Protein targets prediction and analysis. Analysis by DRAR-CPI servers revealed that 392 human proteins have potential interaction with thymol. In KEGG pathway, we found interesting pathways including glycolysis/gluconeogenesis and fatty acid degradation pathway relative to obesity. Further analysis indicated that targets in these pathways associated with carbon metabolism, tyrosine metabolism, pyruvate metabolism, retinol metabolism, drug metabolism-cytochrome p450, metabolism of xenobiotics by cytochrome p450, biosynthesis of amino acids and chemical carcinogenesis pathways (Fig. 1A). In addition, these targets mainly localized in the cytosol (Fig. 1B) and associated with coenzyme binding, oxidoreductase activity, catalytic activity, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor and alcohol dehydrogenase activity, zinc-dependent (Fig. 1C), and involved in organic substance catabolic process, single-organism biosynthetic process, small molecule metabolic process, xenobiotic process, ethanol metabolic process, pyruvate metabolic process, glucose metabolic process, oxidation-reduction process and ethanol process (Fig. 1D).
Our analysis also showed that module 1 (Fig. 1E) was the most important in the whole PPI network, with the highest score of 19.714. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) is a node in module 1 with the MCODE - score of 12.72, which has the highest complexity in 12 proteins responding to thymol, predicting it may be the central protein in glycolysis. Independent of its glycolytic activity, the encoded protein has additionally been identified to have uracil DNA glycosylase activity in the nucleus [27]. Also, GAPDH harbors a region that confers antimicrobial activity against Staphylococcus bacteria [28]. Studies of a similar protein in mice have assigned a variety of additional functions including nitrosylation of nuclear proteins, the regulation of mRNA stability, and acting as a transferrin receptor on the cell surface of macrophage [29–31]. Module 2 (Fig. 1F) had a score of 4.939 and included ADH1A and ADH1B nodes with the MCODE–score of 11 respectively. Module 3 (Fig. 1G) had a score of 3.636 and was involved in the TPI note. Module 4 had a score of 3.273 and included three nodes involved in it including PKLR, LDHB, and ADH7 (Fig. 1H). Besides, two other proteins (ACAT1 and FBP1) were not present in the module. The functional annotation of these representative proteins and reported experimental correlation with thymol were described as Table 1.
Table 1
Function annotation of potential targets of thymol
PDB ID
|
Protein Name
|
Predicted Docking Score
|
Protein Function and Reported Connection with Diseases
|
Reported Experimental Correlation with thymol
|
1ZNQ
|
Glyceraldehyde-3-phosphate dehydrogenase
|
-22.3993
|
catalyzes the reversible oxidative phosphorylation in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD) in carbohydrate metabolism. Suppresses proliferation and invasion of lung and esophageal squamous cell carcinomas [40].
|
No
|
1HSO
|
Alcohol dehydrogenase 1A
|
-23.3497
|
catalyzes the oxidation of alcohols to aldehydes. Variant confers susceptibility to esophageal squamous-cell carcinoma [41].
Polymorphisms in ADH1B associated with the increased risk of gastric cancer [42].
|
No
|
1HSZ
|
Alcohol dehydrogenase 1B
|
-22.79
|
Same as ADH1A
|
No
|
1D1T
|
Alcohol dehydrogenase class 4 mu/sigma chain
|
-25.5514
|
Same as ADH1A and ADH1B, however, the enzyme is inefficient in ethanol oxidation, but it is the most active as a retinol dehydrogenase, thus it may participate in the synthesis of retinoic acid, a hormone important for cellular differentiation. A single nucleotide polymorphism ADH7 is associated with the risk of squamous cell carcinoma of the head and neck [43].
|
No
|
1I0Z
|
L-lactate dehydrogenase B chain
|
-25.1132
|
catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+ in a post-glycolysis process. Correlates With unfavorable survival in hepatocellular carcinoma [44], and promotion of pancreatic cancer progression [45].
|
No
|
2VGB
|
Pyruvate kinase PKLR
|
-24.1893
|
Catalyzes the transphosphorylation of phohsphoenolpyruvate into pyruvate and ATP, which is the rate-limiting step of glycolysis.
