Shengui Sansheng Pulvis maintains blood-brain barrier integrity by vasoactive intestinal peptide after ischemic stroke.

Background Shengui Sansheng Pulvis (SSP) has about 300 years history used for stroke treatment, and evidences suggest it has beneficial effects on neuro-angiogenesis and cerebral energy metabolic amelioration post-stroke. However, its protective action and mechanisms on blood-brain barrier (BBB) is still unknown. Purpose Based on multiple neuroprotective properties of vasoactive intestinal peptide (VIP) in neurological disorders, we investigate if SSP maintaining BBB integrity is associated with VIP pathway in rat permanent middle cerebral artery occlusion (MCAo) model. Methods Three doses of SSP extraction were administered orally. Evaluations of motor and balance abilities and detection of brain edema were performed, and BBB permeability were assessed by Evans blue (EB) staining. Primary brain microvascular endothelial cells (BMECs) were subjected to oxygen-glucose deprivation, and incubated with high dose SSP drug-containing serum and VIP-antagonist respectively. Transendothelial electrical resistance (TEER) assay and Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4.4 kDa) and fluorescein isothiocyanate (FITC)-dextran (70 kDa) were used to evaluate the features of paracellular junction. Western blot detected the expressions of Claudin-5, ZO-1, Occludin and VE-cadherin, matrix metalloproteinase (MMP) 2/9 and VIP receptors 1/2, and immunofluorescence staining tested VIP and Claudin-5 expressions. Results Our results show that SSP significantly reduces EB infiltration in dose-dependent manner in vivo and attenuates TRITC- dextran and FITC-dextran diffusion in vitro, and strengthens endothelial junctional complexes as represented by decreasing Claudin-5, ZO-1, Occludin and VE-cadherin degradations and MMP 2/9 expression, as well as promoting TEER in BMECs after ischemia. Moreover, it suggests that SSP notably enhances VIP and its receptors 1/2 expressions. VIP-antagonist exacerbates paracellular barrier of BMECs, while the result is reversed after incubation with high dose SSP drug-containing serum. Additionally, SSP also improve brain edema and motor and balance abilities after ischemic stroke. Conclusions we firstly demonstrate that the ameliorated efficacy of SSP on BBB permeability is related to the enhancements of VIP and its receptors, suggesting SSP might be an effective therapeutic agent on maintaining BBB integrity post-stroke.

Ginseng-Angelica-Sansheng-Pulvis Boosts Neurogenesis Against Focal Cerebral Ischemia-Induced Neurological Deficiency.

BACKGROUND: The traditional Chinese medicine Ginseng-Angelica-Shanseng-Pulvis (GASP) has been used to treat stroke for 300 years. This present study investigated if it can induce increases in neurogenesis following cerebral ischemic injury. METHODS: Rats following middle cerebral artery occlusion were orally treated with high, medium, and low doses of a standardized GASP extract. RESULTS: After 14 days, treatment with GASP improved regional blood flow and infarction volume by magnetic resonance imaging scanning, enhanced Ki67+ expression in the subventricular zone, increased brain-derived neurotrophic factor (BDNF) secretion, Nestin, and bone morphogenetic protein (BMP) 2/4 expressions in the hippocampus in a dose-dependent manner. Interestingly, low-dose treatment with GASP downregulated doublecortin and Notch1 expressions in the hippocampus, as well as upregulated glial fibrillary acidic protein expression in the subgranular zone and hairy and enhancer of split (Hes) 5 expression in the hippocampus, while treatment with middle and high doses of GASP reversed these results. Meanwhile, the consumed time was shortened in the basket test and the adhesive removal test and the spending time on exploring novel objects was prolonged by GASP treatment whose effects were more obvious at day 14 post-ischemia. CONCLUSION: Our study demonstrates that treatment with GASP increases neurogenesis and ameliorates sensorimotor functions and recognition memory. We hypothesize that these effects are thought be mediated by an effect on the BMP2/4 pathway and Notch1/Hes5 signal. Due to its beneficial efficacy, GASP can be recognized as an alternative therapeutic agent for ischemic stroke.

