD, n = 5). (A–C) The expression of AQP4 in (A) cortex, (B) hippocampus, and (C) striatum with immunofluorescence staining. (D,E) Quantitative analysis for the results of (A–C). One-way analysis of variance (ANOVA) was used for multiple group experiments comparisons and followed by Dunnett test for comparison of two groups within the multiple groups. ^★p < 0.01, ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^●p < 0.01, ^■p < 0.001, compared with the Emodin+GRb1 group.

FIGURE 5

Immunofluorescence staining of Cx43 in I/R rSD, n = 5). (A–C) The expression of Cx43 in (A) cortex, (B) hippocampus, and (C) striatum with immunofluorescence staining. (D,E) Quantitative analysis for the results of (A–C). One-way analysis of variance (ANOVA) was used for multiple group experiments comparisons and followed by Dunnett test for comparison of two groups within the multiple groups. ^★p < 0.01, ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^●p < 0.01, ^■p < 0.001, compared with the Emodin+GRb1 group.

FIGURE 6

The AQP4 mRNA expression in different groups after I/R in rats (mean ± SD, n = 5). (A) Cortex; (B) hippocampus; (C) striatum. Quantitative analysis for the results of groups at 6 h, 24 h, 3d, and 7d, respectively. One-way analysis of variance (ANOVA) was used for multiple group experiments comparisons and followed by Dunnett test for comparison of two groups within the multiple groups. ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^●p < 0.01, compared with the Emodin+GRb1 group.

FIGURE 7

The Cx43 mRNA expression in different groups after I/R in rats (mean ± SD, n = 5). (A) Cortex; (B) hippocampus; (C) striatum. Quantitative analysis for the results of groups at 6 h, 24 h, 3d, and 7d, respectively. One-way analysis of variance (ANOVA) was used for multiple group experiments comparisons and followed by Dunnett test for comparison of two groups within the multiple groups. ^∗p < 0.05, compared with Sham group. ^∗p < 0.001, compared with Sham group. ^▲p < 0.05, ^△ p < 0.01, ^♦p < 0.001, compared with I/R group. ^●p < 0.01, compared with the Emodin+GRb1 group.