The expression profiles of signature genes from CD103+LAG3+ tumour-infiltrating lymphocyte subsets predict breast cancer survival.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs), including T and B cells, have been demonstrated to be associated with tumour progression. However, the different subpopulations of TILs and their roles in breast cancer remain poorly understood. Large-scale analysis using multiomics data could uncover potential mechanisms and provide promising biomarkers for predicting immunotherapy response. METHODS: Single-cell transcriptome data for breast cancer samples were analysed to identify unique TIL subsets. Based on the expression profiles of marker genes in these subsets, a TIL-related prognostic model was developed by univariate and multivariate Cox analyses and LASSO regression for the TCGA training cohort containing 1089 breast cancer patients. Multiplex immunohistochemistry was used to confirm the presence of TIL subsets in breast cancer samples. The model was validated with a large-scale transcriptomic dataset for 3619 breast cancer patients, including the METABRIC cohort, six chemotherapy transcriptomic cohorts, and two immunotherapy transcriptomic cohorts. RESULTS: We identified two TIL subsets with high expression of CD103 and LAG3 (CD103+LAG3+), including a CD8+ T-cell subset and a B-cell subset. Based on the expression profiles of marker genes in these two subpopulations, we further developed a CD103+LAG3+ TIL-related prognostic model (CLTRP) based on CXCL13 and BIRC3 genes for predicting the prognosis of breast cancer patients. CLTRP-low patients had a better prognosis than CLTRP-high patients. The comprehensive results showed that a low CLTRP score was associated with a high TP53 mutation rate, high infiltration of CD8 T cells, helper T cells, and CD4 T cells, high sensitivity to chemotherapeutic drugs, and a good response to immunotherapy. In contrast, a high CLTRP score was correlated with a low TP53 mutation rate, high infiltration of M0 and M2 macrophages, low sensitivity to chemotherapeutic drugs, and a poor response to immunotherapy. CONCLUSIONS: Our present study showed that the CLTRP score is a promising biomarker for distinguishing prognosis, drug sensitivity, molecular and immune characteristics, and immunotherapy outcomes in breast cancer patients. The CLTRP could serve as a valuable tool for clinical decision making regarding immunotherapy.

Plasma and cerebrospinal fluid pharmacokinetics of hydroxysafflor yellow A in patients with traumatic brain injury after intravenous administration of Xuebijing using LC-MS/MS method.

Hydroxysafflor yellow A (HSYA) is the most pharmaceutically relevant compound in Xuebijing (XBJ) for traumatic brain injury (TBI) treatment. We aimed to investigate biofluids pharmacokinetics of HSYA from XBJ to ensure the drug safety and to guide the clinical use.A sensitive, rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was applied to investigate pharmacokinetics of HSYA in TBI patients after intravenous administration of XBJ. Non-compartmental methods using DAS 3.0 software were applied to analyse the pharmacokinetic parameters.A similar half-life (Plasmat1/2: 14.55 ± 3.51 h vs. CSFt1/2: 15.73 ± 3.63) was observed. HSYA reached the peak level rapidly, but exhibited a strongly slow absorption phase from blood to cerebrospinal fluid (CSF, PlasmaTmax: 0.69 ± 0.26 h vs. CSFTmax: 4.0 ± 2.62 h). HSYA exhibited much higher Cmax (PlasmaCmax: 9342.76 ± 2489.23 µg/L vs. CSFCmax: 98.08 ± 14.51 µg/L) and AUC0-t (PlasmaAUC0-t: 57490.5 ± 5560.3 µg h/L vs. CSFAUC0-t: 1851.6 ± 269.1 µg h/L), yet a shorter CL (PlasmaCL: 0.02 ± 0.002 L/h/kg vs. CSFCL: 0.55 ± 0.01 L/h/kg) in plasma than in CSF. The AUCCSF/AUCplasma of HSYA was almost 3.37%.In summary, the results demonstrate that part of HSYA come across blood-brain barrier after XBJ administration. This study provides evidence for better understanding the pharmacokinetics and potential for clinical guidance of XBJ for TBI treatment.

