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. 2021 Nov 28:2021:3303014.
doi: 10.1155/2021/3303014. eCollection 2021.

Combined Metabolomics and Network Toxicology to Explore the Molecular Mechanism of Phytolacca acinose Roxb-Induced Hepatotoxicity in Zebrafish Larvae in Vivo

Dan Cao  1   2 , Chongjun Zhao  1   2 , Zhiqi Li  1   2 , Qiqi Fan  1   2 , Meilin Chen  1 , Yangyu Jiang  1 , Haiyan Wang  1 , Hanjun Ning  3 , Ruichao Lin  1   2 , Jian Li  1
Affiliations

Combined Metabolomics and Network Toxicology to Explore the Molecular Mechanism of Phytolacca acinose Roxb-Induced Hepatotoxicity in Zebrafish Larvae in Vivo

Dan Cao et al. Evid Based Complement Alternat Med. .

Abstract

Phytolacca acinosa Roxb (PAR), a traditional Chinese medicine, has been widely used as a diuretic drug for a long period of time for the treatment edema, swelling, and sores. However, it has been reported that PAR might induce hepatotoxicity, while the mechanisms of its toxic effect are still unclear. In this study, network toxicology and metabolomic technique were applied to explore PAR-induced hepatotoxicity on zebrafish larvae. We evaluated the effect of PAR on the ultrastructure and the function of the liver, predictive targets, and pathways in network toxicology, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS. PAR causes liver injury, abnormal liver function, and apoptosis in zebrafish. The level of arachidonic acid in endogenous metabolites treated with PAR was significantly increased, leading to oxidative stress in vivo. Excessive ROS further activated the p53 signal pathway and caspase family, which were obtained from KEGG enrichment analysis of network toxicology. The gene levels of caspase-3, caspase-8, and caspase-9 were significantly increased by RT-PCR, and the level of Caps3 protein was also significantly up-regulated through western blot. PAR exposure results in the liver function abnormal amino acid metabolism disturbance and motivates hepatocyte apoptosis, furthermore leading to liver injury.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1
(a) Lethal curve from zebrafish induced by PAR from 96 hpf to 120 hpf. (b) The level of ALT and AST in zebrafish.
Figure 2
Figure 2
Histopathology of zebrafish liver in both control group and PAR-treated group. (a) Control (100×, 400×). (b) PAR (100×, 4 00×).
Figure 3
Figure 3
In vivo cell death and apoptosis results. (a) Control group. (b) PAR group.
Figure 4
Figure 4
(a) Visualization of the overall metabolite profile difference between the PAR-treated group and the control group including the OPLS-DA predictive/discriminate score plot (a1), 1000 permutation tests (a2), and V-plot (a3); (b) differential metabolic pathways between the PAR-treated group and the control group.
Figure 5
Figure 5
Network construction of the “PAR-component-target (note: the orange nodes represent the PAR component and green nodes represent the targets of the PAR component).
Figure 6
Figure 6
Analysis results of network toxicology. (a) Venn diagram of 79 potential targets of PAR, which were intersected by “ingredient targets” and “toxic targets”. (b) PPI analysis of core targets from PAR. (c) PPI analysis of common targets of PAR. (d) KEGG pathway analysis by David database.
Figure 7
Figure 7
(a, b) Gene expression of caspase-3, caspase-8, and caspase-9 was examined by QT-PCR in groups treated with PAR. (c) Protein expression of activated Caps3 was examined by western blot in the zebrafish larvae group treated with PAR, n = 80. ∗indicates p < 0.05 versus the control group, ∗∗indicates p < 0.01 versus the control group, by one-way ANOVA.
Figure 8
Figure 8
Molecular mechanism of PAR-induced hepatotoxicity.
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