An Intersectional Study of LncRNAs and mRNAs Reveals the Potential Therapeutic Targets of Buyang Huanwu Decoction in Experimental Intracerebral Hemorrhage.

BACKGROUND/AIMS: Both experimental and clinical studies have revealed satisfactory effects of the traditional Chinese formula Buyang Huanwu decoction (BYHWD) in improving post-intracerebral hemorrhage (ICH) neurological deficiencies. However, the multifaceted mechanisms of BYHWD in ICH treatment are not comprehensively understood. The present study explored various therapeutic targets of BYHWD by using lncRNA and mRNA transcriptomics. METHODS: LncRNA and mRNA microarrays were used to identify differentially expressed genes. ICH-induced upregulated genes (ICH vs sham) and BYHWD-induced downregulated genes (BYHWD vs ICH) were first identified. The intersection between these 2 sets was determined to identify ICH-induced highly expressed genes that were reversed by BYHWD. Then, the genes downregulated after ICH and the genes upregulated after BYHWD treatment were used to generate another set of intersections. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were subsequently performed to determine relative biological functions and signaling transduction pathways according to genes within the intersections. Quantitative real-time PCR was used to validate changes in gene expression observed using the microarray. Finally, a lncRNA-mRNA co-expression network was established to identify links among the genes within the intersections. RESULTS: A total of 18 differentially expressed lncRNAs and 33 differentially expressed mRNAs were identified using 2 lncRNA arrays (ICH vs sham and BYHWD vs ICH). The altered genes were enriched in the hemoglobin complex, oxygen transport and oxygen transporter and were closely associated with pyruvate metabolism. The co-expression network consisted of 53 nodes and 595 connections (308 positive interactions and 287 negative interactions). CONCLUSION: The hemoglobin complex, oxygen transport, oxygen transporter activity and pyruvate metabolism are possible therapeutic targets of BYHWD in ICH treatment. The present study provides the basis and direction for future investigations to explore the mechanisms by which BYHWD protects against long-term neurological deficiencies after ICH.

Astragaloside IV Improves Vasodilatation Function by Regulating the PI3K/Akt/eNOS Signaling Pathway in Rat Aorta Endothelial Cells.

Coronary heart disease (CHD) remains a major public health burden. Endothelial-dependent coronary artery vasoreactivity is a significant indicator of vascular function. Endothelial dysfunction is characterized by decreased nitric oxide (NO) bioavailability and predicts late cardiovascular events. Astragaloside IV (AGIV) is the main active component of the herb Astragalus membranaceus. Although it shows a significant protective effect against vascular endothelial dysfunction, the mechanisms of AGIV promoting the vascular dilation have not been elucidated. This study investigated the vasodilator effect of AGIV on rat aortic rings and the underlying effect of AGIV via the PI3K/Akt/eNOS signaling pathway. We measured the relaxation of isolated RARs after different concentrations of AGIV treatment. Rat aorta endothelial cells were cultured with different doses of AGIV, dimethylsulfoxide, and NG-nitro L-arginine methyl ester. The expression of phosphorylated (p)-Akt and -endothelial nitric oxide synthase (p-eNOS) were tested by Western blot analysis. The messenger (m)RNA expression of eNOS was quantified by real-time polymerase chain reaction. AGIV exerted a vasodilator effect on the aortic rings and increased the NO content in a concentration-dependent manner. The vasorelaxation was suppressed by an eNOS inhibitor. AGIV regulated the PI3K/Akt/eNOS signaling pathway via phosphorylation of Akt at Ser473 and dephosphorylation of eNOS at Thr495. The mRNA expression of eNOS was remarkably upregulated by AGIV. AGIV significantly induced the dilation of the aortic rings, leading to the vasodilator response by enhancing the eNOS release via the PI3K/Akt/eNOS signaling pathway.

Gancao (Glycyrrhizae Radix) provides the main contribution to Shaoyao-Gancao decoction on enhancements of CYP3A4 and MDR1 expression via pregnane X receptor pathway in vitro.

