| Drug-induced liver injury(DILI)is one of the most common adverse drug reactions in clinical practice.According to epidemiological studies at home and abroad,the incidence of DILI presents an upward trend,and the disease burden brought by DILI cannot be ignored.It is of great significance to study and improve the clinical diagnosis and treatment level of DILI.However,due to the lack of specific diagnostic indicators,the current clinical diagnosis of DILI mainly adopts the strategy of exclusion method,and there are great challenges for accurate clinical diagnosis and evaluation.Therefore,based on clinically accessible biological specimens such as patient serum,DILI-related biomarkers were found,and the correlation between these potential biomarkers and DILI clinical phenotype and histopathological phenotype was investigated.Subsequently,some biomarkers and their combinations that can better distinguish DILI patients with different pathogenesis,damage target cell types,and different pathological damage degrees were screened and verified in the cohort of the external verification center,which has potential important value for assisting in improving the level of clinical diagnosis and treatment.Furthermore,based on these biomarker combinations with better distinguishing and diagnostic capabilities,the biological pathway enrichment analysis was performed,to provide a new perspective for understanding the potential mechanism in the complex clinical phenotype of DILI,as well as a reference for promoting clinical translational medicine research of DILI.Therefore,it is an important direction worthy of in-depth study.The main research contents and results in this paper are as follows:1.Establishment of DILI cohort and specimen collectionReferring to the diagnostic criteria of DILI clinical diagnosis and treatment guidelines issued by the American College of Gastroenterology,Chinese Medical Association,and China Association of Chinese Medicine,a screening center cohort(198 cases)and an external verification center cohort(21 cases)were established respectively,and 219 eligible DILI patients and their serum samples were included.The median age of DILI patients included in the study was 49.0 years old,and the ratio of males to females was 1: 2.4.According to the evaluation criteria of the current diagnosis and treatment guidelines,the clinical classification(R-value classification)of DILI patients included in the study belongs to hepatocellular type,cholestatic type,and mixed type,of which the number and composition ratio is 75 cases(34.2%),97 cases(44.3%)and 47 cases(21.5%)respectively;the number and composition ratio of mild(grade ?),moderate(grade ?)and severe(grade ?)liver injury were 127 cases(58.0%),27 cases(12.3%)and 65 cases(29.7%),respectively.Among the 219 DILI patients,107 cases had liver tissue puncture and pathological examination results,accounting for 48.8% of the total cases.Among them,the number and composition ratio of hepatocellular type,cholestatic type,and mixed type were 58cases(54.2%),16 cases(14.9%),and 17 cases(15.9%),respectively,16 cases(14.9%)were not classified;according to liver tissue inflammatory activity grade,the number,and composition ratio of G1 ? G4 were 14 cases(13.1%),36 cases(33.6%),34 cases(31.8%),and 13 cases(12.1%),respectively,and the remaining 10 cases(9.3%)were not classified;according to the liver fibrosis stage,the number of cases and composition ratio of S0 ? S4 were 16 cases(14.9%),22 cases(20.6%),33 cases(30.8%),10 cases(9.3%)and 4 cases(3.7%)respectively,and the remaining 22 cases(20.6%)were not staged.2.Untargeted metabonomic detection of DILI serum based on UPLC-Q-TOF-MSUPLC-Q-TOF-MS was used to detect the collected serum samples of DILI patients.Methodological investigation showed that quality control(QC)samples had a good aggregation degree,which indicated that the sample processing and instrument detection methods were reliable and met the basic requirements of metabolomics detection and analysis.A total of 22,459 compounds were detected from the metabolomics detection data of serum samples from 219 DILI patients.By searching,aligning,and identifying the Human Metabolome Database(HMDB),a total of 2,237 metabolites were found,which were roughly divided into phospholipids and sphingolipids(309,13.8%),fatty acids(161,7.2%),steroids(122,5.4%),sugars(45,2.0%),peptides and amino acids(656,29.3%),vitamins(90,4.0%),nucleosides(59,2.6%),and ubiquinones(25,1.1%),etc.A total of 2237 data sets of relative abundances of metabolites and their inter-group differences were established,which provided data preparation for subsequent multivariate statistical analysis and biomarker