Correlates the promotion colorectal cancer liver colonization [46].
|
No
|
1HTI
|
Triosephosphate isomerase
|
-22.7411
|
Catalyzes the isomerization of glyceraldehydes 3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) in glycolysis and gluconeogenesis. Variant (Arg189Gln) causes neurologic deficits [47].
|
No
|
2F2S
|
Acetyl-CoA acetyltransferase
|
-22.6297
|
ACAT1 catalyzes the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA. As a therapeutic target for Alzheimer's disease [48].
ACAT1 suppresses diet-induced obesity [49].
|
No
|
1FTA
|
Fructose-1,6-bisphosphatase 1
|
-21.4765
|
FBP-1 catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate.
Opposes renal carcinoma progression [50], and regulates obesity [51].
|
No
|
Molecular docking. To confirm the activity of thymol on lipid synthesis and metabolism in vivo, we chosen nematodes as experimental models due to their advantages such as a short cycle, a fast reproduction rate, and high homology with human genes. Homologous genes of thymol’s targets in nematodes were identified by Blast analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). These analyses revealed GPD-1, GPD-2, GPD-3, GPD-4, H24K24.3, TPI-1, PYK-1, PYK-2, 1DH-1, KAT-1, T02G5.7 and FBP-1 in nematodes. However, their 3D structures remain unknown except for TPI-1. Therefore, we constructed the 3D structure of the remaining protein by homology modeling technology. Homology modeling is a technique that can build a 3D structure for proteins based on the 3D structure of a similar protein. A homology model can provide an alternative for subsequent receptor-ligand analyses such as molecular docking.
In our study, we constructed models of these homology proteins by SWISS-MOL and examined interactions between thymol and these proteins through Autodock 4.0 (Fig. 2). Docking analysis revealed that thymol potentially interacts with the different amino acids of these proteins (except 1DH-1) in nematodes (Table 2).
Thymol reduces fat deposition due to high glucose exposure in nematodes. Fat accumulation induced by high-dose glucose (1 mM or 5 mM) in nematodes is inheritable across several generations [24]. So we chose 1 mM or 5 mM of glucose to induce fat deposition in nematodes. ORO allows for the quantification of total lipid levels, while Nile red facilitates the evaluation of lipid distribution among tissues such as the hypodermis, intestine, and the germ line. When used in conjunction, ORO and Nile Red enable the researchers to determine how genotype and environmental changes affect lipid accumulation and where in the worm these changes are occurring [32]. The statistic result of ORO and Nile Red staining suggests both 1 mM and 5 mM of glucose are capable of inducing fat accumulation in the second generation of nematodes, and 5 mM-induced fat accumulation is more significant than 1 mM-induced in nematode (Fig. 3B). Total lipid levels and lipid droplets were visualized by staining with ORO (Fig. 3A) and Nile Red (Fig. 3C) respectively. Futher, the treatment of thymol can reduce lipid content. Importantly, 1 µg/mL and 5 µg/mL thymol treatment significantly reduced lipid deposition in glucose-induced nematodes, indicating thymol can reduce fat content.
Thymol restores the change of transcriptional level of genes in β-oxidation induced by high-dose glucose. Our experimental data verified thymol could reduce lipid deposition in worms. We mentioned above that glucose-induced fat accumulation is on the one hand due to the synthesis of acetyl-CoA from glucose through glycolysis, which is the substrate of lipid biosynthesis, on the other hand is due to the inhibition of fat degradation by the activation of acetyl-CoA carboxylase (ACC) triggered by the rising blood glucose level. Therefore, we suspect that the reduction of fat accumulation by thymol may be achieved by regulating the upstream of glycolysis pathway and downstream of fatty acid β oxidation. To confirm this idea, we determined the transcriptional level of key genes correlating with β-oxidation including carnitine palmitoyltransferase (cpt-1), acyl-CoA oxidase (aco), fatty acid binding protein (fabp) in the process of β-oxidation and tryptophan hydroxylase (tph-1) in the upstream pathway of β-oxidation. The result of qRT-PCR suggested that the decreased level of cpt-1, aco, fabp and tph-1 in nematodes exposed by 1 mM glucose were upregulated after the exposure of thymol (Fig. 4). These findings suggest the reduction of fat deposition caused by thymol is related to the oxidation of fatty acids.