Shengui Sansheng San Ameliorates Cerebral Energy Deficiency via Citrate Cycle After Ischemic Stroke.

Cerebral energy deficiency is a key pathophysiologic cascade that results in neuronal injury and necrosis after ischemic stroke. Shengui Sansheng San (SSS) has been used to treat stroke for more than 300 years. In present study, we investigated the therapeutic efficacy and mechanism of SSS extraction on cerebral energy deficiency post-stroke. In permanent middle cerebral artery occlusion (pMCAo) model of rats, it suggested that SSS extraction in dose-dependent manner improved neurological function, cerebral blood flow (CBF), 18F-2-deoxy-glucose uptake and the density and diameter of alpha smooth muscle actin (α-SMA) positive vasculature in ipsilateral area, as well as decreased infarcted volume. Meanwhile, the metabolomics study in cerebrospinal fluid (CSF) was performed by using 5-(diisopropylamino)amylamine (DIAAA) derivatization-UHPLC-Q-TOF/MS approach. Eighty-eight endogenous metabolites were identified, and mainly involved in citrate cycle, fatty acid biosynthesis, aminoacyl-tRNA biosynthesis, amino acids metabolism and biosynthesis, etc. The remarkable increase of citrate in CSF after treatment with three dosages indicated that the therapeutic mechanism of SSS extraction might be related with citrate cycle. Simultaneously, it showed that high dosage group significantly increased peripheral blood glucose level, the expressions of glucose transporter (GLUT) 1, GLUT3, and monocarboxylic acid transporter 1 (MCT1), which contributed to the transportation of glucose and lactate. By the regulations of phosphorylated pyruvate dehydrogenase E1-alpha (p-PDHA1), acetyl CoA synthetase and citrate synthetase (CS), the levels of citrate and its upstream molecules (pyruvate and acetyl CoA) in peri-infarction zone further enhanced, which ultimately caused the massive yield of adenosine triphosphate (ATP). Our study first demonstrated that SSS extraction could ameliorate cerebral energy deficiency after ischemia by citrate cycle, which is characterized by the enhancements of glucose supply, transportation, utilization, and metabolism.

Droplet Impact on the Super-Hydrophobic Surface with Micro-Pillar Arrays Fabricated by Hybrid Laser Ablation and Silanization Process.

A super-hydrophobic aluminum alloy surface with decorated pillar arrays was obtained by hybrid laser ablation and further silanization process. The as-prepared surface showed a high apparent contact angle of 158.2 ± 2.0° and low sliding angle of 3 ± 1°. Surface morphologies and surface chemistry were explored to obtain insights into the generation process of super-hydrophobicity. The main objective of this current work is to investigate the maximum spreading factor of water droplets impacting on the pillar-patterned super-hydrophobic surface based on the energy conservation concept. Although many previous studies have investigated the droplet impacting behavior on flat solid surfaces, the empirical models were proposed based on a few parameters including the Reynolds number (Re), Weber number (We), as well as the Ohnesorge number (Oh). This resulted in limitations for the super-hydrophobic surfaces due to the ignorance of the geometrical parameters of the pillars and viscous energy dissipation for liquid flow within the pillar arrays. In this paper, the maximum spreading factor was deduced from the perspective of energy balance, and the predicted results were in good agreement with our experimental results with a mean error of 4.99% and standard deviation of 0.10.

Combined treatment of sodium ferulate, n-butylidenephthalide, and ADSCs rehabilitates neurovascular unit in rats after photothrombotic stroke.