BACKGROUND: Chinese herbal formula Shaoyao Gancao decoction (SGD) is often used as an adjuvant with chemotherapeutic agents to treat cancer. Due to the herb-drug interactions, the alternations of drug metabolic enzyme and drug transporters induced by SGD deserve to be explored. We aimed to investigate the effect of SGD on the pregnane X receptor (PXR)-mediated transcriptional regulation of cytochrome P450 3A4 (CYP3A4) and drug transporter multidrug resistance protein 1 (MDR1) in vitro. Besides, we assessed the contribution of constituent herbs to SGD on the regulation of CYP3A4 and MDR1. METHODS: The dual luciferase reporter gene system containing the hPXR expression plasmid and the reporter gene plasmid of CYP3A4 or MDR1 was co-transfected to HepG2 and Caco2 cells. Luciferase activities were determined using a Dual-luciferase reporter assay kit. The gene expression of CYP3A4 and MDR1 in the hPXR-transfected LS174T cells were assessed by real-time qPCR. Finally, the contribution of constituent herbs from SGD was evaluated. RESULTS: SGD, Shaoyao and Gancao concentration-dependently increased promoter activities of CYP3A4 and MDR1 in vitro. Moreover, SGD, Shaoyao and Gancao up-regulated CYP3A4 and MDR1 mRNA in hPXR-transfected LS174T cells. As the herbal constituent of SGD, Gancao possesses significantly higher levels of metabolic enzyme and drug transporters compared with Shaoyao. CONCLUSION: SGD tends to enhance CYP3A4 and MDR1 expression via PXR pathway, especially Gancao provides the main contribution. This study highlights a potential in vitro mechanism for SGD on the regulation of drug metabolic enzymes and drug transporters.

Hyperbaric oxygen for experimental intracerebral haemorrhage: Systematic review and stratified meta-analysis.

OBJECTIVE: Hyperbaric oxygen (HBO) is widely used in treating various neurological diseases. However, HBO for treatment of intracerebral haemorrhage (ICH) remains controversial, in either animal or clinical studies. Therefore, we conducted this systematic review and meta-analysis on studies describing the efficacy of HBO in animal models of ICH. METHODS: Studies were identified by searching mainstream databases through November 2015. The efficacy of HBO in animal models of ICH was assessed by changes in the brain water content (BWC), neurobehavioural outcome (NO) or both. Subgroup analyses were performed according to different design characteristics. RESULTS: In total 15 studies met our inclusion criteria. HBO can reduce the BWC (-0.982, 95% CI, -1.148 to -0.817; P < 0.01; 57 comparisons), and improve NO (-0.767, 95% CI, -1.376 to -0.159; P < 0.01; eight comparisons). HBO was most effective in reducing BWC when given 72 h after ICH for a 4- to 5-day consecutive treatment at the chamber pressure of 3.0 atmosphere absolute. Efficacy was higher with phenobarbital anaesthesia, the blood infusion model and in rabbits. CONCLUSION: Although HBO was found to be effective in experimental ICH, additional confirmation is needed due to possible publication bias, poor study quality and the limited number of studies conducting clinical trials.

Buyang huanwu decoction promotes angiogenesis via vascular endothelial growth factor receptor-2 activation through the PI3K/Akt pathway in a mouse model of intracerebral hemorrhage.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: BYHWD promoted neurological recovery and angiogenesis after ICH in mice by enhancing VEGFR2 phosphorylation through the PI3K/Akt signaling pathway.

Electroacupuncture improves recovery after hemorrhagic brain injury by inducing the expression of angiopoietin-1 and -2 in rats.

