Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective treatments. Angiogenesis following ICH is an important response mediating brain recovery and repair. Phosphorylation of vascular endothelial growth factor receptor 2 (pVEGFR2) via PI3K/Akt signaling plays a key role in mediating cellular processes involved in repair, such as mitogenesis, angiogenesis, and vascular permeability. This study aimed to investigate the potential effects of Buyang Huanwu Decoction (BYHWD), a traditional Chinese medicine formula, on angiogenesis by VEGFR2 activation through the phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway in a mouse model of ICH. Adult male Kunming mice (n = 50) were randomly assigned into sham and ICH-operated groups and treated with one of the followings SU5416 (VEGFR2 inhibitor), BYHWT and BYHWT + SU5416. ICH was induced in mice by injecting collagenase (type VII) into the right globus pallidus of the mouse brain. BYHWD (4.36 g/kg) was administrated in mice by intragastric infusion. Neurological function was evaluated in mice by a modified Neurological Severity Scores (mNSS) as well as corner turn and foot-fault tests. Angiogenesis was examined by intraperitoneal injection of 5-bromodeoxyuridine (BrdU) in mice to quantify new brain vessel growth. SU5416 treatment and assessment of VEGFR2 phosphorylation as well as alterations in PI3K/Akt signaling were performed to determine whether the effect of BYHWD on angiogenesis was partly mediated by phosphorylation of VEGFR2 via the PI3K/Akt signaling pathway. We show that BYHWD treated mice exhibited (i) significantly better recovery from neurological dysfunction, (ii) increased BrdU+ nuclei in vWF+ dilated brain vessels and (iii) higher VEGFR2 phosphorylation immunoreactivity in brain microvessels (P <0.05), (iv) higher expression of PI3K and pAkt at the protein level (P <0.05) when compared to untreated ICH mice. These beneficial effects were reversed by SU5416 (P <0.05). BYHWD promoted neurological recovery and angiogenesis after ICH in mice by enhancing VEGFR2 phosphorylation through the PI3K/Akt signaling pathway.

Background: Xuefu Zhuyu decoction (XFZYD) has been extensively utilized to treat traumatic brain injury (TBI). However, the bioactive compounds and the underlying mechanisms have not yet been elucidated. Objectives: This study aimed to investigate the bioactive constituents of XFYZD that are absorbed in the blood and the mechanisms in treating TBI. Methods: The study presents an integrated strategy in three steps to investigate the material basis and pharmacological mechanisms of XFZYD. The first step involves: (1) performing metabolomics analysis of XFZYD to obtain the main functions and targets; (2) screening the blood-entry ingredients and targets of XFZYD from databases; (3) obtaining the potential components targeting the key functions by integrated analysis of metabolomics and network pharmacology. The second step involves screening pharmacological effects with active ingredients in vitro. In the third step, the effects of the top active compound were validated in vivo, and the mechanisms were explored by protein antagonist experiments. Results: Metabolomics analysis revealed that XFZYD treated TBI mice mainly through affecting the functions of blood vessels. We screened 62 blood-entry ingredients of XFZYD by network pharmacology. Then, we focused on 39 blood-entry ingredients related to vascular genes enriched by XFZYD-responsive metabolites. Performing the natural products library, we verified that hydroxysafflor yellow A (HSYA), vanillin, ligustilide, paeoniflorin, and other substances promoted endothelial cell proliferation significantly compared to the control group. Among them, the efficacy of HSYA was superior. Further animal studies demonstrated that HSYA treatment alleviated neurological dysfunction in TBI mice by mNSS and foot fault test, and decreased neuronal damage by HE, nissl, and TUNEL staining. HSYA increased the density of cerebral microvessels, raised the expression of angiogenesis marker proteins VEGFA and CD34, and activated the PI3K/Akt/mTOR signaling pathway significantly. The angiogenic effects disappeared after the intervention of PI3K antagonist LY294002. Conclusion: By applying a novel strategy of integrating network pharmacology of constituents absorbed in blood with metabolomics, the research screened HSYA as one of the top bioactive constituents of XFZYD, which stimulates angiogenesis by activating the PI3K/Akt/mTOR signaling pathway after TBI.

