Study on Dihydromyricetin Improving Aflatoxin Induced Liver Injury Based on Network Pharmacology and Molecular Docking

Abstract

Aflatoxin B1 (AFB1) is a toxic food/feed contaminant and the liver is its main target organ, thus it poses a great danger to organisms. Dihydromyricetin (DHM), a natural flavonoid compound, can be used as a food additive with high safety and has been shown to have strong hepatoprotective effects. In this experiment, PPI network and KEGG pathway analysis were constructed by network pharmacological analysis technique using software and platforms such as Swiss, String, and David and Cytoscape. We screened AFB1 and DHM cross-targets and pathways of action, followed by molecular docking based on the strength of binding affinity of genes to DHM. In addition, we exposed AFB1 (200 μg/kg) to mice to establish a liver injury model. Histological observation, biochemical assay, oxidative stress indicator assay, TUNEL staining and Western blot were used to evaluate the liver injury. Network pharmacological results were screened to obtain 25 cross-targets of action and 20 pathways of action. It was found that DHM may exert anti-hepatic injury effects by inhibiting the overexpression of Caspase-3 protein and increasing the expression of Bcl-2 protein. DHM (200 mg/kg) was found to reduce AFB1-induced liver indices such as alanine aminotransferase (ALT) and aspartate acyltransferase (AST), and attenuate hepatic histopathological damage through animal models. Importantly, DHM inhibited malondialdehyde (MDA) formation in liver tissue and attenuated AFB1-induced oxidative stress injury by increasing glutathione-S-transferase (GST) glutathione (GPX) catalase (CAT) and superoxide dismutase (SOD). Meanwhile, DHM also restored the expression of anti-apoptotic protein Bcl-2 and antioxidant proteins, Nrf2, Keap1 and its downstream HO-1, and down-regulated the expression of pro-apoptotic proteins Bax and Caspase-3 in AFB1-induced liver tissues. The results confirmed that liver injury caused by AFB1 exposure could be alleviated by DHM, providing valuable guidance for in-depth study of DHM in the treatment of liver-related diseases, and laying the foundation for in-depth development and utilization of DHM.

Keywords: aflatoxin; dihydromyricetin; liver injury; network pharmacology; oxidative stress.

Figure 1

(A) Chemical structure formula of AFB1. (B) Chemical structure formula of DHM.

Figure 2

Network pharmacology-based prediction of multi-targets and pathways for the treatment of liver injury as well as functional annotations and enrichment pathways represented in the form of bubble diagrams. (A) Venn diagram of important compounds and their targets. (B) Network diagram. (C) Top 25 genes classified by degree method. (D) Expression of 25 target genes in human genes. (E) Bar graph of the top ten genes. (F) GO in biological processes (G) GO in cellular components (H) Molecular functions of GO (I) KEGG pathway analysis.

Figure 3

Binding affinity of genes to their compounds and prediction of which related signalling pathways (A) HIF1A (19). (B) SRC (19). (C) CASP3 (22). (D) VEGFA (19). Docking complexes are indicated where genes have a strong binding affinity to their compounds. (E) VEGF signalling pathway. (F)TNF signalling pathway. (G) P13K -AKT signalling pathway. (H) NF-κB signalling pathway.

Figure 4

Protective effect of dihydromyricetin on AFB1-induced liver injury in mice. (A) Flow of drug administration in each group of mice (n = 8). (B) Body weight of mice in each group. (C) Effect of AFB1 on liver index in mice. (D) Liver injury in mice in each group. (n = 8). (E) Liver tissue H&E staining 200×, boxed magnification of H&E staining × 400, histological micrograph of a liver section stained by Masson. All data are expressed as mean ± standard deviation (n = 8). ^# represents statistically significant difference compared with the control group; * represents statistically significant difference compared with the AFB1 group. ^## p < 0.01, * p < 0.05.

Figure 5

Effects of DHM on AFB1-induced liver function and amelioration of AFB1-induced oxidative stress. (A) Serum ALT activity of mice in each group. (B) Serum AST activity of mice in each group. (C) GPX activity in the liver of mice in each group. (D) CAT activity in the liver of each group of mice. (E) Concentration of MDA in the liver of each group of mice. (F) SOD activity in the liver of mice in each group. (G) GST activity in the liver of mice in each group. All data are expressed as mean ± standard deviation (n = 6). ^# represents statistical differences compared to the control group. * epresents statistical differences compared to the AFB1 group. ^## p < 0.01, ^### p < 0.001, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Effect of DHM on AFB1-induced apoptosis. TUNEL staining of (A) the control group, (B) the AFB1 group, and (C) the DHM group. Relative levels of fluorescence intensity were quantified (D). The presence of TUNEL-positive cells was assessed using an image analyser. And pointed out in detail with arrows. All data are expressed as mean ± standard deviation. ^# represents a statistically significant difference compared with the control group, * represents a statistically significant difference compared with the AFB1 group. ^### p < 0.001, *** p < 0.001.

Figure 7

DHM can regulate the Nrf2/HO-1 signalling pathway and attenuate apoptosis to exert protective effects. (A) Protein expression of Nrf2, HO-1, Keap1, Cleaved-caspase-3, Caspase-3, Bax, and Bcl-2 was analysed using western blot analysis. (B) Relative protein expression was quantified by ImageJ analysis. All data are expressed as mean ± standard deviation (n = 3). ^# represents a statistically significant difference compared to the control group, * represents a statistically significant difference compared to the AFB1 group. ^## p < 0.01, ^### p < 0.001, * p < 0.05, ** p < 0.01.