Uncovering the Pharmacological Mechanisms of Gexia-Zhuyu Formula (GXZY) in Treating Liver Cirrhosis by an Integrative Pharmacology Strategy

Abstract

Liver cirrhosis (LC) is a fibrotic lesion of liver tissue caused by the repeated progression of chronic hepatitis. The traditional Chinese medicine Gexia-Zhuyu formula (GXZY) has a therapeutic effect on LC. However, its pharmacological mechanisms on LC remain elucidated. Here, we used the network pharmacology approach to explore the action mechanisms of GXZY on LC. The compounds of GXZY were from the traditional Chinese medicine systems pharmacology (TCMSP) database, and their potential targets were from SwissTargetPrediction and STITCH databases. The disease targets of LC came from GeneCards, DisGeNET, NCBI gene, and OMIM databases. Then we constructed the protein-protein interaction (PPI) network to obtain the key target genes. And the gene ontology (GO), pathway enrichment, and expression analysis of the key genes were also performed. Subsequently, the potential action mechanisms of GXZY on LC predicted by the network pharmacology analyses were experimentally validated in LC rats and LX2 cells. A total of 150 components in GXZY were obtained, among which 111 were chosen as key compounds. The PPI network included 525 targets, and the key targets were obtained by network topological parameters analysis, whereas the predicted key genes of GXZY on LC were AR, JUN, MYC, CASP3, MMP9, GAPDH, and RELA. Furthermore, these key genes were related to pathways in cancer, hepatitis B, TNF signaling pathway, and MAPK signaling pathway. The in vitro and in vivo experiments validated that GXZY inhibited the process of LC mainly via the regulation of cells proliferation and migration through reducing the expression of MMP9. In conclusion, through the combination of network pharmacology and experimental verification, this study offered more insight molecular mechanisms of GXZY on LC.

Keywords: Gexia-Zhuyu formula; liver cirrhosis; network pharmacology; pharmacological mechanisms; traditional Chinese medicine.

FIGURE 1

Flowchart of the study. To explore the pharmacological mechanisms of Gexia-Zhuyu formula (GXZY) in liver cirrhosis (LC) treatment. On the left network pharmacology analysis part, we obtained the common target genes of GXZY and LC, and constructed network analysis to get key target genes and key compounds. Then we analyzed the enrichment and expression of key target genes in the database. On the right of experimental validation, rat animal experiments proved that GXZY inhibiting LC by affecting liver injury, inflammatory and collagen fibers. Cell experiments showed that GXZY inhibited the proliferation and migration of human hepatic stellate cells (HSC). Finally, the experiments of Western Blotting and qRT-PCR were used to verify the expression of key target genes.

FIGURE 2

Identification of the key target genes and their expression. (A). Preliminary hub network. The yellow circles represent weak interacting target genes, and the red circles represent strong interacting target genes. (B). Hub network. The red circles represent related target genes, and the purple circles represent core target genes. (C). Key module. The purple circles represent key target genes. (D). The expression of key genes between normal people and patients with liver cirrhosis.

FIGURE 3

The GO and KEGG functional enrichment of key target genes. (A). Enriched KEGG pathways of key target genes. (B). Enriched biological process of key target genes. (C). Enriched cellular component of key genes. (D). Enriched molecular function of key genes.

FIGURE 4

GXZY affected the liver weight, body weight, and serological liver function of CCl4 induced rat model. (A). The weight of rats induced by CCl4 decreased at the 8 weeks. (B). GXZY increased the body weight and decreased liver index of rats at the 12th week compared with the model group. (C). ALT, AST, and ALP of serological index rats were increased after 8 weeks CCl4 modeling. (D). GXZY decreased ALT, AST, and ALP of serological index rats at the 12th week compared with the model group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 versus the control group, and ^# p < 0.05, ^# p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus the model group.

FIGURE 5

GXZY improved the liver tissue injury of rat induced by CCl4. (A). The representative image of the liver surface shows the roughness, color, and texture of the liver at eighth week and 12th week (n = 6). (B). The representative images of HE staining shows the injury changes of the hepatic portal area, hepatic cord, and central vein at eighth week and 12th week. The images are presented at low power (× 200, Scale bars = 100 μm, n = 3).

FIGURE 6

GXZY improved hepatic collagen deposition and decreased the myofibroblasts and monocytes/macrophages in liver tissue of rat model induced by CCl4. (A). The model group rats had more collagen fiber deposits in the liver at the eighth week compared with the control group. (B). The GXZT group rats had low collagen fiber deposits in the liver at the 12th week compared with the model group. (C). Quantification of liver collagen in rats in the eighth week. (D). Quantification of liver collagen in rats in the 12th week. The fibrosis quantification of the sirius red staining representative images was performed in 5 random fields of each mouse under ×200 magnification (Scale bars = 100 μm, n = 6). ^** p < 0.01 versus the control group, and ^## p < 0.01 versus the model group. (E). Immunofluorescence histochemistry about correlation and colocalization analysis of α-SMA and CD68 in rat liver tissues.

FIGURE 7

GXZY reduced the expression of inflammatory factors and Mmp9 in rat liver. (A). The mRNA expression levels of Tnf in rat. (B). The mRNA expression levels of Il6 in rat. (C). The mRNA expression levels of Mmp9 in rat. (D). The protein expression levels of Mmp9 in rat. ^* p < 0.05 versus the GXZY group.

FIGURE 8

GXZY suppressed proliferation and migration and expression of key target genes of LX-2 cells. (A). LX-2 cells were treated dose-increased GXZY, and the cell viability was detected with CCK-8. (B). The representative images of cell migration assay for LX-2 cells. LX2 cells were treated with GXZY (500 μg/ml) for 0, 24, and 48 h. (C). The area ratio at 24/0 and 48/0 h statistical analysis of cell migration assay for LX-2 cells. (D). The mRNA expression levels of MMP9, CASP3, and GAPDH for LX-2 cells. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 versus the control group.