Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun 8:14:1159094.
doi: 10.3389/fphar.2023.1159094. eCollection 2023.

Exploring the mechanism of JiGuCao capsule formula on treating hepatitis B virus infection via network pharmacology analysis and in vivo/vitro experiment verification

Xu Cao  1   2 , Ningyi Zhang  1 , Hening Chen  1 , Wei Wang  1   3 , Yijun Liang  1 , Jiaxin Zhang  1   2 , Ruijia Liu  1 , Shuo Li  1 , Yuhao Yao  1 , Qian Jin  1 , Ziwei Guo  1 , Yue Chen  1 , Yuanyuan Gong  1 , Xiaoke Li  1   2 , Xiaobin Zao  1   4 , Yong'an Ye  1   2
Affiliations

Exploring the mechanism of JiGuCao capsule formula on treating hepatitis B virus infection via network pharmacology analysis and in vivo/vitro experiment verification

Xu Cao et al. Front Pharmacol. .

Abstract

The JiGuCao capsule formula (JCF) has demonstrated promising curative effects in treating chronic hepatitis B (CHB) in clinical trials. Here, we aimed to investigate JCF's function and mechanism in diseases related to the hepatitis B virus (HBV). We used mass spectrometry (MS) to identify the active metabolites of JCF and established the HBV replication mouse model by hydrodynamically injecting HBV replication plasmids into the mice's tail vein. Liposomes were used to transfect the plasmids into the cells. The CCK-8 kit identified cell viability. We detected the levels of HBV s antigen (HBsAg) and HBV e antigen (HBeAg) by the quantitative determination kits. qRT-PCR and Western blot were used to detect the genes' expression. The key pathways and key genes related to JCF on CHB treatment were obtained by network pharmacological analysis. Our results showed that JCF accelerated the elimination of HBsAg in mice. JCF and its medicated serum inhibited HBV replication and proliferation of HBV-replicating hepatoma cells in vitro. And the key targets of JCF in treating CHB were CASP3, CXCL8, EGFR, HSPA8, IL6, MDM2, MMP9, NR3C1, PTGS2, and VEGFA. Furthermore, these key targets were related to pathways in cancer, hepatitis B, microRNAs in cancer, PI3K-Akt signaling, and proteoglycans in cancer pathways. Finally, Cholic Acid, Deoxycholic Acid, and 3', 4', 7-Trihydroxyflavone were the main active metabolites of JCF that we obtained. JCF employed its active metabolites to perform an anti-HBV effect and prevent the development of HBV-related diseases.

