OBJECTIVE To explore the effective components and potential targets of the Tibetan medicine Gentianopsis paludosa against liver fibrosis based on spectrum-effect relationship, network pharmacology, and molecular docking.

METHODS HPLC was used to establish the fingerprints of 10 batches of Gentianopsis paludosa medicinal materials. Custer analysis and principal component analysis were applied to evaluate the quality of samples from different batches. A total of 130 eight-week-old SPF male C57BL/6J mice were divided into control group, model group, positive control group, and Gentianopsis paludosa treatment groups(S1–S10), with 10 mice in each group. Except for the control group, mice in all other groups were intraperitoneally injection with 25% CCl₄ olive oil solution 3 times per week for 8 weeks to establish the liver fibrosis model. From the 3rd week of modeling, the positive control group was intragastrically given silymarin at 54.6 mg·kg^–1 daily. The treatment groups were intragastrically administered extracts of different batches of Gentianopsis paludosa at a crude drug dose of 3.094 g·kg⁻¹ daily for 6 consecutive weeks, while the model group and control group were given an equal volume of solvent at the same time. After the final administration, the serum liver function and liver fibrosis markers of mice were measured. Liver tissues were subjected to histopathological analysis and scored by the Metavir system. The expression levels of α-smooth muscle actin(α-SMA) and Collagen Ⅰwere detected by immunohistochemistry, so as to comprehensively evaluate its anti-liver-fibrosis efficacy. The liver tissue fibrosis pathological scores and protein expression levels of α-SMA and Collagen Ⅰ were taken as pharmacodynamic indices, and the peak areas of 10 common chromatographic peaks were used as independent variables. Grey relational analysis(GRA, relational degree>0.70) and partial least-squares regression(PLSR, VIP>1 and negative regression coefficient) were respectively applied to screen the chromatographic peaks associated with each index. The components corresponding to the obtained chromatographic peaks were identified as the active components of Gentianopsis paludosa against liver fibrosis. The intersection of screening results from the 2 methods was obtained, and then the intersections results of the 3 indices were merged. The potential active components of Gentianopsis paludosa against liver fibrosis screened via spectrum-effect relationship were taken as research objects. Component targets were predicted using databases including TCMSP, PubChem, and disease targets for anti-liver-fibrosis were obtained from the GeneCards database. Intersection targets were identified using Venny platform, and a protein-protein interaction(PPI) network was constructed using the String database and Cytoscape 3.9.1 software to screen core targets. GO and KEGG enrichment analyses were performed on the intersection targets. Molecular docking between the potential active components and core targets was carried out using AutoDock Vina software to preliminarily explore the potential action targets of Gentianopsis paludosa against liver fibrosis.

RESULTS The HPLC fingerprint similarities of 10 batches of Gentianopsis paludosa medicinal materials ranged from 0.756 to 0.997. A total of 10 common peaks were identified, 4 of which were assigned as isoorientin, swertisin, luteolin, and apigenin by reference standards. The 10 batches of Gentianopsis paludosa were clustered into 3 categories. Spectrum-effect correlation analysis showed that the common peaks F1(isoorientin), F3(swertisin), F4(unidentified) and F8(apigenin) were closely associated with the anti-liver fibrosis efficacy of Gentianopsis paludosa. Network pharmacology prediction revealed that the core targets of the identified components(isoorientin, swertisin, and apigenin) against liver fibrosis were TP53, AKT1, IL6, STAT3, TNF, and MAPK8, which were mainly enriched in multiple signaling pathways including AGE-RAGE, HIF-1, IL-17, and TNF. Molecular docking results showed that 3 components exhibited the best binding affinity to the core targets TP53 and AKT1, with binding energies all <−7.0 kcal·mol⁻¹.

CONCLUSION This study found through spectrum-effect relationship analysis that isoorientin, swertisin, apigenin, and F4(unidentified) are potential active components of Gentianopsis paludosa against liver fibrosis. Combined with the results of network pharmacology and molecular docking, it is preliminarily confirmed that Gentianopsis paludosa may exert its anti-fibrotic effects by acting on core targets such as TP53 and AKT1, and regulating multiple signaling pathways including AGE-RAGE, HIF-1, IL-17, and TNF.