Naoqing formula alleviates cerebral ischemia/reperfusion injury induced inflammatory injury by regulating Csf3 mediated JAK/STAT pathway and macrophage polarization

BACKGROUND: Upon cerebral ischemia/reperfusion injury (CIRI), the brain tissue experiences excessive inflammatory responses, which fuel the activation of immune cells, thereby intensifying cellular damage and inflammatory reactions. Naoqing formula (NQ), a traditional Chinese medicinal compound formulated with musk as the primary component, has been extensively utilized in China for the clinical treatment of ischaemic stroke (IS). PURPOSE: The precise pharmacological mechanism underlying NQ's efficacy in managing IS remains elusive. In this study, we investigate the protective effect and molecular mechanism of NQ against CIRI. METHODS: C57BL/6 mice were utilized to investigate the protective effects of NQ (130, 260 and 520mg/kg) against middle cerebral artery occlusion (MCAO) induced CIRI and the underlying mechanism. Employing molecular biology techniques, transcriptomics, proteomics, and network pharmacological analyses, the study assessed the role of NQ in the inflammatory response of neuronal cells by establishing a model for neuronal cell and microglia inflammatory injury induced by oxygen-glucose deprivation/reperfusion (OGD/R) and lipopolysaccharide (LPS) stimulation. RESULTS: NQ demonstrated significant efficacy in mitigating neuronal damage and cerebral infarction induced by CIRI, achieved through the enhancement of cortical blood flow. Transcriptomic and network pharmacological analyses revealed that NQ mitigated the inflammatory damage caused by CIRI by modulating the Csf3-mediated JAK/STAT pathway. Proteomic analysis further corroborated this finding, indicating that NQ reduced the impact of CIRI by regulating macrophage polarization. Notably, in CIRI mice treated with NQ, there was a notable downregulation of Csf3, JAK2, STAT3, and STAT6, along with a co-localization of Csf3 and CD206. These observations suggested that NQ inhibited the activation of the JAK/STAT pathway and exerted its anti-inflammatory effects by orchestrating the transition of macrophages from the M1 phenotype to the M2 phenotype, triggered by Csf3. Consistent with the in vivo findings, NQ also inhibited the activation of the JAK/STAT pathway in neuronal cells and microglial polarization in vitro, thereby protecting against OGD/R- and LPS-induced inflammatory injury. CONCLUSION: This study confirmed that NQ prevented CIRI induced inflammatory injury by inhibiting Csf3-mediated activation of the JAK/STAT pathway and modulating Csf3-mediated macrophage polarization. This study provided a new perspective on the use of NQ in the treatment of IS.