Development of a sensitive and rapid UHPLC-MS/MS method for simultaneous quantification of nine compounds in rat plasma and application in a comparative pharmacokinetic study after oral administration of Xuefu Zhuyu Decoction and nimodipine.

Xuefu Zhuyu Decoction (XFZYD) is a traditional Chinese medicine prescription used for the clinical treatment of traumatic brain injury (TBI). The purpose of this work was to develop a sensitive and rapid UHPLC-MS/MS method to simultaneously study the pharmacokinetics of nimodipine and eight components of XFZYD, namely, amygdalin, hydroxysafflor yellow A, rutin, liquiritin, narirutin, naringin, neohesperidin and saikosaponin A, in rats with and without TBI. Multiple reaction monitoring was highly selective in the detection of nine analytes and the internal standard without obvious interference. The calibration curves displayed good linearity (r > 0.99) over a wide concentration range. The mean absolute recoveries of the nine analytes were 85-106%, and all matrix effects were in the range 80-120%. The intra- and inter-day precision and accuracy were acceptable (RSD, <15%; RE%, ±20%). The validated method was successfully applied to compare the pharmacokinetics in four experimental groups, including control rats orally administered XFZYD and TBI model rats orally administered XFZYD, XFZYD and nimodipine, or nimodipine alone. The results showed that herb-drug interactions occurred between XFZYD and nimodipine in the treatment of TBI, nimodipine affected the pharmacokinetics of XFZYD, and XFZYD affected the absorption, distribution and excretion of nimodipine in vivo.

The Effect of Traditional Chinese Medicine Zhike-Houpu Herbal Pair on Depressive Behaviors and Hippocampal Serotonin 1A Receptors in Rats After Chronic Unpredictable Mild Stress.

OBJECTIVE: Zhike-Houpu herbal pair (ZKHPHP) is a well-known Chinese medicine to treat gastrointestinal motility dysfunction. Recently, many researchers have found that some of the compounds of ZKHPHP such as meranzin hydrate and magnolol have antidepressant effects. However, little is known about the antidepressant mechanism of ZKHPHP. Therefore, the main aim of the study is to evaluate the antidepressant-like effects of ZKHPHP and its possible mechanism of action on 5-hydroxytryptamine receptor 1A (HTR1A) in the hippocampus CA1 region in rats exposed to chronic unpredictable mild stress. METHODS: Male Sprague Dawley rats were randomly divided into the following six groups: normal, model, ZKHPHP (3 g/kg), ZKHPHP (10 g/kg), ZKHPHP (20 g/kg), and ZKHPHP (30 g/kg); n = 8 per group. We exposed the rats to chronic unpredictable mild stress and then assessed antidepressant-like effects of ZKHPHP by measuring weight change, observing the open-field test, and measuring sucrose water consumption. The antidepressant mechanism was examined by measuring the effect of ZKHPHP on HTR1A protein expression and HTR1A mRNA expression in the hippocampus CA1 region by using immunohistochemistry analysis, Western blotting, and real-time reverse transcription-polymerase chain reaction. RESULTS: ZKHPHP (10 or 20 g/kg) reduced the incidence of depressive-like behaviors and increased HTR1A protein and HTR1A mRNA expression in the hippocampus CA1 in rats displaying depressive behavior, whereas ZKHPHP (3 or 30 g/kg) had no obvious effect on the measured depression indicators. CONCLUSIONS: These data show that ZKHPHP has antidepressant-like effects based on a chronic unpredictable mild stress-induced depression model in rats. ZKHPHP may be attractive as an antidepressant because of its beneficial effects on depression and the absence of gastrointestinal dysregulation, which is a frequently observed unintended effect of many commonly used antidepressive medications.

Rhein exhibits antioxidative effects similar to Rhubarb in a rat model of traumatic brain injury.

