Dalbergia odorifera T.C. Chen leaf extract promotes microglial energy expenditure to phagocytize neutrophils after cerebral ischemia-reperfusion

Abstract

Background and purpose: Cerebral ischemia-reperfusion injury stands as a significant contributor to disability globally, highlighting the urgent requirement for therapeutic interventions aimed at repairing the central nervous system. Worldwide interest in the use of Chinese traditional medicine for health promotion and disease prevention has increased significantly. Recently, Dalbergia odorifera T.C. Chen leaf has been listed in local food safety standard of Hainan Province (standard number: DBS46/003‒2021). Previous research findings from our team have demonstrated Dalbergia odorifera T.C. Chen leaf extract lead the genuine alleviation of brain edema following cerebral ischemia-reperfusion injury, albeit with the underlying mechanism still remaining elusive.

Study design and methods: D. odorifera leaf extract were prepared via ethanol extraction. We investigated the protective effects of D. odorifera leaf extract administered via gastric infusion using the middle cerebral artery occlusion/reperfusion model. The assessment covered the impacts on infarction volume in the brain, permeability of the blood-brain barrier, and neurological evaluations, which were determined by means of 2,3,5-triphenyltetrazolium chloride staining and Evans blue dye. Furthermore, to assess cerebral inflammation and neuronal apoptosis, techniques such as hematoxylin and eosin (H&E) staining and Nissl staining were applied. In order to delve deeper into the alterations occurring in brain tissue following treatment with D. odorifera leaf extract, we employed advanced methodologies including single-cell RNA sequencing (scRNA-seq), immunofluorescence analysis, and cerebral blood flow assessments. Assisted cellular experimental data help verify that the performed accuracy of animal experimental results. The energy metabolic capacity on ATP production, oxygen-consuming capacity, and glucose uptake capacity was estimated with intracellular ATP measurement, 2-NBDG uptake, glutamic acid assay, and oxygen consumption test method.

Results: Following cerebral ischemia-reperfusion, neutrophils migrate into the blood vessels at the ischemic site, with their presence closely linked to the severity of neuroinflammatory damage. Administration of D. odorifera leaf extract was found to alleviate cerebral edema, augment neurological function, and diminish infarct size after cerebral ischemia-reperfusion injury. scRNA-seq revealed that D. odorifera leaf extract enhance the disturbance of interaction between microglial cell and neutrophil. Further insights from scRNA-seq studies indicate that D. odorifera leaf extract enhances phagocytosis function by reprogramming energy metabolism, shifting it from glycolysis to oxidative phosphorylation. Additionally, D. odorifera leaf extract supports oxidative phosphorylation, leading to increased ATP production, which in turn fuels microglial phagocytosis. Consequently, this mechanism enhances the phagocytic activity and clearance of neutrophils in the ischemic brain. Activation of these processes was linked and dependent on GAS6-AXL, MERTK, TYRO3 signaling pathway. To sum up, our findings reveal that D. odorifera leaf extract modulates energy metabolism pathways to bolster microglial-mediated phagocytosis of neutrophils subsequent to ischemic stroke. These discoveries may pave the way for novel therapeutic approaches in stroke management by focusing on the modulation of microglial metabolic reprogramming.

Conclusion: Our results demonstrated that the neutrophil aggregation pathological processes were reduced greatly by D. odorifera leaf extract under on cerebral ischemia reperfusion. The regulation mechanism of D. odorifera leaf extract could be exerted by altering the functional states of microglial differentiation, and concurrently facilitates the phagocytosis of neutrophils by microglia, thereby leading to Simultaneously, it enhances the phagocytic activity of microglia toward neutrophils, thereby contributing to a decreased accumulation of neutrophils within the brain. Additional research has shown that D. odorifera leaf extract reprogramme energy metabolism to promote oxidative phosphorylation, thereby furnishing microglial phagocytosis with ample energy. Above results add to the present stage evidences that D. odorifera leaves has better application prospects in cerebral ischemia‒reperfusion injury.

Keywords: Cerebral Ischemia-reperfusion; Dalbergia odorifera T.C. Chen Leaf Extract; Energy expenditure; Phagocytize; scRNA-seq.