Serum cytokine profiling analysis for zheng differentiation in chronic hepatitis B

Background: Liver-gallbladder dampness-heat (LGDH) and liver kidney yin deficiency (LKYD) syndromes are Chinese medicine (CM) zhengs in chronic hepatitis B (CHB) patients. This study aims to investigate the changes in cytokines and their profiles accompanied by different biological responses in LGDH and LKYD in CHB. Methods: During 2010-2012, a total of 138 morning fasting venous blood samples were obtained from participants in Shuguang Hospital, Shanghai University of Traditional Chinese Medicine in Shanghai, China. First, serum samples from 20 health controls (HCs) and 40 CHB patients (20 LGDH, 20 LKYD) were collected to detect the profiles of cytokines by multiplex biometric ELISA-based immunoassay. Random forest (RF) with a fivefold cross-validation was used to analyze the significant cytokines. Then the significant cytokines were validated using serum samples from an independent cohort of 60 CHB patients (30 LGDH, 30 LKYD) and 18 HCs. Results: There were different profiles of cytokines in LGDH and LKYD. Twenty-three significantly differentially expressed cytokines were detected, among which three cytokines, interleukin (IL)-17, macrophage inflammatory protein (MIP)-1 alpha, and MIP-1 beta, with the largest Gini scores were identified by RF, and further evaluated for their significant changes in serum levels. A receiver-operator characteristic analysis revealed that the logistic regression panel could differentiate LGDH from LKYD (P < 0.001; AUC = 0.827). A functional pathway analysis showed that cytokine-cytokine receptor interaction, cytosolic DNA-sensing pathway, and chemokine signaling pathway overlapped between LGDH and LKYD, whereas Toll-like receptor signaling pathway, intestinal immune network for IgA production, NOD-like receptor signaling pathway, and Jak-STAT signaling pathway were only enriched in LGDH. Conclusions: There were characteristic cytokines profiles in LGDH and LKYD with different inflammatory and immune responses. IL-17, MIP-1 alpha, and MIP-1 beta might be involved in the differentiation of LGDH and LKYD in CHB.