Molecular mechanism of Cigu Xiaozhi formula interfering with HSC-T6 cell activation by regulating HIF-1α signaling pathway based on computer aided drug design / 药学学报

ABSTRACT

In this study, we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design, and predicted and verified its possible targets and related signaling pathways. The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Encyclopaedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases, and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis. GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform, and Cytoscape software was used to construct the "potential active ingredient-key target-pathway" network. The active components and target proteins were subjected to molecular docking analysis by Auto Dock software. According to the results of molecular dynamics simulation and binding free energy calculation, the top 5 active components with degree were scored. The active components stigmasterol and β-sitosterol were subjected to molecular docking. CoCl2 was used to induce HSC-T6 cells to construct hypoxia model in vitro. The cell viability was detected by CCK-8 assay, and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100 µmol·L-1 CoCl2 for 24 h. Under hypoxia condition, HSC-T6 cells were activated, the wound healing rate was significantly increased, and the fluorescence signal of activation marker protein α-smooth muscle actin (α-SMA) was significantly enhanced. However, 6% drug-containing serum could inhibit the activation of HSC-T6 cells, and the wound healing rate was significantly decreased, and the fluorescence signal of α-SMA was significantly weakened. Further studies showed that the expressions of hypoxia-inducible factor-1α (HIF-1α), α-SMA and key proteins of Hedgehog (Hh) signaling pathway in HSC-T6 cells were up-regulated under hypoxia, while the expressions of HIF-1α, α-SMA, Patched-1 (Ptch-1) and glioma related oncogene homology-1 (Gli-1) were down-regulated in 6% drug-containing serum group, the YC-1 group and the cyclopamine group. These results indicated that HIF-1α and Hh signaling pathways were involved in the activation of HSC-T6 cells, and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells, and the mechanism may be related to the inhibition of HIF-1α expression and the blocking of Hh signaling pathway. In conclusion, Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1α and Hh signaling pathway, and exert an anti-hepatic fibrosis effect. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine, in compliance with the Institutional Animal Care Guidelines.