Multi-omics reveals Fuyuan Xingnao Decoction's therapeutic mechanism in cerebral infararction via gut microbiota and JAK2-STAT3 pathway.

Ethnopharmacological Relevance: Cerebral infarction is a cerebrovascular disorder characterized by ischemic and hypoxic necrosis of brain tissue resulting from vascular occlusion, leading to subsequent neurological deficits. Fuyuan Xingnao Decoction (FYXN) has been traditionally used for the prevention and treatment of cerebral infarction at Longhua Hospital, affiliated with Shanghai University of Traditional Chinese Medicine. However, its targeted intervention in the brain remains to be fully elucidated.

Aim Of The Study: This study aims to investigate the therapeutic effects of FYXN on ischemic stroke and its underlying mechanisms, with particular emphasis on its regulation of the JAK2-STAT3 signaling pathway and its impact on the activity of brain microvascular endothelial cells.

Materials And Methods: In this study, we employed metabolomics, network pharmacology, transcriptomics, and proteomics to explore the practical components, potential targets, and pathways of FYXN in the treatment of ischemic cerebral infarction. The gut microbiota of rats with ischemic cerebral infarction was analyzed using 16 S rRNA sequencing, and the levels of short-chain fatty acids were quantified. TTC, H&E, Nissl, and Luxol Fast Blue (myelin staining) were used to assess pathological changes in brain tissue. The expression of JAK2-STAT3 pathway proteins after cerebral infarction was verified by western blotting and immunohistochemistry. The proliferative activity of FYXN on rat and human brain microvascular endothelial cells was validated by CCK8.

Results: In the FYXN, flavonoids had the highest content (24.09 %), followed by terpenoids (12.8 %), phenylpropanoids (12.5 %), and amino acids (11.28 %). Network pharmacology analysis revealed that FYXN shared 131 overlapping targets with cerebral infarction. Animal experiments showed that FYXN significantly reduced the infarct area in the MCAO model (p < 0.05), improved the Zea Longa score (p < 0.05), and enhanced limb grip strength. Pathological staining revealed that it improved neuronal damage and significantly increased the density of Nissl bodies (P < 0.05). Compared with the sham group, the richness and diversity indices of gut microbiota in the MCAO group were significantly reduced (p < 0.05, Cohen's d > 0.8). Butyrate levels were significantly negatively correlated with neuronal damage (R = -0.6, p = 0.041), and butyrate could decrease the Zea Longa neurological function score (p < 0.05, Cohen's d > 0.8), increase rat limb grip strength (p < 0.01), and no obvious demyelination was observed, which was consistent with the trend of nimodipine. Transcriptomics, proteomics, and protein-protein interaction network analysis predicted that FYXN had essential targets in the treatment of cerebral infarction, including JAK2, STAT3, and EGFR. Rat brain tissue protein detection showed that compared with the MCAO group, both the FYXN-L and FYXN-H groups significantly up-regulated JAK2 (fold change >2), p-STAT3 (fold change >2), and VEGF (fold change >2) expression while reducing the expression level of caspase-3. In vitro, the CCK8 assays demonstrated that FYXN significantly promoted the proliferation of rat and human brain microvascular endothelial cells (p < 0.01).

Conclusion: FYXN treatment may ameliorate the cerebral infarct area and neurological deficits in experimental stroke models, plausibly via modulation of gut fatty acid metabolism and potential activation of the JAK2-STAT3 pathway, which provides a theoretical scientific basis for future clinical research.