Exploring the mechanisms of neurotoxicity caused by fuzi using network pharmacology and molecular docking

Abstract

Safety has always been an important issue affecting the development of traditional Chinese medicine industry, especially for toxic medicinal materials, the establishment of risk prevention and control measures for toxic herbs is of great significance to improving the use of traditional Chinese medicine in clinical. Fuzi is a kind of traditional Chinese medicine and its toxicity has become the most important obstacle of limit in clinical using. In this paper, network pharmacology and molecular docking technology were used to analyze the main toxic components of Fuzi, the key targets and the mechanism of neurotoxicity. We carried out CCK-8 and WB assays, and detected LDH release and SDH activity. It was verified that aconitine caused neurotoxicity through a variety of pathways, including MAPK signaling pathway, pathways related to Akt protein, destruction of cell membrane integrity, damage of mitochondrial function affecting energy metabolism and apoptosis. What's more, this study confirmed that aconitine could produce neurotoxicity by promoting apoptosis of hippocampus neuron and decreasing its quantity through Nissl Staining and TUNEL assay. This paper found and confirmed multiple targets and various pathways causing neurotoxicity of Fuzi, in order to provide reference for clinical application and related research.

Keywords: aconitine; fuzi; molecular docking; network pharmacology; neurotoxicity.

FIGURE 1

The workflow of this study.

FIGURE 2

Chemical structure of aconitine.

FIGURE 3

Target screening and establishment of PPI network. (A) Potential targets of neurotoxicity caused by Fuzi and PPI network. (B) PPI network of neurotoxicity targets caused by Fuzi. (The color of the node is marked from red to yellow according to the Degree value in descending order.) (C) Relationship network between candidate toxic compounds and core targets of Fuzi. (Blue triangles represent candidate toxic compounds and red circles represent core targets).

FIGURE 4

GO biological process and KEGG pathway enrichment analysis. (A) The bubble diagram of GO enrichment analysis of 133 intersection targets, including the top 10 significant enrichment terms of BP, CC and MF. (B) The bar plot diagram of KEGG pathway enrichment analysis of 133 intersection targets (top 20).

FIGURE 5

Molecular docking patterns of aconitine and core protein molecules. (The yellow lines represent the hydrogen bond interaction force, which is the main force promoting molecule binding with the active site.)

FIGURE 6

Cell Viability of SH-SY5Y cells after 24 and 48 h aconitine treatment. (*p < 0.05, **p < 0.01 versus control group.)

FIGURE 7

Western blot analysis after 48 h aconitine treatment. (A) Effects of aconitine on the protein secretion of MAPK pathway in SH-SY5Y cells. (B) Effects of aconitine on the secretory levels of p-Akt, Akt and Cleaved-caspase-3 in SH-SY5Y cells.

FIGURE 8

Effects of aconitine on mitochondrial function. (A) The release of LDH of SH-SY5Y cells after 24 h aconitine treatment. (*p < 0.05, **p < 0.01 versus control group.) (B) The SDH activity of SH-SY5Y cells after 24 h aconitine treatment. (*p < 0.05, **p < 0.01 versus control group).

FIGURE 9

Treatment of aconitine for 7 days reduced the activity of hippocampus neurons. (A) Representative images magnified 40 and 100 times in hippocampus. (B) Quantitatively analyzed the number of Nissl’s bodies in control and experimental group (×100). (*p < 0.05 versus control group).

FIGURE 10

Treatment of aconitine for 7 days increased apoptosis of hippocampus neurons. (A) Representative images magnified 40 and 100 times in hippocampus. (B) Quantitatively analyzed the number of apoptotic cells in control and experimental group (×100). (**p < 0.01 versus control group.)