screening.3.Screening of metabonomic markers related to pathological damage target cell typesAt present,the R-value typing method based on serological enzyme biochemical indexes is widely used in clinical guidelines at home and abroad to classify DILI into target cell injury types,such as hepatocellular type,cholestatic type,and mixed type.Different clinical types have different clinical treatment schemes and prognoses.However,this paper analyzed 75 cases with pathological typing in the screening cohort,and found that the consistency between the results of serological R-value typing and the results of pathological actual injury types was poor(Kappa = 0.044 << 0.2),strongly suggesting that serological R-value typing cannot meet the clinical requirements for accurate differentiation and diagnosis of DILI target cell injury types.Based on the serum metabonomic data of 75 cases with pathological classification,the metabonomic classification model of target cell injury types was established based on pathological classification.The sensitivity and specificity of the model reached63.6%,73.6%,54.6% and 98.4%,81.8%,72.0%,respectively;in contrast,the sensitivity and specificity of serological R-value typing were only 45.4%,43.4%,36.4%and 73.4%,50.0%,76.5%,respectively.The data from the external verification center showed that the classification diagnosis model had a better recognition effect than the R-value classification.These results suggest that the classification model of target cell injury types established based on serum metabolomics screening had better accuracy,which was generally superior to the serological R-value typing method.It is expected to carry out further study and develop a new low-invasive auxiliary diagnosis method,which can be used to replace or reduce liver tissue puncture examination to a certain extent,with a good research prospect.4.Screening of metabonomic markers related to the degree of inflammation or fibrosis of liver tissueLiver biopsy can provide important information for clinical diagnosis and evaluation of DILI,but there are contraindications and compliance problems,so it is of great significance to find new alternative biomarkers.Therefore,based on the serum metabonomic data of cases with the pathological examination of liver tissue inflammation and fibrosis,two groups of biomarkers that can reflect the degree of liver tissue inflammation and judge liver fibrosis were screened,respectively.The results showed that 41 serum metabolites were highly correlated with the degree of liver inflammation(G1 ? G4).Then a metabonomic classification model for the degree of liver injury was established,and the classification model was correlated with pathological examination results(Kappa = 0.321).In addition,43 serum metabolites were highly correlated with liver tissue fibrosis(S0 ? S4).The diagnostic model of liver fibrosis established by these metabolites also had a good correlation with pathological examination results(kappa = 0.741)and could significantly improve the APRI score(aspartate aminotransferase-to-platelet ratio index)and FIB-4 index(fibrosis 4 score,4-factor liver fibrosis index)in diagnosing liver fibrosis(AUC = 0.953,0.955).Similar results were observed in external verification centers.Therefore,biomarkers based on serum metabolomics screening can better monitor and judge the true degree of liver tissue pathological damage,further suggesting that it is expected to become an auxiliary diagnostic method to reduce or replace liver puncture.5.Comparative study of DILI with different pathogenesis based on serum metabolomeCurrently,the pathogenesis of DILI is usually divided into intrinsic type,idiosyncratic type,and indirect type in clinical guidelines.The above classification is mainly based on the empirical consensus opinions of expert groups,and there is still a lack of systematic representation and presentation of objective experimental indicators.Therefore,this paper systematically compares and investigates the objective authenticity of the differences among the three types of DILI from the level of serum metabolomes.The results showed that the intrinsic and idiosyncratic DILI samples showed obvious differences in unsupervised PCA analysis and were divided into two groups,suggesting that are significantly different at least at the metabolome level.This result is consistent with the international consensus opinion in recent 30 years that DILI is divided into two different pathogenesis: intrinsic type and idiosyncratic type.The results of this paper well support the mainstream view of distinguishing intrinsic DILI from idiosyncratic DILI.Furthermore,indirect DILI samples were added to PCA analysis,and