BACKGROUND: Angiopoietin (Ang) is one of the major effectors of angiogenesis, playing a critical role in neurovascular remodeling after stroke. Acupuncture has been widely used for treating stroke in China for a long time. Recently, we have demonstrated that electroacupuncture (EA) can accelerate intracerebral hemorrhage (ICH)-induced angiogenesis in rats. In the present study, we investigated the effect of EA on the expression of Ang-1 and Ang-2 in the brain after ICH. METHODS: ICH was induced by stereotactic injection of collagenase type VII into the right globus pallidus. Adult male Sprague-Dawley rats were randomized into the following four groups: sham-operation (SHAM), stroke-no electroacupuncture (SNE), stroke-EA at the Zusanli acupoint (SEZ), and stroke-EA at a nonacupoint (SEN). EA was applied to the bilateral Zusanli (ST36) acupoint in the SEZ group and a nonacupoint in the SEN group. The expression of Ang-1 and Ang-2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Some Ang-1 and Ang-2 immunoreactive microvessels with a dilated outline were detected in the perihematomal tissues after ICH, and the vessels extended into the clot from the surrounding area since day 7. The expression of Ang-1 increased notably as long as 2 weeks after ICH, while Ang-2 immunoreactivity declined at about 7 days following a striking upregulation at 3 days. EA at the Zusanli (ST36) acupoint upregulated the expression of Ang-1 and Ang-2 at both the protein and mRNA levels. However, EA at a nonacupoint had little effect on the expression of Ang-1 and Ang-2. CONCLUSIONS: Our data suggest that EA at the Zusanli (ST36) acupoint exerts neuroprotective effects on hemorrhagic stroke by upregulation of Ang-1 and Ang-2.

N6-methyladenosine profiling reveals that Xuefu Zhuyu decoction upregulates METTL14 and BDNF in a rat model of traumatic brain injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese herbal formula Xuefu Zhuyu decoction (XFZYD) is a classic formula in the category of invigorating blood circulation and resolving blood stasis. It has been proven to improve the neurological and ethological prognosis of traumatic brain injury. XFZYD promotes synaptic and axonal regeneration after traumatic brain injury, which is functionally modulated by the N6-methyladenosine (m6A) modification of RNA. However, the epigenetic effects of XFZYD on m6A modification remain unknown. AIM OF THE STUDY: To explore how XFZYD protects against traumatic brain injury induced by controlled cortical impact (CCI) injury by altering RNA m6A modification. MATERIALS AND METHODS: The modified neurological severity scoring and Morris water maze were performed to evaluate the neuroprotective effects of XFZYD for 14 days and screen the dose. Then, dot blot, western blotting, and methylated RNA immunoprecipitation sequencing (MeRIP-Seq) were used to explore changes in RNA m6A modification in the perilesional cortex. The Metascape platform was used to analyze the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway of the differential m6A-tagged genes. Furthermore, MeRIP-qPCR was conducted to quantify differences in the hub differential m6A modification gene brain-derived neurotrophic factor (Bdnf). RESULTS: XFZYD significantly ameliorated the neurological deficits, spatial learning, and memory impairments in rats post-CCI on day 14. XFZYD enhanced the m6A level, and the expression of METTL14 and YTHDC2 in the perilesional cortex of CCI rats. In all three groups, the 3'-untranslated regions and coding sequence were primarily enriched for m6A peaks. XFZYD reversed the increased proportion of 3'-untranslated regions, and the decreased proportion of coding sequence and 5'-untranslated regions post-CCI. Moreover, XFZYD markedly downregulated 41 elevated m6A-tagged transcripts and upregulated 119 decreased m6A-tagged transcripts following CCI. Gene ontology and KEGG pathway analysis revealed that XFZYD-regulated m6A-tagged transcripts were predominantly enriched in synapse assembly, synaptic plasticity, learning or memory, and MAPK signaling pathway. Then, the hub-regulated m6A-tagged gene BDNF was identified. Both the m6A methylation level and the protein level of BDNF were ascended by XFZYD treatment. CONCLUSION: XFZYD improves neurological deficits, spatial learning and memory impairments in rats post-TBI probably through increasing the expression of METTL14 and BDNF in the cortex. Our study highlights a novel post-transcriptional regulation mechanism mediated by herbal medicine for traumatic brain injury treatment.

Temporal profile of angiogenesis and expression of extracellular matrix-related genes in rat brains following experimental intracerebral hemorrhage.