BACKGROUND: The brain is secondarily harmed by pathological, physiological, and biological reactions that are caused by traumatic brain injury (TBI). Rhein, a significant composition of Rhubarb, is a well-known traditional Chinese treatment method and has a strong oxidation-resisting characteristic, but Rhein's mechanism remains unclear. METHODS: This study aimed to identify Rhein in the brain tissues of TBI model of rats, and confirm whether Rhein induced an antioxidative effect similar to its parent medicine, Rhubarb. First, the ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was applied to identify Rhein in the brain tissue of the controlled cortical impact (CCI) rats after intra-gastric administration of Rhubarb. Further, for the purpose of calculating the oxidant stress of the CCI rats, the malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione disulfide (GSSG), as well as the proportion of glutathione (GSH)/GSSG were measured in the brain tissues. RESULTS: The results showed that Rhein was absorbed in the brain tissues of CCI rats. Rhubarb and rhein elevated the SOD, CAT activities, GSH level, and GSH/GSSG ratio, and diminished the MDA and GSSG levels. CONCLUSION: The data demonstrated that Rhubarb and Rhein had the potential to be used as a neuroprotective drug for TBI, and that Rhein induced an antioxidative effect similar to its parent medicine, Rhubarb.

Qualitative analysis of major constituents from Xue Fu Zhu Yu Decoction using ultra high performance liquid chromatography with hybrid ion trap time-of-flight mass spectrometry.

Xue Fu Zhu Yu Decoction, a famous formula that has been used for treating many blood stasis-caused diseases for many centuries, comprises 11 kinds of traditional Chinese medicines. A convenient, efficient, and rapid analytical method was developed to simultaneously determine the major compounds in this decoction. An ultra-high performance liquid chromatography with hybrid ion trap time-of-flight mass spectrometry method was used to rapidly separate and detect the major constituents of the decoction. Using this technique, we identified or tentatively identified 34 compounds, including 21 flavonoids, 5 terpenoids, 3 organic acids, 2 lactones, 1 alkaloid, 1 amino acid, and 1 cyanogenic glycoside. The MS analysis of these constituents was described in detail. Findings may contribute to future metabolic and pharmacokinetic studies of this medicine.

Impact of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucoside on cognitive deficits in animal models of Alzheimer's disease: a systematic review.

BACKGROUND: The efficacy of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) treatment on cognitive decline in individuals with Alzheimer's disease (AD) has not been investigated. Therefore, we systematically reviewed the effect of TSG on cognitive deficits in a rodent model of AD. METHODS: We identified eligible studies published from January 1980 to April 2015 by searching seven electronic databases. We assessed the study quality, evaluated the efficacy of TSG treatment, and performed a stratified meta-analysis and meta-regression analysis to assess the influence of study design on TSG efficacy. RESULTS: Among a total of 381 publications, 18 fulfilled our inclusion criteria. The overall methodological quality of these studies was poor. The meta-analysis revealed a statistically significant benefit of TSG on acquisition memory (standardized mean difference [SMD] = -1.46 (95 % CI: -1.81 to -1.10, P < 0.0001) and retention memory (SMD =1.93 (95 % CI: 1.40 to 2.46, P < 0.0001) in experimental models of AD. The stratified analysis revealed a significantly higher effect size for both acquisition and retention memory in studies that used mixed sex models and a significantly higher effect size for acquisition memory in studies that used transgenic models. CONCLUSIONS: Our meta-analysis highlights a significantly better treatment effect in rodent AD models that received TSG that in those that did not. These findings indicate a potential therapeutic role of TSG in AD therapy. However, additional well-designed and detailed experimental studies are needed to evaluate the safety of TSG.

An ultra high performance liquid chromatography with tandem mass spectrometry method for plasma and cerebrospinal fluid pharmacokinetics of rhein in patients with traumatic brain injury after administration of rhubarb decoction.

Damage of blood-brain barrier is a common result of traumatic brain injury. This damage can open the blood-brain barrier and allow drug passage. An ultraperformance liquid chromatography with tandem mass spectrometry method was established to determine the concentration of rhein in the biofluids (plasma and cerebrospinal fluid) of patients with a compromised blood-brain barrier following traumatic brain injury after rhubarb administration. Furthermore, the pharmacokinetic profiles were analyzed. A triple-quadruple tandem mass spectrometer with electrospray ionization was used for rhein detection. The mass transition followed was m/z 283.06→239.0. The calibration curve was linear in the concentration range of 10-8000 ng/mL for the biofluids. The intra- and interday precisions were less than 10%. The relative standard deviation of recovery was less than 15% in biological matrices. The pharmacokinetic data showed that rhein was rapidly transported into biofluids, and exhibited a peak concentration 1 h after rhubarb administration. The elimination rate of rhein was slow. The AUCcerebrospinal fluid /AUCplasma (AUC is area under curve) of rhein was approximately 17%, indicating that portions of rhein could pass the impaired blood-brain barrier. The method was successfully applied to quantify rhein in the biofluids of all patients. The data presented can help to guide clinical applications of rhubarb for treating traumatic brain injury.

