Abstract
Ethnopharmacological relevance
Jiawei Dingzhi Wan (DZW), a formula from Gujin Yijian (1576), was traditionally used for forgetfulness. However, it remains unclear whether DZW alleviates chronic cerebral hypoperfusion (CCH)-induced vascular cognitive impairment (VCI) and what the underlying mechanisms are.Aim of the study
To investigate the main active components and mechanisms by which DZW ameliorates CCH-induced VCI.Materials and methods
A 2-vessel occlusion (2VO) rat model was used to assess the effect of DZW (4 and 2 g/kg) on CCH-induced VCI. Cerebral blood flow (CBF), cognitive function (Morris Water Maze), and histopathology were evaluated. Immunofluorescence (IF) was used to evaluate the number of cerebral microvessels. In vitro, a thoracic aorta ring was used to test vasoconstriction. Network pharmacology, untargeted metabolomics, and targeted sphingolipomics were used to explore the mechanisms. Related protein levels were determined by Western blot. High-performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ-MS) was used to quantitatively analyze the main components of DZW and the components absorbed into the plasma after intragastric administration of DZW in rats. Drug affinity responsive target stability (DARTS) analysis identified plasma-absorbed components and their direct binding targets. Wound healing and aortic ring assays assessed effects on angiogenesis and vasoconstriction.Results
DZW (4 g/kg and 2 g/kg) significantly increased cortical CBF by approximately 63% and 44%, respectively, compared to the model group. DZW also significantly shortened escape latency in the Morris Water Maze test and increased the number of platform crossings in the probe trial. DZW significantly increased the expression of CD31 in CCH rats, suggesting that DZW can promote angiogenesis. Additionally, the intestinal absorption solution of DZW (20-640 μL) had a contractile effect on thoracic aorta vessels, both with and without endothelialization. Network pharmacology and untargeted metabolomics revealed that sphingolipid signaling is a strongly correlated pathway. Further targeted sphingolipomics found that the levels of sphingosine (Sph) and sphingosine-1-phosphate (S1P) increased, while the levels of ceramide (Cer 18:0, Cer 18:1, Cer 20:0, Cer 22:0, and Cer 24:0) decreased after DZW (4 g/kg) treatment, which was associated with the S1P synthesis enzyme ACER2. Our study found that DZW promotes angiogenesis through the S1PR3/VEGF/VEGFR2 signaling pathway and regulates vasoconstriction through the S1PR3/ROCK1 signaling pathway. Subsequently, 25 components in DZW and 13 components absorbed into the plasma were identified as the main components by high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ-MS) analysis. DARTS-based immunoblot analysis confirmed that among the 13 plasma-absorbed components, in addition to 3-butylphthalide and ginsenoside Re, 11 other compounds directly bound to ACER2 and affected its expression. All 13 components absorbed into the plasma directly bound to S1PR3 and affected its expression. Further research has shown that paeoniflorin, ferulic acid, ginsenoside Rg1, ginsenoside Re, ligustilide, and 3-butylphthalide (20 μM) can promote vascular endothelial cell migration in the wound healing assay, while paeoniflorin, polygalaxanthone III, tenuifolin, and senkyunolide I (100 μM) were found to induce vasoconstriction in thoracic aortic ring assays.Conclusion
This study demonstrates for the first time that DZW ameliorates CCH-induced VCI through ACER2 and S1P-mediated dual vascular regulation, including both microvascular angiogenesis and aortic vasoconstriction. We identified six angiogenesis and four vasoconstrictive components of DZW. These findings provide novel mechanistic insights into the multi-component therapeutic strategy of TCM for CCH-induced VCI.