Ginsenoside Rg1 treats ischemic stroke by regulating CKLF1/CCR5 axis-induced neuronal cell pyroptosis

Background Ischemic stroke, a severe and life-threatening neurodegenerative condition, currently relies on thrombolytic therapy with limited therapeutic window and potential risks of hemorrhagic transformation. Thus, there is a crucial need to explore novel therapeutic agents for ischemic stroke. Ginsenoside Rg1 (Rg1), a potential neuroprotective agent, exhibits anti-ischemic effects attributed to its anti-inflammatory, anti-oxidant, and anti-apoptotic properties. Nevertheless, the precise underlying mechanism of action remains to be fully elucidated. Purpose This study aimed to explore whether Rg1 exerts anti-ischemic stroke effects by inhibiting pyroptotic neuronal cell death through modulation of the chemokine like factor 1 (CKLF1)/ C-C chemokine receptor type 5 (CCR5) axis. Methods In this study, the MCAO model was used as an ischemic stroke model, and experimental tests were performed after 6 hours of ischemia. The anti-ischemic effect of Rg1 was examined by TTC staining, nissl-staining and neurobehavioral tests. In the in vitro experiments, PC12 cells were subjected to stimulation with CKLF1′s mimetic peptide C27 to assess the potential of CKLF1 to induce focal neuronal cell death. Additionally, the impact of CKLF1 mimetic peptide C27, antagonistic peptide C19, and CCR5 inhibitor MVC on PC12 cells subjected to oxygen-glucose deprivation (OGD) and subsequently treated with Rg1 was investigated. In vivo, Rg1 treatment was examined by quantitative real-time PCR (qPCR), ELISA, immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and co-immunoprecipitate (Co-IP) assays to perspective whether Rg1 treatment reduces CKLF1/CCR5 axis-induced pyroptotic neuronal cell death. In addition, to further explore the biological significance of CKLF1 in ischemic stroke, CKLF1 ?/? rats were used as the observation subjects in this study. Results The in vitro results suggested that CKLF1 was able to induce neuronal cells to undergo pyroptosis. In vivo pharmacodynamic results showed that Rg1 treatment was able to significantly improve symptoms in ischemic stroke rats. In addition, Rg1 treatment was able to inhibit the interaction between CKLF1 and CCR5 after ischemic stroke and inhibited CKLF1/CCR5 axis-induced pyroptosis. The results of related experiments in CKLF1 ?/? rats showed that Rg1 lost its therapeutic effect after CKLF1 knockdown. Conclusion Our findings indicate that the activation of the NLRP3 inflammasome is initiated by the CKLF1/CCR5 axis, facilitated through the activation of the NF-κB pathway, ultimately resulting in the pyroptosis of neuronal cells. Conversely, Rg1 demonstrates the capability to mitigate neuronal cell damage following CKLF1-induced effects by suppressing the expression of CKLF1. Thus, CKLF1 represents a crucial target for Rg1 in the context of cerebral ischemia treatment, and it also holds promise as a potential target for drug screening in the management of ischemic stroke.

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