Blood-brain barrier (BBB) disruption is a key pathophysiological factor of intracerebral hemorrhage (ICH). The level of zonula occludens-1 (ZO-1) has been closely associated with the degree of BBB damage, and is an indicator of BBB destruction. The aim of the present study was to evaluate the effects of rhubarb on BBB function in a rat model of ICH. ICH was induced in rats by treatment with type VII collagenase. Sham-operated rats were administered with an equal volume of saline. Following the administration of rhubarb decoction (20 g/kg), neurobehavioral function evaluation and Evans blue extravasation assays were performed at days 1, 3 and 5 after ICH. ZO-1 expression in the brain of ICH-induced rats were analyzed via reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses. The results suggested that rhubarb significantly ameliorated neurological symptoms and attenuated BBB permeability. The results of immunohistochemistry and RT-PCR studies indicated that the expression of ZO-1 expression was robust in the sham-operated group and was weak in the vehicle-treated group at day 3. The present data indicated that rhubarb effectively attenuated ICH-induced BBB damage in rats, raising the possibility that rhubarb or its active components may be considered useful as neuroprotective drugs for ICH. The protective mechanisms appeared to involve the preservation of BBB integrity and elevation of ZO-1 protein expression levels.

Objectives. Xuefu Zhuyu decoction (XFZYD), a traditional Chinese medicine (TCM) formula, has been demonstrated to be effective for the treatment of traumatic brain injury (TBI). However, the underlying pharmacological mechanisms remain unclear. This study aims to explore the potential action mechanisms of XFZYD in the treatment of TBI and to elucidate the combination principle of this herbal formula. Methods. A network pharmacology approach including ADME (absorption, distribution, metabolism, and excretion) evaluation, target prediction, known therapeutic targets collection, network construction, and molecule docking was used in this study. Results. A total of 119 bioactive ingredients from XFZYD were predicted to act on 47 TBI associated specific proteins which intervened in several crucial pathological processes including apoptosis, inflammation, antioxidant, and axon genesis. Almost each of the bioactive ingredients targeted more than one protein. The molecular docking simulation showed that 91 pairs of chemical components and candidate targets had strong binding efficiencies. The “Jun”, “Chen”, and “Zuo‐Shi” herbs from XFZYD triggered their specific targets regulation, respectively. Conclusion. Our work successfully illuminates the “multicompounds, multitargets” therapeutic action of XFZYD in the treatment of TBI by network pharmacology with molecule docking method. The present work may provide valuable evidence for further clinical application of XFZYD as therapeutic strategy for TBI treatment.

As a bioactive absorbed compound of rhubarb, Rhein is applied for the treatment of brain injury. However, the underlying pharmacological mechanisms remain unclear. In this study, we aimed to explore antineuroinflammatory functions and underlying mechanisms of Rhein in vitro. BV2 microglia cells were chosen and irritated by LPS. The influence of Rhein on cell viability was determined using MTT assay. We finely gauged the proinflammatory cytokines of TNF‐α and IL‐1β through tests of immunofluorescence staining, ELISA, RT‐qPCR, and western blot. Additionally, mediators including IL‐6, IL‐12, iNOS, and IL‐10 were surveyed by ELISA. Furthermore, protein levels of the underlying signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF‐κB) were tested adopting western blot. We found that Rhein reduced the secretion of pivotal indicators including TNF‐α and IL‐1β, effectively restraining their mRNA and protein expression in LPS‐activated BV2 microglial cells. Besides, Rhein treatment demoted the production of IL‐6, IL‐12, and iNOS and promoted the excretion of IL‐10. Subsequent mechanistic experiments revealed that Rhein obviously downregulated the phosphorylation levels of PI3K, Akt, p38, and ERK1/2 and simultaneously upregulated the PTEN expression. In addition, Rhein antagonized the increase of TLR4, p‐IκBα, and NF‐κB. In summary, Rhein suppresses neuroinflammation via multiple signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF‐κB) in LPS‐stimulated BV2 microglia cells. This study highlights a natural agent for prevention and treatment of neuroinflammation.