Exploration Of Biomarkers For Predicting Hematoma Expansion After Intracerebral Hemorrhage

Part 1 Exploration of biomarkers for predicting hematoma expansion based on conventional laboratory testsObjective: Intracerebral hemorrhage(ICH)is a severe cerebrovascular disease with high rates of disability and mortality.Hematoma expansion(HE)occurs in about 1/3 of patients after ICH,and often accompanies with deterioration of the disease.HE is proved to be a strong predictor of poor post-ICH outcome.Effective and reliable prediction of HE is of great significance for early control of HE and improvement of prognosis.Predictive biomarkers based on conventional laboratory tests are convenient,fast,economical,stable and accessible.We aimed to explore biomarkers for predicting HE based on conventional laboratory tests and investigate the association between the biomarker and post-ICH prognosis,so as to develop effective interventions for ICH.Methods: We retrospectively analyzed consecutive acute ICH patients from January 2012 to November 2020 with admission laboratory tests and baseline computed tomography(CT)within 6 hours from symptom onset and follow-up CT within 48 hours from the initial CT.Univariate and multivariate regression analyses were used to explore the potential biomarkers of HE.The results were verified in a prospective validation cohort from December 2020 to November 2021.The relationship between the selected biomarker and 3-month outcome after ICH was also investigated and mediation analysis was undertaken to determine the role of HE in the effect of selected biomarker on poor outcomes.Results: Of 734 ICH patients,163(22.2%)patients presented HE.Higher direct bilirubin was associated with HE [adjusted odds ratio(OR)of per 1.0 μmol/L change 1.082,95% confidence interval [CI] 1.011-1.158,P=0.023],and the direct bilirubin level influenced the risk of HE in a dose-response relationship.When the direct bilirubin was regarded as dichotomous variable with the cut-off value of 5.65 μmol/L in the validation cohort,the direct bilirubin > 5.65 μmol/L was a predictor of HE(adjusted OR 4.476,95% CI 1.404-14.270,P=0.011).Higher direct bilirubin was also associated with poor 3-month outcome (adjusted OR of per 1.0 μmol/L change 1.145,95% CI 1.058-1.245,P=0.001).Mediation analysis revealed that HE partially mediated association of higher direct bilirubin with poor outcome.Conclusions: Direct bilirubin is a predictor of HE and poor 3-month outcome after ICH.Its involvement in oxidative stress response and brain injury may be the underlying mechanisms associated with HE.As a small molecule product of oxidative stress metabolism,the signal molecules regulating the metabolism of direct bilirubin involved in the upstream pathway deserve further exploration.Interventions aiming at direct bilirubin to improve post-ICH prognosis have potential clinical significance and feasibility.Part 2 Exploration of biomarkers for predicting hematoma expansion based on proteomicsObjective: Hematoma expansion(HE)is proved to be a strong predictor of poor outcome after intracerebral hemorrhage(ICH).The pathological mechanism of HE is not completely clear,and effective treatments are relatively limited.Plasma proteomics is a reliable and powerful method for screening biomarkers.We aimed to explore the expression of regulatory proteins involved in direct bilirubin metabolism and investigate novel laboratory biomarkers for HE using Tandem Mass Tags(TMT)-based quantitative proteomics technique.Exploring the biological function,mechanism and related pathways of biomarkers can provide basis and a new direction for further investigating the mechanism of HE and looking for feasible therapeutic intervention targets.Methods: We prospectively collected clinical data of acute ICH patients from December 2020 to November 2021 with baseline CT within 6 hours from symptom onset.Blood samples were collected from patients who met the requirements after admission and the plasma was separated.All patients conducted follow-up CT within 24 to 48 hours from the initial CT.Blood samples within 24 hours from symptom onset were selected for analysis.Blood samples from five patients with HE and five matched patients without HE were selected,and TMT proteomics technique was used to identify the differentially expressed proteins between two groups.Bioinformatics analysis of differentially expressed proteins was performed using the gene ontology(GO)database and the Kyoto encyclopedia of genes and genomes(KEGG)database.Candidate biomarkers were screened based on the possible mechanism of HE,referring to bioinformatics analysis and literature review.Univariate and multivariate analyses were performed to verify the selected biomarkers in the whole eligible cohort.In addition,correlation analysis was conducted to explore its correlation with hematoma expansion volume and the area under the receiver operating characteristic curve was calculated to explore its ability to predict HE.Results: In the proteomic screening phase,a total of 297 proteins were differentially expressed,including 140 up-regulated proteins and 157 down-regulated proteins.Among the differentially expressed proteins,biliverdin reductase,which is involved in the porphyrin metabolic pathway and a key regulatory protein of direct bilirubin,was significantly upregulated.Referring to fold change,pathways associated with HE and pathways with higher rich level as well as literature review,complement C3,syndecan-4(SYND4),peroxiredoxin-2(Prx-2)and platelet basic protein(PBP)were selected as candidate biomarkers,and were further verified.The levels of the four proteins were higher in patients with HE compared with patients without HE and the difference was statistically significant(P<0.05).They also positively correlated with the absolute volume of HE and had ability to predict HE.In multivariate analyses,the four proteins were classified as categorical variables and were associated with HE after adjusted for gender,age and time from symptom onset to initial CT.Conclusions: TMT proteomic technology is able to reveal the plasma proteomic characteristics of HE after ICH.The level of direct bilirubin can reflect the level of a regulated response to oxidative stress in the body under pathological stimulation.Complement C3,SYND4,Prx-2 and PBP are feasible biomarkers of HE.Pathological processes and molecular pathways related to immunity and cell adhesion may be involved in HE.PI3K-Akt signaling pathway and pathways related to neurodegenerative diseases may be associated with HE.The pathological mechanism of HE still needs to be further explored,and it is expected to make up for the defects of existing therapies and develop reliable and effective new therapeutic targets.