BACKGROUND: Angiogenesis is essential for the repair process after intracerebral hemorrhage (ICH). METHODS: Given the importance of the extracellular matrix (ECM) in angiogenesis, we analysed the temporal profile of angiogenesis in rat brains on days 4, 7, and 21 after ICH. To this end, we compared the expression of ECM-related genes between ICH-induced and sham-operated groups using a complementary DNA (cDNA) array. We further measured protein expression using western blot and immunohistochemistry assays. Fluorescein isothiocyanate (FITC)-dextran was injected into the tail vein to examine the angioarchitecture in the perihematomal region. RESULTS: Among the 88 ECM-related genes, we identified 42, 50, and 38 genes that were significantly upregulated on days 4, 7, and 21 after ICH, respectively (P < 0.05). Particularly, collagens, integrins, and matrix metalloproteinases (MMPs) were significantly increased on day 4 post-ICH and continued to increase at the other time points. Western blot and immunohistochemistry analyses showed a comparable trend in the upregulation of MMPs. Compared to the sham group, FITC-dextran labelling demonstrated decreased perfusion and increased vascular permeability in the perihematomal region in the ICH group. Doxycycline, an MMP inhibitor, significantly reduced angiogenesis (P < 0.05). CONCLUSIONS: The results of this study indicate that MMPs are involved in modulating angiogenesis following ICH.

Buyang Huanwu Decoction: A Traditional Chinese Medicine, Promotes Lactate-Induced Angiogenesis in Experimental Intracerebral Hemorrhage.

Identifying the underlying mechanisms and exploring effective therapies for intracerebral hemorrhage (ICH) are urgently needed. Here, we aim to elucidate the potential roles and underlying mechanisms of Buyang Huanwu decoction (BYHWD) in ICH. In the first set of experiments, rats were randomly divided into five groups: Sham, ICH, ICH + sodium oxamate (OXA), ICH + BYHWD, and ICH + BYHWD + OXA. The lactate level around the hematoma was evaluated. PCNA+/vWF+ nuclei were observed. Additionally, an online bioinformatics analysis tool was used to predict the BYHWD druggable targets related to angiogenesis. Then, we validated these predictions. In the second set, exogenous sodium L-lactate (Lac) was infused into the intact brains of rats. Rats were randomly divided into three groups: Sham, Lac, and Lac + YC-1. The numbers of PCNA+/vWF+ nuclei and the expression of HIF-1α and VEGF were evaluated. In the first set of experiments, compared with the ICH group, the BYHWD group exhibited significantly increased numbers of PCNA+/vWF+ nuclei, and neurological dysfunction was markedly improved. Bioinformatics analysis revealed that the improvements caused by BYHWD indicated a role for the HIF-1α pathway. The HIF-1α and VEGF protein levels were upregulated after BYHWD administration. Moreover, we verified that lactate was involved in the predicted mechanisms. In the second set, lactate facilitated angiogenesis and HIF-1α and VEGF expression. Co-infusion with a HIF-1α inhibitor, YC-1, significantly inhibited these effects. Our data suggest that the pharmacological effects of BYHWD involve lactate-induced angiogenesis, these data may provide new evidence for its use in ICH.

Temporal metabolomic alteration in rat brains of experimental intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is the top lethal and disabling form of stroke. The pathophysiology of ICH is not fully understood yet. Metabolites are indicators and regulators of cellular processes. However, the overall brain metabolic pattern and the temporal alterations after ICH remain unknown. METHODS: A total of 40 male rats were randomly assigned to sham group and ICH group. ICH was induced by collagenase Ⅶ. Body weight was assessed. Neurological deficits were evaluated by modified neurological severity score. Then, the perihematomal brain tissues were collected for metabolites detection using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The metabolic profiles were displayed by principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA) and cluster analysis. The significant differential metabolites were screened by fold change > 2.0, the false discovery rate (FDR) < 0.05 and Variable Importance of Projection (VIP) > 1. Next, the relevant metabolic pathways were discerned by MetaboAnalyst website. A metabolite-protein interaction network was subsequentially constructed to further annotate the function of differential metabolites. RESULTS: Rats suffered from compromised body weight increasement and impaired neurological function. The metabolomics profiles of brain tissues in the post-ICH rats were markedly different from those in the sham group on days 3 and 14. Thirty-four metabolites (bilirubin, uric acid, 6-Methylnicotinamide et al.) were abnormally upregulated in the acute stage, while 27 metabolites were disturbed in the recovery stage, including bilirubin, uric acid, and histamine et al. Seven and three metabolic pathways altered in the acute and recovery stage, respectively. Metabolite-protein interaction analysis revealed that the disturbed metabolites may participate in ICH pathophysiology by altering amino acid metabolism, peroxisome proliferators-activated receptor signaling pathway, fatty acid metabolism and urea cycle in the acute stage, while influencing amino acid metabolism, urea cycle and peroxisome in the recovery stage. CONCLUSIONS: Our study mapped the pathological metabolomics profiles of the post-ICH rat brains in the acute and recovery phases. This work will assist in discovering novel therapeutic targets and treatments for ICH.