The remodelling of structural and functional neurovascular unit (NVU) becomes a central therapeutic strategy after cerebral ischaemic stroke. In the present study, we investigated the effect of combined therapy of sodium ferulate (SF), n-butylidenephthalide (BP) and adipose-derived stromal cells (ADSCs) to ameliorate the injured NVU in the photochemically induced thrombotic stroke in rats. After solely or combined treatment, the neovascularization, activation of astrocytes, neurogenesis, expressions of vascular endothelial growth factor (VEGF) and claudin-5 were assessed by immunohistochemical or immunofluorescence staining. In order to uncover the underlying mechanism of therapeutic effect, signalling of protein kinase B/mammalian target of rapamycin (AKT/mTOR), extracellular signal-regulated kinase 1/2 (ERK1/2), and Notch1 in infarct zone were analysed by western blot. 18 F-2-deoxy-glucose/positron emission tomography, magnetic resonance imaging, Evans blue staining were employed to evaluate the glucose metabolism, cerebral blood flow (CBF), and brain-blood barrier (BBB) permeability, respectively. The results showed that combined treatment increased the neovascularization, neurogenesis, and VEGF secretion, modulated the astrocyte activation, enhanced the regional CBF, and glucose metabolism, as well as reduced BBB permeability and promoted claudin-5 expression, indicating the restoration of structure and function of NVU. The activation of ERK1/2 and Notch1 pathways and inhibition of AKT/mTOR pathway might be involved in the therapeutic mechanism. In summary, we have demonstrated that combined ADSCs with SF and BP, targeting the NVU remodelling, is a potential treatment for ischaemic stroke. These results may provide valuable information for developing future combined cellular and pharmacological therapeutic strategy for ischaemic stroke.

Shengui Sansheng San extraction is an angiogenic switch via regulations of AKT/mTOR, ERK1/2 and Notch1 signal pathways after ischemic stroke.

BACKGROUND: As a traditional Chinese herbal formula, Shengui Sansheng San (SSS) has been employed for stroke treatment more than 300 years. PURPOSE: We hypothesize that SSS extraction is an angiogenic switch in penumbra post-stroke, and corresponding mechanisms are investigated. METHODS: In present study, rats were subjected to permanent middle cerebral artery occlusion model (MCAo) and were treated with low, middle and high doses of SSS extraction. We assessed neurological function and survival rate, and measured infarct volume by 2,3,5-triphenyltetrazolium chloride staining on day 7 after ischemia. von Willebrand factor (vWF), stromal cell-derived factor-1 alpha (SDF-1α) /chemokine (C-X-C motif) receptor 4 (CXCR4) axis, vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) as well as protein kinase B (AKT)/mammalian target of rapamycin (mTOR) /hypoxia-inducible factor-1 alpha (HIF-1α), extracellular signal-regulated kinase 1/2 (ERK1/2) and Notch1 signaling pathways were respectively investigated by immunofluorescence assay or western blotting in vivo and oxygen-glucose-deprived (OGD) brain microvascular endothelial cells (BMECs); simultaneously, wound healing of BMECs and tube formation assay were administrated. RESULTS: Compared to MCAo group, SSS extraction could significantly improve neurological functional scores, survival rate and cerebral infarct volume, enhance vWF+ vascular density and perimeter, SDF-1α/CXCR4 axis, VEGF expression, as well as activate AKT/mTOR/HIF-1α and ERK1/2 and inhibit Notch1 pathways in penumbra. In vitro, containing SSS extraction serum increased BMEC migration, capillary formation and VEGF expression via up-regulations of AKT/mTOR and ERK1/2 pathways in OGD BMECs, but ERK inhibitor (U0126) reversed the result of VEGF expression in high dose of SSS group. Additionally, VEGFR2 and Notch1 expressions were suppressed by containing SSS extraction serum. All results were in dose dependent manner. CONCLUSION: Our study firstly demonstrates that SSS extraction is an angiogenic switch. Due to suppressed VEGFR2/Notch1 cascades and activated AKT/mTOR and ERK1/2 signals in BMECs, a feedback loop of angiogenic homeostasis is established. Furthermore, the comprehensive mediations of SDF-1α/CXCR4 axis, AKT/mTOR/HIF-α, ERK1/2 and Notch1 pathways in penumbra contribute to the improvements of neurological function, survival rate and infarct volume post-stroke.