Synergism of salvianolic acid B and ginsenoside Rg1 magnifies the therapeutic potency against ischemic stroke

Abstract

Even though considerable progress has been made to reduce insult, ischemic stroke is still a significant cause of mortality and morbidity in the world, and new therapeutic strategies are urgently needed. In the present study, the magnesium salt of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) combination as a multicomponent strategy against stroke was evaluated. The synergistic effect of Sa1B and Rg1 was evaluated by Bliss independence analysis on the middle cerebral artery occlusion model. The infarct volume, neuroethology, cerebral structure, and neurocyte number were evaluated by 3,5-triphenyltetrazolium chloride staining, Longa score, Garcia score, hematoxylin-eosin staining, and Nissl staining, respectively. Metabolomics was used to search for potential biomarkers and explore the mechanism of Sa1B/Rg1. First, the superior effects of SalB/Rg1 than SalB or Rg1 at the same dose were evaluated. Compared with SalB ( P < 0.001) or Rg1 ( P < 0.01), SalB/Rg1 significantly decreased infarct volume through 3,5-triphenyltetrazolium chloride staining and protected the structural integrity of cortex and striatum. The superior effect of SalB/Rg1 on neurological behavior was also detected compared with SalB or Rg1 significantly. Accompanying behavioral improvement, a considerable increase of SalB/Rg1 on neurons detected by Nissl staining was found on the cortex compared with SalB ( P < 0.05) or Rg1 ( P < 0.01). Second, the synergistic effect between SalB and Rg1 was strictly verified by Bliss independence analysis ( P < 0.01) based on infarct volume. Finally, alleviation of cerebral metabolic disorders may be the possible mechanism of SalB/Rg1. Our study provided a multicomponent strategy against ischemic stroke, with not only dose reduction but also improved efficacy relative to single agents.

Graphical abstract

Fig. 1

SalB/Rg1 significantly reduced infarct volume and improved neuronal structure comparing SalB or Rg1 at the same dose. (a) Comparison among SalB/Rg1, SalB, and Rg1 at the same dose. (b) Representative pictures of the brain with TTC stain. (c) Quantification of cerebral infarct volume with TTC stain. (d) Representative hematoxylin–eosin staining images from whole rat brain (up) and subregions of the cortex (up) and striatum (down) in rats. Data were expressed with the mean ± SEM. ***P < 0.001 versus sham group; ###P < 0.001 versus MCAO group; &&&P < 0.001 versus SalB group; $$$P < 0.001 versus Rg1 group. Scale bar: 50 μm. MCAO, middle cerebral artery occlusion; SalB/Rg1, salvianolic acid B/ginsenoside Rg1; TTC, 3,5-triphenyltetrazolium chloride.

Fig. 2

Superior efficiency of SalB/Rg1 than SalB or Rg1 treatment at the same dose on neurological behavior and neurocyte protection at the same dose. (a) Garcia score. (b) Longa score. (c) Representative images of Nissl staining. (d) Quantitative results on neurocytes at the cortex (left) and striatum (right). Data were expressed with the mean ± SEM, n = 5–10 per group, *P < 0.05, **P < 0.01, ***P < 0.001 versus sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus MCAO group; &P < 0.05 versus SalB group; $P < 0.05, $$P < 0.05 versus Rg1 group. Scale bar: 50 μm. MCAO, middle cerebral artery occlusion; SalB/Rg1, salvianolic acid B/ginsenoside Rg1.

Fig. 3

SalB and Rg1 reduced infarct volume synergistically. (a) Experiment design according to the Bliss independence model. (b) Representative brain pictures with TTC stain. (c) Quantification of cerebral infarct volume according to TTC stain. (d) Synergistic effect between SalB and Rg1 according to the Bliss independence statistical model. (e) Representative hematoxylin–eosin staining images from whole rat brain (up) and subregions of the cortex (up) and striatum (down) in rats. Data were expressed as the mean ± SEM, n = 5–10 per group, ***P < 0.001 versus sham group; ###P < 0.001 versus MCAO group; &&P < 0.01 versus SalB group; $$$P < 0.001 versus Rg1 group. Scale bar: 50 mm. MCAO, middle cerebral artery occlusion; SalB/Rg1, salvianolic acid B/ginsenoside Rg1; TTC, 3,5-triphenyltetrazolium chloride.

Fig. 4

Superior efficiency of SalB/Rg1 than SalB or Rg1 treatment on neurological behavior and neurocyte protection at the dose according to Bliss independence statistical model design. (a) Garcia score. (b) Longa score. (c) Representative images of Nissl staining. (d) Quantitative results of neurocytes in the cortex (left) and striatum (right). Data were expressed with the mean ± SEM, n = 5–10 per group, *P < 0.05, **P < 0.01, ***P < 0.001 versus sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus MCAO group; &P < 0.05, &&P < 0.01 versus SalB group; $P < 0.05, $$P < 0.01 versus Rg1 group. Scale bar: 50 μm. MCAO, middle cerebral artery occlusion; SalB/Rg1, salvianolic acid B/ginsenoside Rg1.

Fig. 5

The differential metabolites were reversed by SalB/Rg1 after MCAO and prediction of the cerebroprotection mechanism of SalB/Rg1. (a) PLS-DA diagram of sham, MCAO, and SalB/Rg1 groups in positive ion mode. (b) PLS-DA diagram of sham, MCAO, and SalB/Rg1 groups in negative ion mode. (c) Venn diagram of differential metabolites in sham group, and SalB/Rg1 group compared with MCAO group. (d) Heat map analysis of metabolites disturbance among sham, MCAO, and SalB/Rg1 groups. (e) Comparing sham group to MCAO group in Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. (f) Comparing the SalB/Rg1 group to the MCAO group in the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. (g) ROC analysis of GPCho (16 : 0/16 : 0) in the sham group and MCAO group. (h) ROC analysis of GPCho (16 : 0/16 : 0) in the MCAO group and SalB/Rg1 group. AUC, area under the curve; GPCho, glyceryl phosphoryl choline; GPEtn, glycerol-phosphoethanolamine; MCAO, middle cerebral artery occlusion; MG, methylglyoxal; PC, phosphatidylcholine; PE, phosphatidyl ethanolamine; PLS-DA, partial least squares discriminant analysis; ROC, receiver operating characteristic curve; SalB/Rg1, salvianolic acid B/ginsenoside Rg1.