The impact of ginsenosides on cognitive deficits in experimental animal studies of Alzheimer's disease: a systematic review.

BACKGROUND: The efficacy of ginsenoside treatment on cognitive decline in individuals with Alzheimer's disease (AD) has yet to be investigated. In this protocal, we conducted a systematic review to evaluate the effect of ginsenosides on cognitive deficits in experimental rodent AD models. METHODS: We identified eligible studies by searching seven electronic databases spanning from January 1980 to October 2014. We assessed the study quality, evaluated the efficacy of ginsenoside treatment, and performed a stratified meta-analysis and meta-regression analysis to assess the influence of the study design on ginsenoside efficacy. RESULTS: Twelve studies fulfilled our inclusion criteria from a total of 283 publications. The overall methodological quality of these studies was poor. The meta-analysis revealed that ginsenosides have a statistically significant positive effect on cognitive performance in experimental AD models. The stratified analysis revealed that ginsenoside Rg1 had the greatest effect on acquisition and retention memory in AD models. The effect size was significantly higher for both acquisition and retention memory in studies that used female animals compared with male animals. CONCLUSIONS: We conclude that ginsenosides might reduce cognitive deficits in AD models. However, additional well-designed and well-reported animal studies are needed to inform further clinical investigations.

Determination of hydroxysafflor yellow A in biological fluids of patients with traumatic brain injury by UPLC-ESI-MS/MS after injection of Xuebijing.

A simple, novel, specific, rapid and reproducible ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in biological fluids (plasma, urine and cerebrospinal fluid) of patients with traumatic brain injury after intravenous injection of Xuebijing (XBJ). Liquid-liquid extraction was performed, and separation was carried out on an Acquity UPLC™ BEH C18 column, with gradient elution using a mobile phase composed of methanol and 0.1% formic acid at a flow rate of 0.3 mL/min. A triple quadrupole tandem mass spectrometer with electrospray ionization was used for the detection of HSYA. The mass transition followed was m/z 611.0 → 491. The retention time was less than 3.0 min. The calibration curve was linear in the concentration range from 2 to 6125 ng/mL for cerebrospinal fluid, plasma and urine. The intra- and inter-day precisions were <10%, and the relative standard deviation of recovery was <15% for HSYA in biological matrices. The method was successfully applied for the first time to quantify HSYA in the biological fluids (especially in cerebrospinal fluid) of patients with traumatic brain injury following intravenous administration of XBJ.

18F-FP-CIT dopamine transporter PET findings in the striatum and retina of type 1 diabetic rats.

PURPOSE: Noninvasive methods used in clinic to accurately detect DA neuron loss in diabetic brain injury and diabetic retinopathy have not been reported up to now. 18F-FP-CIT is a promising dopamine transporter (DAT) targeted probe. Our study first applies 18F-FP-CIT PET imaging to assess DA neuron loss in the striatum and retina of T1DM rat model. METHODS: T1DM rat model was induced by a single intraperitoneal injection of streptozotocin (STZ) (65 mg kg-1, ip). 18F-FP-CIT uptake in the striatum and retina was evaluated at 4 weeks, 8 weeks and 12 weeks after STZ injection. The mean standardized uptake value (SUVmean) and the maximum standardized uptake value (SUVmax) were analyzed. Western blot was performed to confirm the DAT protein levels in the striatum and retina. RESULTS: PET/CT results showed that the SUV of 18F-FP-CIT was significantly reduced in the diabetic striatum and retina compared with the normal one from 4-week to 12-week (p < 0.0001). Western blots showed that DAT was significantly lower in the diabetic striatum and retina compared to the normal one for all three time points (p < 0.05). The results from Western blots confirmed the findings in PET imaging studies. CONCLUSIONS: DA neuron loss in the striatum and retina of T1DM rat model can be non-invasively detected with PET imaging using 18F-FP-CIT targeting DAT. 18F-FP-CIT PET imaging may be a useful tool used in clinic for DR and diabetic brain injury diagnosis in future. The expression level of DAT in striatum and retina may act as a new biomarker for DR and diabetic brain injury diagnosis.

