Background: Adequate collateral circulation can remarkably improve patient prognoses for patients experiencing ischemic stroke. Hypoxic preconditioning enhances the regenerative properties of bone marrow mesenchymal stem cells (BMSCs). Rabep2 (RAB GTPase binding effector protein 2) is a key protein in collateral remodeling. We investigated whether BMSCs and hypoxia-preconditioned BMSCs (H-BMSCs) augment collateral circulation poststroke, particularly through Rabep2 regulation. Methods: BMSCs or H-BMSCs (1×10 ⁶ ) were delivered intranasally in ischemic mice with distal middle cerebral artery occlusion at 6 hours poststroke. Two-photon microscopic imaging and vessel painting methods were used to analyze collateral remodeling. Blood flow, vascular density, infarct volume, and gait analysis were assessed to evaluate poststroke outcomes. Expressions of proangiogenic marker VEGF (vascular endothelial growth factor) and Rabep2 were determined by Western blotting. Western blot, EdU (5-ethynyl-2’-deoxyuridine) incorporation, and tube formation assays were conducted on cultured endothelial cells treated with BMSCs. RESULTS: BMSCs were more effectively transplanted in the ischemic brain after hypoxic preconditioning. The ipsilateral collateral diameter was increased by BMSCs and strengthened by H-BMSCs ( P <0.05). BMSCs increased peri-infarct blood flow and vascular density and reduced infarct volume, gait deficits ( P <0.05), and furthermore by H-BMSCs ( P <0.05). VEGF and Rabep2 protein expression was increased by BMSCs ( P <0.05), which was enhanced by preconditioning ( P <0.01). Additionally, BMSCs increased Rabep2 expression, proliferation, and tube formation of endothelial cells in vitro ( P <0.05). H-BMSCs enhanced these effects ( P <0.05), which were annulled by Rabep2 knockdown. CONCLUSIONS: BMSCs increased collateral circulation and improved poststroke outcomes, through the upregulation of Rabep2. These effects were enhanced by hypoxic preconditioning.

In the process of ischemic stroke (IS), cellular macroautophagy/autophagy and apoptosis play a vital role in neuroprotection against it. Therefore, regulating their balance is a potential therapeutic strategy. It has been proved that hydroxysafflor yellow A (HSYA) has anti-inflammatory and antioxidant effects, which can both protect neurons. By exploring bioinformatics combined with network pharmacology, we found that HIF1A and CASP3, key factors regulating autophagy and apoptosis, may be important targets of HSYA for neuroprotection in an oxygen glucose deprivation and reperfusion (OGD/R) model. In this study, we explored a possible new mechanism of HSYA neuroprotection in the OGD/R model. The results showed that OGD/R increased the expression of HIF1A and CASP3 in SH-SY5Y cells and induced autophagy and apoptosis, while HSYA intervention further promoted the expression of HIF1A and inhibited the level of CASP3, accompanied by an increase in autophagy and a decrease in apoptosis in SH-SY5Y cells. The inhibition of HIF1A diminished the activation of autophagy induced with HSYA, while the inhibition of autophagy increased cell apoptosis and blocked the neuroprotective effect of HSYA, suggesting that the neuroprotective effect of HSYA should be mediated by activating the HIF1A/BNIP3 signaling pathway to induce autophagy. These results demonstrate that HSYA may be a promising agent for treating IS.

Objective: To investigate the potential mechanisms of Piper longum L. in the treatment of cerebral ischemic stroke (CIS) and conduct experimental verification. Methods: Network pharmacology was used to identify intersecting targets of Piper longum L. and CIS. A drug-active component-target-disease network and the target protein-protein interaction (PPI) network were subsequently constructed, to screen major components and key targets. The intersecting targets were subjected to comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking and molecular dynamics (MD) simulations were performed to assess the binding energy and stability between the main active components and core targets. Surface plasmon resonance (SPR) was also employed to validate these binding interactions, providing further evidence for the underlying molecular mechanisms. Results: In total, 13 active components were identified in Piper longum L., and 60 intersectional targets were acquired. Among the main active components were piperine, 1,2,5,6-tetrahydrotanshinone, sesamin, N-(2,5-dimethoxyphenyl)-4-methoxybenzamide, and pipernonaline. The key targets predominantly consisted of tumor necrosis factor (TNF), interleukin-6 (IL-6), prostaglandin-endoperoxide synthase 2 (PTGS2), cAMP response element-binding protein (CREB1), and interleukin-1β (IL-1β). These identified targets were enriched in 77 GO terms and involved in multiple signaling pathways, including the TNF and cAMP (cyclic adenosine monophosphate) pathways. The main active components exhibit low binding energies with the key targets. MD simulations further demonstrated the structural stability of the complex formed between the main component, sesamin, and the associated CREB1 target, as well as the significance of key residues ILE240 (isoleucine240), ARG241 (arginine241), and PRO244 (proline244) in the binding interaction. Finally, the SPR method provided experimental evidence of the direct binding between sesamin and CREB1. Conclusions: Piper longum L. may exert pharmacological effects on CIS by acting on targets such as CREB1, PTGS2, and TNF, and by modulating signaling pathways including cAMP and inflammation.