Network pharmacological and experimental validation of the mechanism of Chaihu Guizhi Ganjiang decoction (柴胡桂枝干姜汤) regulating T helper cell 17/regulatory T cell balance to improve autoimmune hepatitis

doi:10.19852/j.cnki.jtcm.2026.01.010

Abstract

Abstract:

OBJECTIVE: To elucidate the therapeutic efficacy and mechanism of action of Chaihu Guizhi Ganjiang decoction (柴胡桂枝干姜汤, CGGD) in autoimmune hepatitis.

METHODS: CGGD components and potential target genes were extracted from previously published databases. The autoimmune hepatitis (AIH)-related regulatory genes were obtained from the DisGeNET database. Intersections were taken, and enrichment analyses were performed on the extracted data. Concanavalin A (ConA)-induced AIH model mice were treated with CGGD via gavage. The results of network pharmacological analysis were experimentally validated.

RESULTS: Network pharmacology revealed 228 genes at the intersection of AIH and CGGD. Kyoto Encyclopedia of Genes and Genomes analysis revealed that CGGD primarily regulates the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) signaling pathway and cellular metabolism in AIH. Gene Ontology enrichment analysis revealed that CGGD modulates inflammation through transcription factor-mediated signaling pathways. As predicted, CGGD attenuated ConA-induced AIH in a dose-dependent manner by activating the PI3K/AKT signaling pathway. Histopathological assessment confirmed the protective effects of CGGD against ConA-induced AIH. Further investigation revealed that CGGD regulated the T helper cell 17 (Th17)/regulatory T cell (Treg) balance by modulating the PI3K/Akt/ nuclear factor kappa-B (NF-κB) pathway.

CONCLUSIONS: This study demonstrated the therapeutic effect of CGGD on AIH through a combination of network pharmacological prediction and experimental validation. Its mechanism of action involves PI3K/Akt/ NF-κB-mediated regulation of Th17/Treg cells.

Key words: autoimmune hepatitis, T helper cell 17, regulatory T cell, phosphoinositide 3-kinase, protein kinase B, NF-kappa B, network pharmacology, Chaihu Guizhi Ganjiang decoction

LIANG Zihao, GAO Jie, SONG Jinyun, ZHENG Qin, ZHAO Hongyu. Network pharmacological and experimental validation of the mechanism of Chaihu Guizhi Ganjiang decoction (柴胡桂枝干姜汤) regulating T helper cell 17/regulatory T cell balance to improve autoimmune hepatitis[J]. Journal of Traditional Chinese Medicine, 2026, 46(1): 110-118.

Figures/Tables 5

Figure 1 Venn diagram of the AIH and CGGD target genes ETCM: Encyclopedia of Traditional Chinese Medicine; TCMSP: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform; AIH: autoimmune hepatitis; CGGD: Chaihu Guizhi Ganjiang decoction.

Figure 2 Enrichment analysis of common target genes of AIH and CGGD A: top five significantly enriched terms (P < 0.05) in the biological process (red), cellular component (green), and molecular function (purple) categories according to GO analysis; B: the top 15 significantly enriched KEGG pathways (P < 0.05). GO: Gene Ontology; NMDA: N-methyl-D-aspartic acid; PI3K: phosphoinositide 3-kinase; AKT: protein kinase B; cAMP: cyclic adenosine monophosphate; Rap 1: ras-proximate-1; NOD: nucleotide-binding Oligomerization; MAPK: mitogen-activated protein kinase; NF-kapper B: nuclear factor kappa-B; Th17: T helper cell 17; AIH: autoimmune hepatitis; CGGD: Chaihu Guizhi Ganjiang decoction; KEGG: kyoto encyclopedia of genes and genomes.

Figure 3 CGGD alleviates ConA-induced liver injury A: histological changes in the liver were assessed using HE staining. Scale bars = 100 μm; A1: Ctrl group; A2: ConA group; A3: CGGD-L group; A4: CGGD-M group; A5: CGGD-H group; B: activities of and AST; C: activities of ALT. Ctrl Group: 200 μL of normal saline was administered daily for one week, followed by an intravenous injection of 10 μL of normal saline via the tail vein on the eighth day; ConA Group: 200 μL of normal saline was administered daily for one week, followed by an intravenous injection of ConA at a dose of 20 mg/kg according to body weight on the eighth day; CGGD-L Group: received ConA by gavage at doses of 2000, mg/kg once daily for one week prior to model induction; CGGD-M Group: received ConA by gavage at doses of 4000 mg/kg once daily for one week prior to model induction; CGGD-H Group: received ConA by gavage at doses of 8000 mg/kg once daily for one week prior to model induction. AST: aspartate aminotransferase; ALT: alanine aminotransferase; CGGD: Chaihu Guizhi Ganjiang decoction. Statistical differences between groups were analyzed using a one-way analysis of variance. Data were presented as mean ± standard error of the mean (n = 6). Compared with the Ctrl group, aP < 0.01; compared with the ConA group, bP < 0.01; compared with the CGGD-L group, cP < 0.01; compared with the CGGD-M group, dP < 0.01.

Table 1 mRNA expression levels of cytokine levels

Group	n	IL-1β	IL-6	TNF-α	IL-10
Ctrl	6	1.00±0.46^a	1.00±0.30^a	1.00±0.22^a	1.00±0.32^c
ConA	6	27.22±5.50	32.98±4.95	17.56±3.34	2.08±0.39
GCCD-L	6	16.67±4.21^a	25.44±2.26^b	8.82±1.93^a	2.23±0.51
GCCD-M	6	13.16±3.05^a	19.32±2.82^a	6.57±1.01^a	2.41±0.55
GCCD-H	6	5.43±2.06^a	13.99±2.95^a	2.83±0.90^a	4.13±0.91^a

Figure 4 CGGD regulates Th17/Treg homeostasis in ConA-induced AIH in mice A: flow cytometry analysis of CD4+IL-17+ (Th17) in mouse spleen; A1: Ctrl group; A2: ConA group; A3: CGGD-L group; A4: CGGD-M group; A5: CGGD-H group; A6: the quantitative frequency analysis of Th17 cells; B: flow cytometry analysis of CD4+Foxp3+ (Treg) cells in mouse spleen; B1: Ctrl group; B2: ConA group; B3: CGGD-L group; B4: CGGD-M group; B5: CGGD-H group; B6: the quantitative frequency analysis of Treg cells. Control Group: 200 μL of normal saline was administered daily for one week, followed by an intravenous injection of 10 μL of normal saline via the tail vein on the eighth day; ConA Group: 200 μL of normal saline was administered daily for one week, followed by an intravenous injection of ConA at a dose of 20 mg/kg according to body weight on the eighth day; CGGD-L, CGGD-M & CGGD-H Group: received ConA by gavage at doses of 2000, 4000, and 8000 mg/kg once daily for one week prior to model induction. Th17: T helper cell 17; Treg: regulatory T cell. CGGD: Chaihu Guizhi Ganjiang decoction. Statistical differences between groups were analyzed using a one-way analysis of variance. Data were presented as mean ± standard error of the mean (n = 6). Compared with the Ctrl group, aP < 0.01; compared with the ConA group, bP < 0.01; compared with the CGGD-L group, cP < 0.01; compared with the CGGD-M group, d P < 0.01.

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