Dataset Information

Identification of novel biomarkers and therapeutic target candidates for stasis-heat symptom pattern of acute intracerebral hemorrhage by quantitative plasma proteomics.

ABSTRACT:

Objective

To explore the novel biomarkers and therapeutic target candidates related to the stasis-heat syndrome of acute intracerebral hemorrhage (AICH).

Methods

Applying an isobaric tagging for relative and absolute quantitation-(iTRAQ-) based quantitative proteomic approach, plasma samples from AICH patients with stasis-heat, and AICH patients with non-stasis-heat and healthy control subjects were collected and analyzed to distinguish differentially expressed proteins (DEPs) correlated to AICH with stasis-heat in this block design. The standard Western blot was applied to verify DEPs. Additionally, DEPs were analyzed via bioinformatic platforms and further approved via Ingenuity Pathway Analysis (IPA).

Results

A total of 26 DEPs were found among AICH with the stasis-heat, AICH with non-stasis-heat, and healthy control group. The seven DEPs compared with the non-stasis-heat group are closely related to the pathogenesis of stasis heat. These proteins showed three different protein expression patterns. The alpha-1-b glycoprotein (A1BG) and copper-protein (CP) were up-regulated in the stasis-heat group, but down-regulated in the non-stasis-heat group. Compared with the non-stasis-heat group, the expression abundance of actinin, alpha 1 (ACTN1), carbonic anhydrase I (CA1), peroxiredoxin 2 (PRDX2), and vinculin (VCL) is higher in the stasis-heat group, while the CD44 is the opposite. These differences reflect that stasis-heat syndrome has more severe inflammatory immune response, coagulation disorders and damage. Bioinformatics analysis revealed that a wide variety of cellular and metabolic processes and some signaling pathways were involved in the pathophysiology of AICH with stasis-heat. AICH with stasis-heat syndrome showed more severe inflammatory reactions, tissue damage, and coagulation disorders than non-stasis heat syndrome.

Conclusions

There are differences in the protein expression patterns between the stasis-heat syndrome and non-stasis-heat syndrome. These differences reflect that stasis-heat syndrome has more severe damage. CD44, CP, ACTN1, CA1, VCL, PRDX2, and A1BG could be the potential biomarkers and therapeutic target candidates of the stasis-heat subtype. This study provides a reasonable explaination for Liangxue Tongyu decoction through anti-inflammatory and brain protection treatment.

SUBMITTER: Lexin W

PROVIDER: S-EPMC9924781 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Publications

Identification of novel biomarkers and therapeutic target candidates for stasis-heat symptom pattern of acute intracerebral hemorrhage by quantitative plasma proteomics.

Lexin Wei W Weiyi L I LI Ting Tian T Ning Zhang Z Shijing Yang Y Dongqing Yang Y Guochun L I LI Fang Y E YE

Journal of traditional Chinese medicine = Chung i tsa chih ying wen pan 20220801 4

<h4>Objective</h4>To explore the novel biomarkers and therapeutic target candidates related to the stasis-heat syndrome of acute intracerebral hemorrhage (AICH).<h4>Methods</h4>Applying an isobaric tagging for relative and absolute quantitation-(iTRAQ-) based quantitative proteomic approach, plasma samples from AICH patients with stasis-heat, and AICH patients with non-stasis-heat and healthy control subjects were collected and analyzed to distinguish differentially expressed proteins (DEPs) cor ...[more]

PMID: 35848980

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Project description:BackgroundThe systematic mechanisms of acute intracerebral hemorrhage are still unknown and unverified, although many recent researches have indicated the secondary insults. This study was aimed to disclose the pathological mechanism and identify novel biomarker and therapeutic target candidates by plasma proteome.MethodsPatients with AICH (n = 8) who demographically matched healthy controls (n = 4) were prospectively enrolled, and their plasma samples were obtained. The TMT-LC-MS/MS-based proteomics approach was used to quantify the differential proteome across plasma samples, and the results were analyzed by Ingenuity Pathway Analysis to explore canonical pathways and the relationship involved in the uploaded data.ResultsCompared with healthy controls, there were 31 differentially expressed proteins in the ICH group (P < 0.05), of which 21 proteins increased while 10 proteins decreased in abundance. These proteins are involved in 21 canonical pathways. One network with high confidence level was selected by the function network analysis, in which 23 proteins, P38MAPK and NFκB signaling pathways participated. Upstream regulator analysis found two regulators, IL6 and TNF, with an activation z-score. Seven biomarker candidates: APCS, FGB, LBP, MGMT, IGFBP2, LYZ, and APOA4 were found. Six candidate proteins were selected to assess the validity of the results by subsequent Western blotting analysis.ConclusionOur analysis provided several intriguing pathways involved in ICH, like LXR/RXR activation, acute phase response signaling, and production of NO and ROS in macrophages pathways. The three upstream regulators: IL-6, TNF, LPS, and seven biomarker candidates: APCS, APOA4, FGB, IGFBP2, LBP, LYZ, and MGMT were uncovered. LPS, APOA4, IGFBP2, LBP, LYZ, and MGMT are novel potential biomarkers in ICH development. The identified proteins and pathways provide new perspectives to the potential pathological mechanism and therapeutic targets underlying ICH.