the results showed that indirect DILI samples did not present an independent group,but had more overlap with idiosyncratic hepatotoxicity(the proportion of overlapping samples was45.4%),suggesting that the newly proposed indirect DILI in 2019 may not be regarded as independent pathogenesis,at least it is difficult to accurately distinguish from idiosyncratic hepatotoxicity.The newly proposed indirect DILI needs more clinical and experimental evidence to support it.In addition,the samples with Polygonum Multiflorum-induced DILI and Psoraleae Fructus/Epimedii Folium-induced DILI were included in PCA analysis,to try to evaluate the possible types of liver injury.The results showed that all DILI samples with Polygonum Multiflorum belonged to the same group as the known idiosyncratic DILI samples,however,it has an obvious separation effect with the known direct DILI samples,suggesting that the liver injury of Polygonum Multiflorum is mainly idiosyncratic rather than intrinsic type.This result is consistent with the previous clinical and experimental research results of the research group and the understanding that the liver injury type of Polygonum Multiflorum is idiosyncratic in the literature published by various research teams at home and abroad.According to the preliminary search of the author,it is the first time that the pathogenesis of Polygonum Multifloruminduced DILI is confirmed to be idiosyncratic heterogeneous from the perspective of metabolomics,which provides an important reference for correctly understanding the liver injury mechanism of Polygonum Multiflorum and carrying out scientific prevention and control.Similarly,we also included Psoraleae Fructus/Epimedii Folium-induced DILI samples into PCA analysis.The results showed that 75% of the samples were distributed in the known idiosyncratic DILI group,and the other 25%were distributed in the known direct DILI group,suggesting that Psoraleae Fructus/Epimedii Folium-induced DILI may be mainly an idiosyncratic damage mechanism,but the direct damage mechanism cannot be excluded.The previous experimental research of the research group and the literature reports of other research teams showed that some components in Psoraleae Fructus had direct hepatocyte toxicity,and its damage target was mitochondria;however,the direct hepatotoxicity of Epimedii Folium is weak,and it is manifested as specific heterogeneous liver injury mediated by immune stress.The results of metabolomics evaluation in this paper are consistent with those reported in the literature and mutually corroborate each other.Through the above analysis of different types of DILI pathogenesis,we put forward targeted risk prevention and control suggestions: for intrinsic/direct hepatotoxicity,the key is risk prevention of drugs;for idiosyncratic and indirect hepatotoxicity,the key point is risk prevention of host factors.6.The biomarkers of different phenotypes of DILI reflect different biological pathways and potential mechanismsUnderstanding the potential mechanism of different types of DILI is helpful for clinical diagnosis and drug selection.Therefore,we analyzed the biomarkers with different phenotypes of DILI,and the results showed that these biomarkers reflected different biological pathways.This provides a new perspective and research basis for revealing and explaining the pathophysiological mechanism and pathological progress of DILI classification.7.There are limitations in this study.The included samples are only cases from two hospitals,the screening center and the external verification center,and a more central,larger sample size and prospective cohort samples are needed for verification.Targeted metabolomics research on key pathways and metabolites is needed.The analysis of the mechanism is mainly based on the enrichment analysis and discussion of biological pathways,which needs to be confirmed by molecular biology.In conclusion,based on the screening central cohort and the external central cohort,this study screened and verified the combination of serum metabolomics student markers related to DILI typing and diagnosis.These biomarker combinations provide new evaluation indexes and reference bases for DILI target cell injury types,injury severity,liver tissue inflammation degree and liver fibrosis,and chronic DILI cirrhosis decompensation.Based on the results of unsupervised multivariate statistical evaluation of the overall metabolome,the differences between different pathogenesis types of DILI are systematically and objectively presented,which provides a unique reference basis for further clinical transformation research of DILI. |
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