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.

Enhanced antitumoral efficacy and immune response following conditionally replicative adenovirus containing constitutive HSF1 delivery to rodent tumors.

BACKGROUND: Oncolytic adenoviruses are promising as anticancer agents but have limited clinical responses. Our previous study showed that heat shock transcription factor 1 (HSF1) overexpression could increase the anti-tumor efficacy of E1B55kD deleted oncolytic adenovirus through increasing the viral burst. Due to the important roles of heat shock proteins (HSPs) in eliciting innate and adaptive immunity, we reasoned that besides increasing the viral burst, HSF1 may also play a role in increasing tumor specific immune response. METHODS: In the present study, intra-dermal murine models of melanoma (B16) and colorectal carcinoma (CT26) were treated with E1B55kD deleted oncolytic adenovirus Adel55 or Adel55 incorporated with cHSF1, HSF1i, HSP70, or HSP90 by intra-tumoral injection. Tumors were surgically excised 72 h post injection and animals were analyzed for tumor resistance and survival rate. RESULTS: Approximately 95% of animals in the Adel55-cHSF1 treated group showed sustained resistance upon re-challenge with autologous tumor cells, but not in PBS, Adel55, or Adel55-HSF1i treated groups. Only 50-65% animals in the Adel55-HSP70 and Adel55-HSP90 treated group showed tumor resistance. Tumor resistance was associated with development of tumor type specific cellular immune responses. Adel55-cHSF1 treatment also showed higher efficacy in diminishing progression of the secondary tumor focus than Adel55-HSP70 or Adel55-HSP90 treatment. CONCLUSIONS: Besides by increasing its burst in tumor cells, cHSF1 could also augment the potential of E1B55kD deleted oncolytic adenovirus by increasing the tumor-specific immune response, which is beneficial to prevent tumor recurrence. cHSF1 is a better gene for neoadjuvant immunotherapy than other heat shock protein genes.

Integrated Analysis of Single-Cell and Bulk RNA-Sequencing Reveals a Tissue-Resident Macrophage-Related Signature for Predicting Immunotherapy Response in Breast Cancer Patients.

Immune checkpoint therapy (ICT) is among the widely used treatments for breast cancer (BC), but most patients do not respond to ICT and the availability of the predictive biomarkers is limited. Emerging evidence indicates that tissue-resident macrophages (RTMs) inhibit BC progression, suggesting that their presence may predict immunotherapy response. A single-cell RNA-sequencing analysis of BC samples was performed to identify five RTM clusters with a mixed phenotype of M1-M2 macrophages. The comprehensive results showed that a high score of each RTM cluster was associated with a high infiltration of CD8+ T cells, M1 macrophages, and dendritic cells, and improved overall survival. In addition, a low score of each RTM cluster was associated with a high infiltration of M0 macrophages, naïve B cells and Tregs, and poor overall survival. Gene signatures from each RTM cluster were significantly enriched in responders compared with nonresponders. Each RTM cluster expression was significantly higher in responders than in nonresponders. The analyses of bulk RNA-seq datasets of BC samples led to identification and validation of a gene expression signature, named RTM.Sig, which contained the related genes of RTM clusters for predicting response to immunotherapy. This study highlights RTM.Sig could provide a valuable tool for clinical decisions in administering ICT.

Effects of Meranzin Hydrate On the LncRNA-miRNA-mRNA Regulatory Network in the Hippocampus of a Rat Model of Depression.

