SnRNA-seq reveals the ameliorative effects of optimized Xueyu Jingshen formula on high altitude cerebral edema by modulating energy metabolism, inflammation and BBB integrity.

BACKGROUND

High-altitude cerebral edema (HACE) is a type of lethal neurological emergency, whose underlying pathogenic mechanisms and core therapeutic targets remain largely unelucidated to date. Optimized Xueyu Jingshen formula (OXJF) has been shown to exert definite clinical efficacy in the intervention of HACE, whereas the underlying molecular mechanisms remain to be systematically and deeply explored.

PURPOSE: This work leverages single-nucleus RNA sequencing (snRNA-seq) to elucidate pathological rewiring of cell-type-specific interactions in HACE, and to uncover molecular targets of OXJF, thereby laying the groundwork for novel clinical therapeutic strategies.

METHODS

We firstly conducted a qualitative analysis of the chemical components of OXJF using UHLPC-Q-TOF-MS. We comprehensively evaluated the pharmacodynamic effects of OXJF firstly by detecting the behavioral changes, brain water content, BBB permeability and pathological damage of HACE mice, combined with the analysis of cerebral microvascular blood flow imaging. Afterwards, we constructed a cerebral cell atlas of HACE mice by using snRNA-seq and achieved systematic identification of various cell types. The proportion of each cell type was quantified and verified through multiplex immunofluorescence. AUCell was employed to ascertain the activity of gene modules, thereby identifying cell population exhibiting specific functional states or transcriptional signatures. Differential genes and signaling pathways enrichment analyses were employed to elucidate vital biomarkers and pathways. To clarify the differentiation patterns of cell subtypes, we introduced pseudotime analysis for relevant investigations. CellChat was performed to decipher cell-cell communication networks based on ligand-receptor pairs analysis. Ultimately, we verified whether mitochondrial Complex I was the pivotal target mediating cellular responses to hypoxic stimulation and OXJF intervention in C8-D1A, HT22, BV2 and MO3.13 cell lines.

RESULTS

A total of 163 chemical components, mainly including flavonoids, organic acids and terpenoids, were successfully identified. Pharmacodynamic experiments certified that OXJF could largely improve cognitive and motor functions, and reduce BWC in HACE mice. Meanwhile, by upregulating the expression of tight junction proteins, OXJF reversed high-altitude-induced increases in BBB permeability, and effectively alleviated cerebral microvascular hemodynamic disorders as well as pathological damage in HACE mice. 92,616 annotated nuclei were mainly identified as astrocyte, excitatory and inhibitory neurons, microglia, oligodendrocyte precursor cells and oligodendrocytes. Cell proportion analysis and multiplex immunofluorescence staining verified that no significant alterations in the proportions of the aforementioned cell populations were observed in both HACE mice and OXJF-treated mice. Gene modules associated with energy metabolism, inflammation and BBB ‌integrity exhibited high AUCell scores, implying their potential involvement as underlying mechanisms through which OXJF ameliorated HACE. Furthermore, key regulatory components within the above modules were identified including Hif1a, Vhl, Ikbkb, Rela, Tlr4, Tnfrsf11a, Irak1, Ndufb8, Nfe2l2, Ndufs4, Vegfa, Pdgfa and Gja1 genes. Cell trajectory analysis indicated that diverse cell types manifested marked heterogeneity in response to HACE and OXJF intervention. Further CellChat analysis unveiled widespread ligand-receptor interactions across cell population, and these interactions were mapped to central signaling molecules including ADGRB, EPHB, PTN, UNC5, CLDN, CypA, JAM, SLIT and GAP. In vitro cellular assays had confirmed that OXJF suppressed reactive oxygen species production and mitochondrial permeability transition pore opening via upregulating the expression of mitochondrial Complex Ⅰ subunits NDUFA9, NDUFS4 and NDUFB8. This effect drove a noteworthy increase in ATP content, which in turn rescued energy supply in C8-D1A, HT22, BV2 and MO3.13 cell lines.

CONCLUSION

This study provides and defines the first systematic snRNA-seq profiles under OXJF-treated HACE mice. The potential mechanisms of OXJF in treating HACE were related to maintaining energy metabolism, inflammation and BBB homeostasis.