Deciphering the Therapeutic Mechanisms of Wuzi Ershen Decoction in Treating Oligoasthenozoospermia through the Network Pharmacology Approach.

BACKGROUND: Infertility affects approximately 15% of couples around the world, and male factors are accounted for 40-50%. Oligoasthenozoospermia is the most common reason for male infertility. Unfortunately, effective drug therapy is still lacking except for assisted reproductive technology (ART). Previous researchers found that Wuzi Ershen decoction (WZESD) can increase sperm count, enhance sperm vitality, and improve semen quality. However, the pharmacological mechanisms remain unclear. METHODS: In this study, we screened compounds and predicted the targets of WZESD based on the TCMSP and BATMAN-TCM database combined with literature searching in the PubMed database. We obtained proteins related to oligoasthenozoospermia through GeneCards and submitted them to STRING to obtain the protein-protein interaction (PPI) network. Potential targets of WZESD were mapped to the network, and the hub targets were screened by topology. We used online platform Metascape and Enrichr for GO and KEGG enrichment analyses. AutoDock Vina was utilized for further verification of the binding mode between compounds and targets. RESULTS: Totally, 276 bioactive compounds were obtained and targeted 681 proteins. 446 oligoasthenozoospermia disease-specific proteins were acquired, and further bioinformatics analysis found that they were mainly involved in the formation of gametes, meiosis, and sperm differentiation. Protein interaction network analysis revealed that target proteins of WZESD were associated with oligoasthenozoospermia disease-specific proteins. The 79 targets of disease-specific proteins, which were anchored by WZESD, mainly participate in the cellular response to the organic cyclic compound, regulation of the apoptotic process, nitricoxide biosynthetic and metabolic process, oxidative stress, and protein phosphorylation regulation, which are the causes for oligoasthenozoospermia. Molecular docking simulation further validated that bioactive compounds originated from WZESD with targeted proteins showed high binding efficiency. CONCLUSIONS: This study uncovers the therapeutic mechanisms of WZESD for oligoasthenozoospermia treatment from the perspective of network pharmacology and may provide a valuable reference for further experimental research studies and clinical applications.

Progressive brain changes in Parkinson's disease: A meta-analysis of structural magnetic resonance imaging studies.

The progressive changes of brain structure in patients with Parkinson's disease (PD) remain controversial. To identify this controversy, a systematic review and meta-analysis of longitudinal magnetic resonance imaging studies in brain volume was performed. The percentage of volume change over time between patients with PD and healthy subjects of each brain region of interest was obtained. In total, 11 studies, comprising 833 cases (463 patients with PD and 370 healthy control subjects), were included for systematic review and meta-analysis. Ten brain regions were involved. Patients with PD in comparison with healthy controls showed significant progressive reductions in whole gray matter and caudate, putamen, accumbens, and amygdala volumes. Significant whole-brain atrophy from PD was also associated with cognitive dysfunction. The annual percentage of volume loss was -1.04% in whole-brain volume with cognitive decline, -1.16% in whole-brain volume in PD compared to PD with cognitive decline, -0.29% for whole gray matter, -0.62% for caudate, -0.97% for putamen, -3.55% for amygdala, and -5.40% for accumbens in patients with PD versus control subjects. Overall, our findings suggest that PD is related to progressive, regional brain atrophy, mainly affecting gray matter. However, due to existing confounding factors, the limited number of included studies, significant heterogeneity, and defective study design, the results should be interpreted with caution. Further confirmation is required by more studies with strict design, large samples, and unified paradigm.

Rhein Suppresses Neuroinflammation via Multiple Signaling Pathways in LPS-Stimulated BV2 Microglia Cells.

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-1ß 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-1ß, 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.

Integrative analysis of transcriptomes highlights potential functions of transfer-RNA-derived small RNAs in experimental intracerebral hemorrhage.

