Investigation into the Potential Mechanism of Radix Paeoniae Rubra Against Ischemic Stroke Based on Network Pharmacology

Abstract

Background: Radix Paeoniae Rubra (RPR), an edible and medicinal Traditional Chinese Medicine (TCM), is extensively employed in therapeutic interventions of cardiovascular and cerebrovascular diseases. However, the curative effect of RPR on ischemic stroke remains ambiguous. This work integrated network pharmacology, molecular docking, and experimental validation to explore the mechanisms of RPR in treating ischemic stroke.

Methods: In this study, we preliminarily elucidated the therapeutic effect and mechanism of RPR on ischemic stroke through network pharmacology, molecular docking analysis, and experimental verification.

Results: The results indicated that RPR improved the neurological deficit scores, decreased the size of infarcts, and reduced brain edema symptoms in the tMCAO mice model. Furthermore, through network pharmacology and molecular docking, four core targets (MAPK3, TNF-α, MAPK14, and JNK) closely related to RPR's treatment of ischemic stroke were identified, exhibiting strong affinity with two key active components of RPR: albiflorin (AF) and β-sitosterol (BSS). The Western blot showed the potential mechanism of RPR treatment for ischemic stroke by regulating the MAPK signaling pathway. Moreover, RPR and its main active ingredients exhibited a significant inhibitory effect on platelets.

Conclusion: In conclusion, this study revealed that RPR alleviates ischemic injury by activating the MAPK signaling pathway, and its protective effect may partly stem from inhibiting platelet activation. This work may provide a scientific basis for the development and utilization of RPR as a natural edible material to prevent ischemic stroke and anti-platelet therapy.

Keywords: Radix Paeoniae Rubra; albiflorin; ischemic stroke; platelet; β-sitosterol.

Figure 1

The network pharmacology analysis of RPR against stroke: (A) “RPR-Component-Target” network. The pink nodes represent the ingredients of RPR, and the blue nodes represent the targets, (B) The Venn diagram of 128 targets intersected by RPR and stroke. (C) The PPI network of the 128 common targets. (D) The core targets of the 128 common targets ranked by degree value. The node size and degree value are positively correlated. (E–G) A cluster analysis identifies the top 3 core seed nodes of the core intersection targets.

Figure 2

“Disease-Pathway-Target-Component-Drug” network. The orange nodes represent signaling pathway involved in stroke, the green nodes represent ingredients of RPR, and the blue nodes represent the common targets between stroke and RPR.

Figure 3

The enrichment analysis of 128 intersected targets: (A) the top 20 GO enrichment terms (BP: biological process, CC: cellular component, and MF: molecular function); (B) Top 20 KEGG enrichment analysis item.

Figure 4

The protective effect of RPR on tMCAO mice: (A) Representative images of TTC staining of mice brain tissue with different dosages of RPR for 3 d of preventive administration, cerebral infarction area circled by yellow dashed, n = 5; (B) The quantification results of brain infarction volume; (C) H&E staining and Nissl staining images of brain tissues, n = 5. Scale bar = 50 μm. (D) Neurological score treated with different dosages of RPR, n = 5; (E,F) Representative Western blots showing ZO-1, Occludin, and Claudin- 5 levels, n = 5; (G,H) IL-6 level and IL-1β level in brain tissues (n = 5). The data are presented as mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Sham group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model group, ns, no significance.

Figure 5

RPR inhibited stroke via the MAPK signaling pathway. Western blotting and quantification analysis of p-p38, p38 (A), p-ERK, ERK (B), p-JNK, JNK (C), and TNF-α (D) in the brain tissue of tMCAO mice. n = 5. The data are presented as mean ± SD. ## p < 0.01 vs. Sham group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model group.

Figure 6

Molecular docking: (A–D) The results of molecular docking of MAPK3 (A), TNF (B), MAPK14 (C), and JNK (D) with AF. (E–H) The results of molecular docking of MAPK3 (E), TNF (F), MAPK14 (G), and JNK (H) with BSS. (I) Heat maps of the docking binding energy of the top 6 core targets with the top 4 active compounds in RPR.

Figure 7

Active compounds of RPR inhibit agonist-induced platelet aggregation and granules release: (A) The effects of RPR and the two active compounds, including AF and BSS on cell viability of platelets. (B–E) The effects of RPR and the two active compounds, including AF and BSS on aggregation induced by thrombin (B1,B2,C) and ADP (D1,D2,E). (F) The effects of RPR and the two active compounds, including AF and BSS, on the release of ATP secretion induced by thrombin (F1) and ADP (F2). (G) The effects of RPR and the two active compounds, including AF and BSS on the release of PF4 induced by thrombin (G1) and ADP (G2); n = 5. The data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p< 0.001, # p < 0.05, ## p < 0.01, ns, no significance.

Figure 8

Active compounds of RPR inhibit platelet clot retraction and adhesion: (A) The effects of RPR and the two active compounds, including AF and BSS on platelet clot retraction within 40 min. (B) Statistics of the volume of clot retraction. (C) The effects of RPR and the two active compounds, including AF and BSS, on platelet adhesion within 45 min. (D) Statistics of the area of platelet adhesion, n = 5. Results were quantified and presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p< 0.001 vs. PBS group, ns, no significance.

Figure 9

The proposed mechanism of RPR therapeutic effects on ischemic stroke. RPR alleviates ischemic stroke by down-regulating MAPK pathways; furthermore, the protective effect may be partly due to the anti-platelet function of its active compounds.