Objective

2

To investigate the characteristics of metabolic reprogramming during cerebral ischemia-reperfusion injury using single-cell transcriptome sequencing， analyze the heterogeneity of microglial populations， and evaluate the interventional effects of Huanglian Jiedutang on metabolic abnormalities and neuroinflammation.

Methods

2

A transient middle cerebral artery occlusion （tMCAO） model was used to establish ischemic stroke in mice. Local cerebral blood flow changes were monitored by laser speckle imaging. Neurological impairment was evaluated using the Zea-Longa score， and histopathological damage in brain tissue was observed by HE and Nissl staining. Animals were divided into a sham group， model group， Huanglian Jiedutang group， and

Ginkgo biloba

extract （GBE） group. After 1 week of acclimatization， intragastric administration was initiated. The sham and model groups received normal saline， the Huanglian Jiedutang group was administered 1.82 g·kg

-1

， and the GBE group was administered 0.432 g·kg

-1

after preparation as a 2.16 mg/mL solution. All groups were treated for 5 consecutive days

（0.2 mL/10 g/day）， and the tMCAO model was established on day 6 after the final administration. At the molecular level， single-cell RNA sequencing was performed on ischemic hemisphere tissue. Non-negative matrix factorization （NMF） was used to cluster microglial subpopulations， combined with differential expression analysis， metabolic reprogramming assessment， and inflammatory factor correlation analysis to elucidate their functional characteristics in ischemia-reperfusion injury. Transcription factor enrichment analysis was further conducted to identify key regulatory nodes. Finally， PCR was used to detect mRNA expression changes of relevant genes to validate the single-cell sequencing results.

Results

2

Compared with the sham group， the model group showed increased neurological function scores （

P

<

0.01）， decreased blood flow levels （

P

<

0.01）， disordered cortical structure， increased cytoplasmic vacuolization， and increased Nissl bodies. Compared with the model group， the Huanglian Jiedutang and GBE groups showed decreased neurological function scores （

P

<

0.01）， increased blood flow levels （

P

<

0.01）， alleviated cortical structural disorder， reduced cytoplasmic vacuolization， and decreased Nissl bodies. Single-cell analysis showed that microglia could be divided into five subpopulations. Among them， clusters 3 and 5 exhibited significant pro-inflammatory phenotypes， with marked activation of hypoxia and NF-

κ

B signaling pathways， and were identified as pro-inflammatory subpopulations. Clusters 1 and 2 were enriched in Wnt/

β

-catenin and transforming growth factor（TGF）-

β

signaling pathways and exhibited prominent anti-inflammatory and reparative characteristics. Meanwhile， glycolysis-related genes， such as HK2， PFKP， and LDHA， were significantly upregulated in the pro-inflammatory subpopulations. Correlation analysis showed that the expression levels of inflamma

tory molecules were positively correlated with glycolysis-related gene expression levels， whereas the expression levels of reparative and anti-inflammatory molecules were negatively correlated with glycolysis-related gene expression levels， indicating that microglia rely on the glycolytic pathway for energy acquisition under ischemic conditions. Further single-cell transcriptome analysis revealed that Huanglian Jiedutang effectively downregulated key genes driving metabolic reprogramming （such as HK2， PFKP， and LDHA）， significantly reduced the proportion of microglial subpopulations accompanied by glycolytic reprogramming， and inhibited their transformation toward a damage phenotype， thereby reducing inflammatory injury. Meanwhile， compared with the sham group， the mRNA expression levels of interleukin （IL）-1

β

， IL-6， tumor necrosis factor（TNF）-

α

， CCL2， CXCL2， and CSF3 were significantly upregulated （

P

<

0.01） in the model group， whereas the mRNA expression levels of endothelial- and pericyte-related functional genes， including RGS5， PECAM1， VEGFB， and NOS3， were significantly downregulated （

P

<

0.01）. In contrast， compared with the model group， the Huanglian Jiedutang and GBE groups showed significantly decreased mRNA expression levels of IL-1

β

， IL-6， TNF-

α

， CCL2， CXCL2， and CSF3 （

P

<

0.01）， and significantly increased mRNA expression levels of endothelial- and pericyte-related functional genes， including RGS5， PECAM1， VEGFB， and NOS3 （

P

<

0.01）.

Conclusion

2

Huanglian Jiedutang exerts neuroprotective effects by regulating the metabolic reprogramming state of microglia and modulating their inflammatory levels， thereby inhibiting neuroinflammatory injury.