Meranzin hydrate (MH) is a frequently used antidepressant drug in China; however it underlying mechanism remains unknown. In this study, we aimed to explore whether MH could ameliorate depression-like behavior in rats by regulating the competitive endogenous RNA (ceRNA) network. We developed a depression-like rat model using an unpredictable chronic mild stress (UCMS) protocol, and the differentially expressed lncRNAs, miRNAs, and mRNAs were identified between the model group and MH group. Then, a ceRNA network responding to MH treatment was constructed by their corresponding relationships in the databases. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to explore molecular mechanisms associated with MH treatment. The study indicated that rats in the model group showed loss of weight and deteriorated behavior in behavior tests compared with rats in the normal group. A total of 826 lncRNAs, 121 miRNAs, and 954 mRNAs were differentially expressed in the hippocampus of UCMS rats after MH treatment. In addition, 13 miRNAs were selected, and 12 of them were validated in the hippocampus by qRT-PCR. Then, we predicted upstream lncRNAs and downstream mRNAs of the validated miRNAs and interacted with the results of microarrays. Eventually, a lncRNA-miRNA-mRNA regulatory network, responding to MH treatment, was constructed based on the 314 lncRNAs, 11 miRNAs, and 221 mRNAs. KEGG pathways suggested that these genes may be highly related to Wnt signaling, axon guidance, and MAPK signaling pathways. All these results suggest that MH may be a potential representative compound for the treatment of depression, and its mechanism of action is related to the ceRNA modification.

Integrated metabolomics and network pharmacology to reveal the mechanisms of hydroxysafflor yellow A against acute traumatic brain injury.

Traumatic brain injury (TBI) has become a leading cause of mortality, morbidity and disability worldwide. Hydroxysafflor yellow A (HSYA) is effective in treating TBI, but the potential mechanisms require further exploration. We aimed to reveal the mechanisms of HSYA against acute TBI by an integrated strategy combining metabolomics with network pharmacology. A controlled cortical impact (CCI) rat model was established, and neurological functions were evaluated. Metabolomics of brain tissues was used to identify differential metabolites, and the metabolic pathways were enriched by MetaboAnalyst. Then, network pharmacology was applied to dig out the potential targets against TBI induced by HSYA. The integrated network of metabolomics and network pharmacology was constructed based on Cytoscape. Finally, the obtained key targets were verified by molecular docking. HSYA alleviated the neurological deficits of TBI. Fifteen potentially significant metabolites were found to be involved in the therapeutic effects of HSYA against acute TBI. Most of these metabolites were regulated to recover after HSYA treatment. We found 10 hub genes according to network pharmacology, which was partly consistent with the metabolomics findings. Further integrated analysis focused on 4 key targets, including NOS1, ACHE, PTGS2 and XDH, as well as their related core metabolites and pathways. Molecular docking showed high affinities between key targets and HSYA. Region-specific metabolic alterations in the cortex and hippocampus were illuminated. This study reveals the complicated mechanisms of HSYA against acute TBI. Our work provides a novel paradigm to identify the potential mechanisms of pharmacological effects derived from a natural compound.

TMT-based proteomics analysis reveals the protective effects of Xuefu Zhuyu decoction in a rat model of traumatic brain injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuefu Zhuyu decoction (XFZYD) is a traditional Chinese herbal prescription. It is effective in treating traumatic brain injury (TBI). However, the underlying molecular mechanisms remain unclear. AIM OF THE STUDY: This study aimed to reveal the possible mechanisms of XFZYD in treating acute TBI through proteomics clues. MATERIALS AND METHODS: Controlled Cortical Impact (CCI) rats were given gavage administration of XFZYD (9 g/kg/d) or distilled water (equal volume) for three days. The Modified Neurological Severity Score (mNSS), brain water content, HE staining, Nissl staining and immunohistochemistry were performed to assess the effects of XFZYD for TBI treatment. Additionally, tandem mass tag-based (TMT) quantitative proteomics technology was applied to detect proteins of brain cortex. Bioinformatics analysis including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Protein-protein interaction (PPI) networks were used to analyze differentially expressed proteins (DEPs). Bioinformatics Analysis Tool for Molecular mechanism of TCM (BATMAN-TCM) was conducted to anchor diseases and pathways. Besides, western blotting and immunofluorescence were exerted to verify related proteins. RESULTS: XFZYD improved neurologic functions, reduced encephaledema and ameliorated cell morphology around the injured area in CCI rats. A total of 6099 proteins were identified with false discovery rate (FDR) < 1%. Overlapping DEPs (105 DEPs) were identified (295 DEPs and 804 DEPs in CCI/Sham or XFZYD/CCI group, respectively). Of these DEPs, 17 were regulated by XFZYD. Bioinformatics analysis showed that the 17 DEPs were predominantly related to platelet activation and PI3K-Akt signaling pathway. Next, PLG and CD34 were verified with molecular biotechnology. CONCLUSIONS: XFZYD exerts therapeutic effects through multi-pathways regulation in the treatment of TBI. This work may provide proteomics clues for the continuation of research on TBI treatment with XFZYD.

