Abstract
OBJECTIVE: Blood-Heat syndrome is a core syndrome of Traditional Chinese Medicine (TCM) in Henoch-Schönlein purpura nephritis (HSPN), yet its biological basis remains unclear. This study aimed to systematically elucidate the scientific basis of Blood-Heat syndrome within the context of HSPN and to identify its objective biomarkers using a multidimensional biological approach. METHODS: In the clinical research part, we divided it into a discovery cohort and a validation cohort. The discovery cohort employed Data-Independent Acquisition (DIA) proteomics technology to analyze serum samples from HSPN patients with Blood-Heat syndrome (n = 15), those without Blood-Heat syndrome (non-Blood-Heat, n = 30), and healthy controls (n = 30). The findings were then validated through ELISA in both the discovery cohort and an independent validation cohort (n = 30 for blood heat syndrome, n = 30 for non-blood heat syndrome). In the basic research component, we established a rat model combining HSPN with Blood-Heat syndrome to replicate the clinical findings. RESULTS: Proteomic analysis identified 87 specific differentially expressed proteins (DEPs) associated with Blood-Heat syndrome. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant enrichment in the sphingolipid signaling pathway (P = 0.02). We further identified a panel of nine core biomarkers (AHSG, HRG, KNG1, HP, AZGP1, PTX3, MAPK1, A1BG, and COL1A1), which demonstrated excellent diagnostic performance in distinguishing between healthy control group and blood-heat syndrome, as well as between blood-heat syndrome and non-blood-heat syndrome (with AUC values all ≥0.7). ELISA validation showed that, compared to the healthy control group and non-Blood-Heat group, the levels of AHSG, HRG, and KNG1 were significantly downregulated in the Blood-Heat group, while the other six markers were significantly upregulated (P < 0.01 for all). This trend was fully replicated in the HSPN Blood-Heat syndrome rat model. CONCLUSION: Based on multidimensional evidence from clinical proteomics and animal model replication, this study suggests that Blood-Heat syndrome in the context of HSPN has a reproducible molecular phenotype. The functional enrichment of its differential proteins involves the sphingolipid signaling pathway, accompanied by an enhanced inflammatory background represented by ERK2 upregulation. Based on these findings, we propose a core scientific hypothesis of "Blood-Heat-related stress-sphingolipid signaling-associated alterations-ERK2-mediated inflammatory amplification," providing a direction for future mechanistic validation and targeted intervention research.