Transfer-RNA-derived small RNAs (tsRNAs) are a novel class of short non-coding RNAs, that possess regulatory functions. However, their biological roles in hemorrhagic stroke are not understood. In this study, by RNA sequencing, we investigated the tsRNA expression profiles of intracerebral hemorrhagic rat brains in the chronic phase. A total of 331 tsRNAs were identified (308 in sham and 309 in intracerebral hemorrhage). Among them, the validation revealed that 7 tsRNAs (1 up-regulated and 6 down-regulated) were significantly changed. Subsequently, we predicted the target mRNAs of the 7 tsRNAs. Through integrative analysis, the predicted targets were validated by mRNA microarray data. Moreover, we confirmed the functions of tsRNAs targeting mRNAs in vitro. Furthermore, using bioinformatics tools and databases, we developed a tsRNA-mRNA-pathway interaction network to visualize their potential functions. Bioinformatics analyses and confirmatory experiments indicated that the altered genes were mainly enriched in several signaling pathways. These pathways were interrelated with intracerebral hemorrhage, such as response to oxidative stress, endocytosis, and regulation of G protein-coupled receptor signaling pathway. In summary, this study systematically revealed the profiles of tsRNAs after an experimental intracerebral hemorrhage. These results may provide novel therapeutic targets following a hemorrhagic stroke in the chronic phase.

Thrombin-induced miRNA-24-1-5p upregulation promotes angiogenesis by targeting prolyl hydroxylase domain 1 in intracerebral hemorrhagic rats.

OBJECTIVE: Thrombin is a unique factor that triggers post-intracerebral hemorrhage (ICH) angiogenesis by increasing hypoxia-inducible factor-1α (HIF-1α) at the protein level. However, HIF-1α mRNA remains unchanged. MicroRNAs (miRNAs) mediate posttranscriptional regulation by suppressing protein translation from mRNAs. This study aimed to determine if miRNAs might be involved in thrombin-induced angiogenesis after ICH by targeting HIF-1α or its upstream prolyl hydroxylase domains (PHDs). METHODS: The study was divided into two parts. In part 1, rats received an injection of thrombin into the right globus pallidus. An miRNA array combined with miRNA target prediction, luciferase activity assay, and miRNA mimic/inhibitor transfection were used to identify candidate miRNAs and target genes. Part 2 included experiments 1 and 2. In experiment 1, rats were randomly divided into the sham group, ICH group, and ICH+hirudin-treated (thrombin inhibitor) group. In experiment 2, the rats were randomly divided into the sham group, ICH group, ICH+antagomir group, ICH+antagomir-control group, and ICH+vehicle group. Western blotting and quantitative real-time polymerase chain reaction were used to determine the expression of protein and miRNA, respectively. The coexpression of miR-24-1-5p (abbreviated to miR-24) and von Willebrand factor was detected by in situ hybridization and immunohistochemical analysis. The angiogenesis was evaluated by double-labeling immunofluorescence. Neurological function was evaluated by body weight, modified Neurological Severity Scores, and corner turn and foot-fault tests. RESULTS: In part 1, it was shown that miR-24, which is predicted to target PHD1, was upregulated (fold-change of 1.83) after thrombin infusion, and that the miR-24 mimic transfection decreased luciferase activity and downregulated PHD1 expression (p < 0.05). miR-24 inhibitor transfection increased PHD1 expression (p < 0.05). In part 2, it was shown that miR-24 was expressed in endothelial cells. The HIF-1α protein level and proliferating cell nuclear antigen-positive (PCNA+) nuclei in vessels were increased, while the PHD1 protein level was decreased after ICH, and these effects were reversed by hirudin (p < 0.05). The antagomiR-24-treated rats exhibited a markedly lower body weight and significantly poorer recovery from neurological deficit compared with those in ICH groups (p < 0.05). AntagomiR-24 intervention also led to lower miR-24 expression, a higher PHD1 protein level, and fewer PCNA+ nuclei in vessels compared with those in ICH groups (p < 0.05). CONCLUSIONS: The present study suggests that thrombin reduces HIF-1α degradation and initiates angiogenesis by increasing miR-24, which targets PHD1 after ICH.