Keywords: JiGuCao capsule formula; UHPLC-MS/MS; experimental verification; hepatitis B virus; network pharmacology.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Flowchart of the study. We started by conducting in vivo experiments using the hepatitis B virus (HBV) replication mouse model. The mice’s weight, serum HBV s antigen (HBsAg), serum HBV e antigen (HBeAg), and pathological condition were detected. Next, cell experiments showed that the JiGuCao capsule formula (JCF) inhibited the expression of HBsAg, HBeAg, and HBV x protein (HBx) and the proliferation of HBV-expressing cell lines in vitro. On the right side of the network-pharmacology-analysis, we obtained the targets of JCF and chronic hepatitis B, and further constructed network analyses to get key target genes and key metabolites. Then we analyzed the enrichment of key target genes in databases.
FIGURE 2
FIGURE 2
Total ion chromatography (TIC) of JCF with its 50% methanol solution form and medicated rat serum form. (A) TIC diagram of 50% methanol solution form of JCF in positive ion mode. (B) TIC diagram of 50% methanol solution form JCF in negative ion mode. (C) TIC diagram of JCF-medicated rat serum in positive ion mode. (D) TIC diagram of JCF medicated rat serum in negative ion mode. (E) The top 10 enriched metabolites of JCF in its 50% methanol solution form (green) and serum form (purple), respectively. (F) The structures of the overlapped metabolites.
FIGURE 3
FIGURE 3
JCF accelerates the elimination of serum HBsAg and associated intrahepatic deposits in HBV replication C57BL/6J mice. (A) C57BL/6J mice weights at days 0, 7, and 14 after HDI, n = 6 in each group. (B) C57BL/6J mice’s serum HBsAg levels at days 0, 7, and 14 after treatment, n = 6 in each group. (C) The representative images of H&E staining and HBsAg IHC of mice’s liver. The images are presented at high power (× 200, Scale bars = 100 μm). *p < 0.05 versus the Control group. ns, not significant.
FIGURE 4
FIGURE 4
JCF inhibits the replication of HBV and the proliferation of HBV-related hepatoma cells in vitro. (A, B) HepG2.2.15 (A) and HepAD38 (B) cells were treated with DMSO (0.1%), JCF (50 0 μg/mL), and ETV (10 μM) for 48 h. The levels of HBsAg and HBeAg in cell supernatants were examined by ELISA, and the mRNA expression levels of HBx were detected by qRT-PCR. (C) CCK-8 assays were used to determine the effects of DMSO (0.1%) and JCF (500 μg/mL) on the proliferation of HepG2.2.15, HepAD38, HepG2, and Huh7 cell lines after 96 h of treatment. (D) CCK-8 assays were used to determine the effects of gradient concentration JCF medicated rat serum on the proliferation of HepG2.2.15, HepAD38 cells, HepG2, and Huh7 cells after 96 h of treatment. Data are presented as the mean ± SD, and the p-value was calculated by one-way ANOVA between 3 groups and Student’s t-test between 2 groups. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
FIGURE 5
FIGURE 5
JCF inhibits HBx-expressed cells’ proliferation. (A, B) Huh7 (A) and HEK-293T (B) cells were transfected with HBV-expressing plasmids for 48 h and then treated with DMSO and JCF for another 48 h. The levels of HBsAg and HBeAg in the supernatants were examined by ELISA. (C, D) Huh7 (C) and HEK-293T (D) cells were transfected with HBx and HBV-expressing plasmids for 48 h, respectively. The HBx expression was detected by Western blotting. (E, F) Huh7 (E) and HEK-293T (F) cells were transfected with HBx and HBV-expressing plasmids for 48 h and then treated with DMSO and JCF for another 96 h. The cells’ viability was detected by CCK-8. Data are presented as the mean ± SD, and the p-value was calculated by the Student’s T-test between the 2 groups. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
FIGURE 6
FIGURE 6
Network pharmacological analysis of JCF on CHB treatment. (A) The OGEs of JCF on CHB treatment. The blue circle represents CHB targets, and the red circle represents JCF putative targets. (B) The PPI network of OGEs in the STRING database. (C) Process of network pharmacological analysis using Cytoscape software based on the topological method. (D) The relationship of key genes, the purple circles represent key target genes, and the black line indicates their relationship.
FIGURE 7
FIGURE 7
The KEGG and BP functional enrichment of OGEs and key genes. (A) Enriched KEGG pathways of OGEs. (B) Enriched GO-BP of OGEs. (C) Enriched KEGG pathways of key genes. (D) Enriched GO-BP of key genes. The red box represents the repetitive key pathways.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Brugman S., Witte M., Scholman R. C., Klein M. R., Boes M., Nieuwenhuis E. E. (2014). T lymphocyte-dependent and -independent regulation of Cxcl8 expression in zebrafish intestines. J. Immunol. 192, 484–491. 10.4049/jimmunol.1301865 - DOI - PubMed
    1. Cao G. Y., Geng S. X., Luo Y., Tian S., Ning B., Zhuang X. S., et al. (2021a). The rapid identification of chemical constituents in Fufang Xiling Jiedu capsule, a modern Chinese medicine, by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry and data mining strategy. J. Sep. Sci. 44, 1815–1823. 10.1002/jssc.202001093 - DOI - PubMed
    1. Cao H., Chen X., Wang Z., Wang L., Xia Q., Zhang W. (2020). The role of MDM2-p53 axis dysfunction in the hepatocellular carcinoma transformation. Cell. Death Discov. 6, 53. 10.1038/s41420-020-0287-y - DOI - PMC - PubMed
    1. Cao X., Zao X., Xue B., Chen H., Zhang J., Li S., et al. (2021b). The mechanism of TiaoGanYiPi formula for treating chronic Hepatitis B by network pharmacology and molecular docking verification. Sci. Rep. 11, 8402. 10.1038/s41598-021-87812-9 - DOI - PMC - PubMed
    1. Chen J., Xu W., Chen Y., Xie X., Zhang Y., Ma C., et al. (2017). Matrix metalloproteinase 9 facilitates hepatitis B virus replication through binding with type I interferon (IFN) receptor 1 to repress IFN/JAK/STAT signaling. J. Virol. 91, 018244–e1916. 10.1128/JVI.01824-16 - DOI - PMC - PubMed

Grants and funding

This research was financially supported by the cooperation project (HX-DZM-202212) from Dongzhimen Hospital affiliated with the Beijing University of Chinese Medicine, the youth training program (SSMYJY-3-2021-12) from Sun Simiao Research Institute affiliated with the Beijing University of Chinese Medicine, the Guangxi Key Research and Development Program (AB20159029), and the Central Government Guides Local Science and Technology Development Fund Projects (ZY21195044).