UPLC-ESI-IT-TOF-MS metabolomic study of the therapeutic effect of Xuefu Zhuyu decoction on rats with traumatic brain injury.

It has been widely reported that Xuefu Zhuyu decoction (XFZYD), a traditional Chinese medicine, is effective in the treatment of traumatic brain injury (TBI). However, the mechanism of the therapeutic process is still not fully understood. Metabolomic technique can be used to explore the mechanisms underlying the treatment of TBI with XFZYD. The purpose of this work was to investigate the metabolic characteristics of blood samples from rats with and without XFZYD treatment and the dynamic changes in metabolite profiles on days 1, 3, 7, 14 and 21 after injury (within the severe phase of TBI) based on untargeted UPLC-ESI-IT-TOF-MS analysis. Pattern recognition, clustering analysis and metabolic pathway analysis were used to analyse the metabolomic data of three groups (a sham-operated group, a TBI model, and an XFZYD-treated TBI model). The results showed that XFZYD reversed the abnormalities in the levels of small-molecule metabolites (such as L-acetylcarnitine, L-tryptophan, indoleacrylic acid, γ-aminobutyric acid, hypotaurine, LysoPC(18:1)(11Z), creatine, L-phenylalanine and L-leucine) in TBI rats through six metabolic pathways (including phenylalanine, tyrosine and tryptophan biosynthesis; phenylalanine metabolism; valine, leucine and isoleucine biosynthesis; taurine and hypotaurine metabolism; tryptophan metabolism; and alanine, aspartate and glutamate metabolism) involved in the therapy process. XFZYD regulated the metabolic disorders of endogenous markers by the possible mechanisms of neuroprotection, energy metabolism, inflammatory response and oxidative stress. This study revealed the holistic and dynamic metabolic changes caused by XFZYD in rats with TBI and provided important research methods and approaches for exploring the multiple metabolites and metabolic pathways involved in the therapeutic effect of XFZYD on TBI.

Identifying circRNA-associated-ceRNA networks in the hippocampus of Aß1-42-induced Alzheimer's disease-like rats using microarray analysis.

Alzheimer's disease (AD) is the most common form of dementia worldwide. Accumulating evidence indicates that non-coding RNAs are strongly implicated in AD-associated pathophysiology. However, the role of these ncRNAs remains largely unknown. In the present study, we used microarray analysis technology to characterize the expression patterns of circular RNAs (circRNAs), microRNAs (miRNAs), and mRNAs in hippocampal tissue from Aß1-42-induced AD model rats, to integrate interaction data and thus provide novel insights into the mechanisms underlying AD. A total of 555 circRNAs, 183 miRNAs and 319 mRNAs were identified to be significantly dysregulated (fold-change ≥ 2.0 and p-value < 0.05) in the hippocampus of AD rats. Quantitative real-time polymerase chain reaction (qRT-PCR) was then used to validate the expression of randomly-selected circRNAs, miRNAs and mRNAs. Next, GO and KEGG pathway analyses were performed to further investigate ncRNAs biological functions and potential mechanisms. In addition, we constructed circRNA-miRNA and competitive endogenous RNA (ceRNA) regulatory networks to determine functional interactions between ncRNAs and mRNAs. Our results suggest the involvement of different ncRNA expression patterns in the pathogenesis of AD. Our findings provide a novel perspective for further research into AD pathogenesis and might facilitate the development of novel therapeutics targeting ncRNAs.