Project description:Background: A reliable distinction between ischemic stroke (IS) and intracerebral hemorrhage (ICH) is required for diagnosis-specific treatment and effective secondary prevention in patients with stroke. However, in resource-limited settings brain imaging, which is the current diagnostic gold standard for this purpose, is not always available in time. Hence, an easily accessible and broadly applicable blood biomarker-based diagnostic test differing stroke subtypes would be desirable. Using an explorative proteomics approach, this pilot study aimed to identify novel blood biomarker candidates for distinguishing IS from ICH. Material and Methods: Plasma samples from patients with IS and ICH were drawn during hospitalization and were analyzed by using liquid chromatography/mass spectrometry. Proteins were identified using the human reference proteome database UniProtKB, and label-free quantification (LFQ) data were further analyzed using bioinformatic tools. Results: Plasma specimens of three patients with IS and four patients with ICH with a median National Institute of Health Stroke Scale (NIHSS) of 12 [interquartile range (IQR) 10.5-18.5] as well as serum samples from two healthy volunteers were analyzed. Among 495 identified protein groups, a total of 368 protein groups exhibited enough data points to be entered into quantitative analysis. Of the remaining 22 top-listed proteins, a significant difference between IS and ICH was found for Carboxypeptidase N subunit 2 (CPN2), Coagulation factor XII (FXII), Plasminogen, Mannan-binding lectin serine protease 1, Serum amyloid P-component, Paraoxonase 1, Carbonic anhydrase 1, Fibulin-1, and Granulins. Discussion: In this exploratory proteomics-based pilot study, nine candidate biomarkers for differentiation of IS and ICH were identified. The proteins belong to the immune system, the coagulation cascade, and the apoptosis system, respectively. Further investigations in larger cohorts of patients with stroke using additional biochemical analysis methods, such as ELISA or Western Blotting are now necessary to validate these markers, and to characterize diagnostic accuracy with regard to the development of a point-of-care-system for use in resource-limited areas.

Project description:Intracerebral hemorrhage- (ICH-) induced secondary brain injury (SBI) is a very complex pathophysiological process. However, the molecular mechanisms and drug targets of SBI are highly intricate and still elusive, yet a clear understanding is crucial for the treatment of SBI. In the current study, we aimed to confirm that nuclear factor-E2-related factor 2 (Nrf2)/Optineurin- (OPTN-) mediated mitophagy alleviated SBI by inhibiting nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation based on the isobaric tag for relative and absolute quantization (iTRAQ) quantification proteomics. Human ICH brain specimens were collected for iTRAQ-based proteomics analysis. Male Nrf2 wild-type (WT) and knockout (KO) mice were employed to establish ICH murine models. The survival rate, hematoma volume, neurofunctional outcomes, blood-brain barrier (BBB) permeability, brain edema, spatial neuronal death, NLRP3 inflammasome, inflammatory response, mitochondrial function, and mitophagy level were evaluated after ICH. The iTRAQ quantification analysis showed that the differentially expressed proteins (DEPs), Nrf2 and NLRP3, were closely associated with the initiation and development of SBI after ICH. The Nrf2 KO mice had a significantly lower survival rate, bigger hematoma volume, worse neurological deficits, and increased BBB disruption, brain edema, and neuronal death when compared with the Nrf2 WT mice after ICH. Furthermore, Nrf2 KO enhanced NLRP3 inflammasome activation and neuroinflammation as evidenced by the NF-κB activation and various proinflammatory cytokine releases following ICH. Moreover, Nrf2 could interact with and modulate the mitophagy receptor OPTN, further mediating mitophagy to remove dysfunctional mitochondria after ICH. Furthermore, OPTN small interfering RNA (siRNA) increased the NLRP3 inflammasome activation by downregulating mitophagy level and enhancing mitochondrial damage in the Nrf2 WT mice after ICH. Together, our data indicated that Nrf2/OPTN inhibited NLRP3 inflammasome activation, possibly via modulating mitophagy, therefore alleviating SBI after ICH.