iTRAQ-based quantitative proteomics reveals the neuroprotection of rhubarb in experimental intracerebral hemorrhage.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhubarb is a traditional Chinese medicine(TCM), that possesses neuroprotective, anti-inflammatory, antibacterial, antioxidative, purgative and anticancer properties, and has been used to treat intracerebral hemorrhage (ICH) and many other diseases. AIMS OF THE STUDY: This study aimed to investigate the changes of brain protein in ICH rats treated with rhubarb and to explore the multi-target mechanism of rhubarb in the treatment of ICH via bioinformatics analysis of differentially expressed proteins (DEPs). MATERIALS AND METHODS: Rats were subjected to collagenase-induced ICH and then treated orally with 3 or 12 g/kg rhubarb daily for 2 days following ICH. After sacrifice, total protein of brain tissue was extracted, and isobaric tag for relative and absolute quantification (iTRAQ)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was employed to quantitatively identify of the DEPs in two treatment groups compared with the vehicle group. The DEPs were analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and STRING databases. Bioinformatics Analysis Tool for Molecular mechanism of TCM (BATMAN-TCM) was used to predict the target of rhubarb and western blotting was used for verification. RESULTS: In total, 1356 proteins were identified with a 1% false discovery rate (FDR). Among them, 55 DEPs were significantly altered in the sham, vehicle, low dose rhubarb group (LDR, 3 g/kg), and high dose rhubarb group (HDR, 12 g/kg). Enrichment analysis of GO annotations indicated that rhubarb mainly regulated expression of some neuron projection proteins involved in the response to drug and nervous system development. The dopaminergic synapse pathway was found to be the most significant DEP in the combined analysis of the KEGG and BATMAN-TCM databases. Based on the results of the STRING analysis, oxidative stress (OS), calcium binding protein regulation, vascularization, and energy metabolism were important in the rhubarb therapeutic process. CONCLUSION: Rhubarb achieves its effects mainly through the dopaminergic synapse pathway in ICH treatment. The ICH-treating mechanisms of rhubarb may also involve anti-OS, calcium binding protein regulation, angiogenic regulation, and energy metabolism improvement. This study adds new evidence to clinical applications of rhubarb for ICH.

Thrombin promotes pericyte coverage by Tie2 activation in a rat model of intracerebral hemorrhage.

Pericyte coverage on the endothelial tubes leads to the formation of a mature and stable microvessel system, which is critical for brain repair after intracerebral hemorrhage (ICH). We report herein that thrombin promotes pericyte coverage by activating Tie2 and the downstream signaling pathway PI3K/Akt in a rat model of ICH. ICH was induced by injection of autologous blood with or without thrombin inhibitor hirudin. Rats were treated with thrombin alone or in combination with a Tie2 inhibitor. The expression of total- and phospho-Tie2, PI3K and phospho-Akt, blood perfusion, pericyte coverage, IgG extravasation, neuron survival and neurological deficits were evaluated by western blot, fluorescein-5-isothiocyanate-dextran staining, immunohistochemistry, Nissl staining and modified neurological severity scores respectively. Induction of ICH resulted in increased phosphorylation of Tie2 on endothelial cells and pericyte coverage, better formation of integral and functional microvessels, more surviving neurons and accelerated motor function recovery, all of which were significantly attenuated by hirudin at 7 and 14 days after ICH induction. Furthermore, thrombin increased phosphorylation of Tie2 and Akt, expression of PI3K, and pericyte coverage, which were however reversed by pharmacological inhibition of Tie2. Our results demonstrated that thrombin promotes pericyte coverage on microvessels following ICH by enhancing activation of Tie2, in which the downstream PI3K/Akt signaling pathway might be involved.

Chaihu-Shugan-San Reinforces CYP3A4 Expression via Pregnane X Receptor in Depressive Treatment of Liver-Qi Stagnation Syndrome.

