慢性肝病中医证候演变的分子特征:动态网络生物标志物分析OA

Objective To elucidate the biological basis of traditional Chinese medicine(TCM)syn-dromes from the perspective of"same syndrome,different diseases"in patients with chronic hepatitis B(CHB),liver cirrhosis(LC),and hepatocellular carcinoma(HCC),thereby provid-ing a complementary approach for the diagnosis and treatment of chronic liver diseases(CLD). Methods To investigate the dynamic characteristics of TCM syndromes in CLD,transcrip-tomic profiling of peripheral blood mononuclear cells(PBMCs)was performed from patients with CHB,LC,or HCC presenting with three TCM syndromes:liver gallbladder dampness heat syndrome(LGDHS),liver depression spleen deficiency syndrome(LDSDS),and liver kidney Yin deficiency syndrome(LKYDS).These participants were recruited at Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine between August 1,2018 and December 31,2021.Differentially expressed genes(DEGs)were identified using the random variance model(RVM)F test with false discovery rate(FDR)correction.Principal component analysis(PCA)and unsupervised hierarchical clustering were applied to visualize sample grouping.Dynamic network biomarkers(DNB)analysis was employed to detect criti-cal transition stages during syndrome evolution,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses to characterize the functional roles and pathway involvement of the DNB members.Random forest(RF)analysis and the area under the receiver operating characteristic(ROC)curves(AUC)were used to rank the importance of candidate genes.External validation was performed using mi-croarray data from an independent CLD cohort(GSE89377)and RNA-seq data from The Can-cer Genome Atlas Liver Hepatocellular Carcinoma(TCGA-LIHC)dataset.Additionally,re-verse transcription quantitative polymerase chain reaction(RT-qPCR)was performed on an independent cohort of LC patients to validate the expression levels of the candidate genes. Results The study included a total of 132 participants.DNB analysis identified LDSDS stage as a critical tipping point during TCM syndrome evolution across CHB,LC,and HCC.The phosphoinositide 3-kinase/protein kinase B(PI3K-AKT)signaling pathway was consistently enriched in the DNB analysis across all three types of CLD,suggesting its potential involve-ment in the critical transition of TCM syndromes.Among the 24 core DNB members of the PI3K-AKT pathway,four genes—integrin subunit beta 1(ITGB1),collagen type IV alpha 1 chain(COL4A1),collagen type IV alpha 2 chain(COL4A2),and DNA damage inducible tran-script 3(DDIT3)—were identified by RF analysis(Gini score>1)and ROC analysis.ROC anal-ysis demonstrated high discriminative ability for distinguishing LGDHS from LKYDS in CHB patients,with AUC of 0.789 1 for ITGB1,0.707 0 for COL4A1,0.714 8 for COL4A2,and 0.894 5 for DDIT3.In the independent CLD cohort(GSE89377),all four genes showed significant stepwise upregulation from normal to CHB,LC,and HCC(all P<0.05).In the TCGA-LIHC dataset,their expression progressively increased with tumor stage.RT-qPCR validation in an independent LC cohort(30 LGDHS vs.30 LKYDS)confirmed that ITGB1,COL4A2,and DDIT3 were significantly upregulated in LKYDS compared with LGDHS(P=0.015 2,0.018 6,and 0.024 7,respectively),whereas COL4A1 showed a non-significant upward trend(P=0.120 1). Conclusion This study introduces a novel approach to understanding the molecular features underlying TCM syndrome evolution in CLD.The PI3K-AKT pathway and four identified genes(ITGB1,COL4A1,COL4A2,and DDIT3)play crucial roles in the transition from excess(LGDHS)to deficiency(LKYDS)via the critical LDSDS stage.These findings offer potential quantitative biomarkers and therapeutic targets for TCM syndrome differentiation and may help arrest syndrome progression in CLD.