Chemokine receptor 5 (CCR5) is one of the main co-receptors of HIV-1, and has been found to be a potential therapeutic target for stroke. Maraviroc is a classic CCR5 antagonist, which is undergoing clinical trials against stroke. As maraviroc shows poor blood-brain barrier (BBB) permeability, it is of interest to find novel CCR5 antagonists suitable for neurological medication. In this study we characterized the therapeutic potential of a novel CCR5 antagonist A14 in treating ischemic stroke mice. A14 was discovered in screening millions compounds in the Chemdiv library based on the molecular docking diagram of CCR5 and maraviroc. We found that A14 dose-dependently inhibited the CCR5 activity with an IC₅₀ value of 4.29 μM. Pharmacodynamic studies showed that A14 treatment exerted protective effects against neuronal ischemic injury both in vitro and vivo. In a SH-SY5Y cell line overexpressing CCR5, A14 (0.1, 1 μM) significantly alleviated OGD/R-induced cell injury. We found that the expression of CCR5 and its ligand CKLF1 was significantly upregulated during both acute and recovery period in focal cortical stroke mice; oral administration of A14 (20 mg·kg⁻¹·d⁻¹, for 1 week) produced sustained protective effect against motor impairment. A14 treatment had earlier onset time, lower onset dosage and much better BBB permeability compared to maraviroc. MRI analysis also showed that A14 treatment significantly reduced the infarction volume after 1 week of treatment. We further revealed that A14 treatment blocked the protein-protein interaction between CCR5 and CKLF1, increasing the activity of CREB signaling pathway in neurons, thereby improving axonal sprouting and synaptic density after stroke. In addition, A14 treatment remarkably inhibited the reactive proliferation of glial cells after stroke and reduced the infiltration of peripheral immune cells. These results demonstrate that A14 is a promising novel CCR5 antagonist for promoting neuronal repair after ischemic stroke. A14 blocked the protein-protein interaction between CKLF1 and CCR5 after stroke by binding with CCR5 stably, improved the infarct area and promoted motor recovery through reversing the CREB/pCREB signaling which was inhibited by activated CCR5 Gαi pathway, and benefited to the dendritic spines and axons sprouting.

This study aims to perform a comprehensive multi-strategy screening of quality markers (Q-markers) by integrating serum pharmacochemistry, chromatographic fingerprinting, and network pharmacology. The objective is to apply these methodologies for the quality control of traditional Chinese medicine (TCM) compound preparations. Firstly, the UPLC-Q-Exactive Orbitrap-MS was used to conduct a comparative analysis of the aqueous extract of Di Huang Yin Zi (DHYZ), the blank group, and the dosing group. Through the Xcalibur software, 10 blood components and 17 metabolites absorbed by DHYZ were identified. Then, the HPLC acetonitrile-formic acid water gradient elution system was used for detection at 330 nm. HPLC analysis was conducted on 16 batches of samples, and 22 common peaks with a similarity greater than 0.9 were established. 8 qualitative chemical components were quantitatively analyzed. Including morroniside, loganin, echinacoside, 5-hydroxymethylfurfural, acteoside, cinnamaldehyde, polygalaxanthone III and 3,6'-disinapoyl sucrose. The sample quality was comprehensively evaluated by using cluster analysis (HCA), Principal component analysis (PCA), and Orthogonal partial least squares discriminant Analysis (OPLS-DA). The results show that the 16 batches can be divided into 4 categories, and the results of PCA and HCA are consistent. The OPLS-DA model is stable and reliable, identifying morroniside (peak 4), echinacoside (peak 9), acteoside (peak 11), and 3,6'-disinapoyl sucrose (peak 13) as key discrimination markers. Finally, using network pharmacology methods, we constructed a component-target-disease-pathway network, thereby identifying 27 components associated with the efficacy of DHYZ. By integrating the above results, 3 active ingredients, morroniside, echinacoside, and 3,6'-disinapoyl sucrose, were identified as Q-markers that can be used for the quality control of DHYZ, providing a reference for establishing a robust quality control system for DHYZ.