Backgrounds. Chaihu-Shugan-San (CSS) is a classic traditional Chinese herbal prescription for treating depression. However, the underlying mechanism of the Chinese syndrome-specific efficacy of CSS is poorly understood. Aim of the Study. From traditional Chinese medicine and pharmacogenetics perspectives, the present study aimed to investigate the antidepressant effects of CSS on a mouse model of Liver-Qi Stagnation (LQS) syndrome and its underlying mechanisms. Methods and Materials. We used two main mouse models of depressive syndromes in the study, including LQS and liver stagnation and spleen deficiency (LSSD) syndrome. Tail suspension and forced swimming tests were used to evaluate the effects of CSS on animal behaviour. The expression level of the CYP450 enzyme from liver microsomes was analysed by western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). More specifically, we analysed the key compounds of CSS that are responsible for CYP450 regulation via bioinformatics. Ultimately, luciferase assays were employed to confirm the prediction in vitro. Results. CSS remarkably reduced the immobile time in LQS rather than in LSSD mice. Although CSS significantly upregulated CYP2C9 in mice with both syndromes, activated translation of CYP3A4 induced by CSS was only observed in the LQS group. Bioinformatics analysis revealed that the unique regulation of CYP3A4 was responsible for the effects of glycyrrhetinic acid (GA) from CSS. Further luciferase assays confirmed the enhancement of CYP3A4 expression via the pregnane X receptor (PXR) pathway in vitro. Conclusions. CSS specifically upregulates the translation of CYP3A4 via the PXR pathway in depressed LQS mice. GA, a bioactive compound that originates from CSS, contributes to this activation. This work provides novel insight into Chinese syndrome-based therapy for depression.

Systematic Analysis of tRNA-Derived Small RNAs Reveals Novel Potential Therapeutic Targets of Traditional Chinese Medicine (Buyang-Huanwu-Decoction) on Intracerebral Hemorrhage.

Although Buyang-Huanwu-Decoction (BYHWD), a famous traditional Chinese medicine, has been utilized to promote the recovery of neurological function in intracerebral hemorrhage (ICH) for centuries, its therapeutic mechanisms remain unclear. tRNA-derived small RNA (tsRNA) is a novel class of short non-coding RNA, possessing potential regulating functions. In the current study, we explored the novel therapeutic targets of BYHWD by tsRNA-sequencing. Rats were randomly divided into three groups: sham, ICH, and BYHWD-treated groups. The modified neurological severity score, corner turn test, foot-fault test, and weight change were used to assess neurological injury. After BYHWD treatment, these behavioral tests were obviously meliorated compared with ICH group in the recovery phase. In the rat brain tissues surrounding the hemorrhagic region, a total of 350 tsRNAs for exact match were identified. 12 of tRNAs (fold change >1.3 and P-value <0.05) were significantly changed in ICH group compared to sham group. Among them, 3 of tRNAs (rno-tRFi-Ser-25a, rno-tRF5-Ala-16a and rno-tRF5-Glu-29a) were markedly regulated by BYHWD treatment and validated with quantitative real-time PCR. Additionally, target prediction and bioinformatics analyses revealed that these tsRNAs could play therapeutic roles through FoxO signaling pathway, positive regulation of long term synaptic depression, autophagy - animal, IL-17 signaling pathway and regulation of cytoskeleton and transforming growth factor beta. In conclusion, tsRNAs are the potential therapeutic targets of BYHWD on ICH treatment. The present study provides novel insights for future investigations to explore the mechanisms, by which BYHWD promotes neurological function recovery after ICH.

Leukemia Inhibitory Factor Decreases Neurogenesis and Angiogenesis in a Rat Model of Intracerebral Hemorrhage.

Neurogenesis and angiogenesis can improve the neurologic function after intracerebral hemorrhage (ICH). Leukemia inhibitory factor (LIF) plays an important role in neurogenesis and angiogenesis. In this study, a rat model of autologous blood-induced ICH was used to evaluate the effect of LIF on the neurogenesis and angiogenesis following ICH. After ICH, LIF-positive neurons and dilated vessels were detected in the peri-hematomal region. It was found that LIF levels increased significantly and peaked 14 days after ICH induction. Double immunofluorescence confirmed that LIF was expressed in neurons and endothelial cells. ICH also led to increases of doublecortin (DCX)- and von Willebrand factor (vWF)-positive cells as well as proliferation of cell nuclear antigen (PCNA)+/DCX+ and PCNA+/vWF+ nuclei. All these ICH-induced increases were significantly attenuated by exogenous LIF infusion. These data suggested that LIF was a negative regulator of neurogenesis and angiogenesis after ICH.