Project description:ObjectiveIntracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and morbidity rates. Our aim was to comprehensively analyze transcriptome and proteome in an experimental ICH model.MethodsAll mice were divided into ICH model (n = 3) and sham groups (n = 3). ICH was induced by collagenase VII. The ipsilateral hemisphere was used for whole transcriptome and proteomics resequencing. After preprocessing, differentially expressed lncRNAs (DElncRNAs), mRNAs (DEmRNAs), miRNAs (DEmiRNAs), and DEproteins between ICH and sham groups were identified. Functional enrichment analysis was performed using the clusterProfiler package, followed by protein-protein interaction (PPI) analysis. After that, the Pearson correlation coefficient between DEmRNAs and DElncRNAs or between DEmRNAs and DEproteins was calculated. DElncRNAs with similar functions were analyzed by the GOSemSim package. After prediction of DEmiRNA-DEmRNA and DElncRNA-DEmiRNA relationships, a competing endogenous RNA (ceRNA) network was constructed. Several DEmRNAs and DElncRNAs were validated in ipsilateral hemisphere tissues of the ICH model and control groups using RT-qPCR and western blot.ResultsBetween the ICH and sham groups, 31 DElncRNAs, 367 DEmRNAs, 35 DEmiRNAs, and 96 DEproteins were identified. DEmRNAs were mainly enriched in inflammation, such as cytokine-cytokine receptor interaction, IL-17, and TNF signaling pathways. A PPI network of DEmRNAs was constructed and hub genes were identified, such as IL6 (degree = 59), TNF (degree = 44), and CXCR2 (degree = 39). 24 DElncRNAs with similar functions were identified, including 15 up- and 9 down-regulated lncRNAs. After integration of DEmiRNA-DEmRNA and DElncRNA-DEmiRNA relationships, we constructed a ceRNA network, composed of 71 DEmRNAs, 17 DEmiRNAs, and 12 DElncRNAs. RT-qPCR and western blot results confirmed that C3, Fga, and Slc4a1 proteins were more lowly expressed and Penk was more highly expressed in ICH than control groups, which could become potential markers for ICH.ConclusionOur findings identified ICH-related DE-RNAs and proteins and potential molecular mechanisms of ICH by transcriptome resequencing and quantitative proteomic analyses.

Project description:Intracerebral hemorrhage (ICH) prognostication during the acute phase is often subjective among physicians and often affects treatment decisions. The present study explores objective imaging parameters using quantitative corticospinal tract (CST) fiber reconstruction during the acute phase of ICH and correlates these parameters with functional outcome and patient recovery. We prospectively enrolled nonsurgical spontaneous supratentorial ICH patients and obtained an MRI scan on day 5 ± 1. Q-space diffeomorphic reconstruction was performed using DSI Studio, and quantitative anisotropy (QA) was calculated. The CST was reconstructed based on QA. The dichotomized modified Rankin Scale score on day 90 (favorable outcome = 0-2) and Barthel Index (favorable recovery = 100 on day 90 or improvement between discharge and day 90 > 60%) were assessed. Thirty-three patients, median age 72 years (interquartile range (IQR) 64-83), 21 female (64%), 21 (64%) with lobar hemorrhage, median ICH volume on admission 15.0 (IQR 7.0-27.4) mL, were included. Sixteen patients (48%) had a favorable outcome and 24 (73%) had a favorable recovery. The mean number of ipsilesional reconstructed CST fiber pathways was higher in patients with favorable outcomes (153 (standard deviation (SD) 103) vs. 60 (SD 39), p = 0.003) and predicted outcome after adjustment (Exp(B) = 1.016 (95% CI = 1.002-1.030)). QA in the ipsilesional posterior limb of the internal capsule showed a trend towards an association with favorable outcome (Exp(B) = 1.194 (95% CI = 0.991-1.439 (adjusted))). The total (ipsilesional + contralesional) number of reconstructed fiber pathways was associated with favorable recovery (Exp(B) = 1.025 (95% CI = 1.003-1.047 (adjusted))). Quantitative tractography parameters assessed in the acute phase of ICH may represent a promising predictor of long-term outcome and recovery. This might facilitate prognostic evaluation and organization of rehabilitation.

Dataset Information

Identification of novel biomarkers and therapeutic target candidates for stasis-heat symptom pattern of acute intracerebral hemorrhage by quantitative plasma proteomics.

Objective

Methods

Results

Conclusions

Publications

Identification of novel biomarkers and therapeutic target candidates for stasis-heat symptom pattern of acute intracerebral hemorrhage by quantitative plasma proteomics.

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