Anorexia means loss of appetite and is a state whereby a desire to eat is either reduced or eliminated resulting in reducing or stopping food intake. Sipjeondaebo-tang (SDT) and Hyangsayukgunja-tang (HYT) are prescriptions known to have appetite-improving effects, but studies on their mechanisms and active components are insufficient. The hypothalamus is the center of appetite control, and various appetite control mechanisms are known. We used mouse hypothalamic neuronal GT1-7 cells as appetite control center cells and analyzed the difference in efficacy between SDT and HYT using microarray and network pharmacology. Microarray analysis showed that SDT and HYT affect the regulation of genes related to appetite control in the digestive tract and central nervous system. Using network pharmacology, we analyzed the differential expression of neuropeptide Y receptors, glucagon, corticotropin-releasing hormone receptors 1, and 5-hydroxytryptamine receptor 4 among the 17 anorexia-related genes selected from the comparative toxicogenomics database and also analyzed the active components that affect gene expression. In conclusion, the appetite-related genes contributed to anorexia control, and the difference in the action mechanism of the two complex prescriptions could be explained.
Anorexia means loss of appetite and is a state whereby a desire to eat is reduced or eliminated resulting in reducing or stopping food intake. Anorexia has various causes such as nausea, vomiting, side effects of medication, psychological factors, and aging. Appetite is the psychological desire to eat, associated with sensory experiences such as the sight and smell of food or cognitive, emotional, social situations, and cultural conventions. Appetite is regulated by interactions between peptide hormones in the digestive tract or adipose tissue and the hypothalamus. The hypothalamus is the center of appetite control, and various appetite control mechanisms are known. The hypothalamus regulates short- and long-term dietary intake by synthesizing numerous anorectic and orexigenic neuropeptides. The function and structure of several hypothalamic peptides, including melanin-concentrating hormone (MCH), cocaine- and amphetamine-regulated transcript (CART), orexins, neuropeptide Y (NPY), melanocortins, and agouti-related peptide (AGRP) have been studied in rodent models. In addition, peripheral neuropeptides, including bombesin, amylin, peptide YY (PYY3-36), ghrelin, and cholecystokinin (CCK), govern essential gastrointestinal processes, such as absorption, secretion, and motility, offer feedback to the central nervous system on nutrition availability, and may help regulate food intake.
Sibjeondaebo-tang (SDT) and Hyangsayukgunja-tang (HYT) are commonly used prescriptions for anorexia but have different components. SDT is a frequently prescribed herbal medicine comprising 10 herbs (Astragali Radix, Panax ginseng radix, Atractylodes Rhizoma Alba, Poria sclerotium, Rehmanniae Radix, Angelicae Gigantis Radix, Paeonia Radix, Cnidii Rhizoma, Glycyrrhizae Radix et Rhizoma, and Cinnamomi Ramulus) in Korea, Japan, and China. SDT is also called Shi-Quan-Da-Bu-Tang in China and Juzen-taiho-to in Japan. SDT is used to treat both qi and blood deficiency syndromes by balancing Yin and Yang, and is also widely used for treating chronic illnesses by restoring physiological function and improving immunity. HYT (named as “Xiang Sha Liu Jun Zi Tang” in Chinese) has been used for various digestive disorders, such as gastric flatulence, anorexia, nausea, and vomiting. HYT is commercially available and comprises 14 herbs: Cyperi Rhizoma, Atractylodis Rhizoma Alba, Poria Sclerotium, Pinelliae Tuber, Citri Unshius Pericarpium, Amomi Fructus Rotundus, Magnoliae Cortex, Amomi Fructus, Ginseng Radix Alba, Aucklandiae Radix, Aipiniae Oxyphyllae Fructus, Glycyrrhizae Radix et Rhizoma, Zingiberis Rhizoma Crudus, and Zizyphi Fructus.
Herbal medicine preparations are widely used owing to their long history and safety. They are effective in treating complex syndrome-type diseases owing to the complex efficacy of their various components. However, these preparations have many drawbacks, such as non-specific and weak efficacy, resulting in efficacy evaluation difficulties. Therefore, microarray and bioinformatic methods should be used that would facilitate the analysis of complex herbal prescriptions. In this study, we used mouse hypothalamic neuronal GT1-7 cells as appetite-regulating cells and analyzed the differences in the related genes by SDT and HYT using microarray.
SDT and HYT were prepared from a chopped herbal mixture by Hanpoong Pharmaceutical Co., Ltd. (Jeonju, Korea) and the Korea Institute of Oriental Medicine, respectively. Briefly, the mixture was added to 125 mL of distilled water and decocted at 90–100℃ for 3 h. The extract was filtered through filter paper with a 5 μm pore size. The filtrate was concentrated using an evaporator, and the remaining mass was vacuum-dried to obtain a powder. The powder was dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments.
GT1-7 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Corning, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO, USA) and 1% antibiotics (100 U/mL penicillin and 100 U/mL streptomycin; Gibco BRL, Carlsbad, MD, USA). The cells were seeded at a density of 2 × 10 4 cells/well on a 96-well plate. GT1-7 cells were incubated at 37°C in a 5% CO 2 humidified incubator. The cells were treated under various concentrations (25, 50, 100, and 200 µg/mL) for 24 h. Cell viability was measured using an EZ-cytox assay kit from DoGenBio (Seoul, Korea) at 450 nm using a microplate reader.
GT1-7 cells were treated with 25 and 200 µg/mL SDT and HYT, respectively. After 24 h, total RNA was isolated using the RNeasy mini kit following the manufacturer’s protocol (Qiagen Inc., Valencia, CA, USA). Subsequently, the purity was quantified by measuring the 260/280 ratio between 1.8 and 2.1. In addition, the integrity number (RIN) was over 7.
Cell samples were collected and subjected to total RNA extraction for use in the Clariom™ S Assay platform for mice. Following the manufacturer’s instructions, the extracted total RNA was converted into cDNA using the GeneChip Whole Transcript (WT) Amplification kit. Subsequently, the sense cDNA was fragmented and biotin-labeled using terminal deoxynucleotidyl transferase (TdT), facilitated by the GeneChip WT Terminal labeling kit. Approximately, 5.5 µg of the biotin-labeled DNA target was hybridized to the Affymetrix GeneChip Array and maintained at a consistent temperature of 45°C for 16 h. After hybridization, the arrays were processed through a wash-and-stain cycle on a GeneChip Fluidics Station 450, followed by scanning with a GCS3000 Scanner (Affymetrix). Probe cell intensity data were generated and converted into a CEL file via the Affymetrix® GeneChip Command Console® Software. Furthermore, the Affymetrix Power Tools and R 3.3.3 software facilitated data analysis, allowing comprehensive examination and interpretation of the microarray data.
To compile a list of genes perceived to be associated with anorexia, we gathered list of genes from the toxicogenomics database (CTD), where genes are curated by their associations with diseases in terms of markers, mechanisms, or therapeutics. We analyzed the fold-change in anorexia-associated genes in the microarray compared with that in the control. Genes with an absolute fold-change value ≥ 1.5 were presumed to be differentially expressed genes (DEGs). To infer the compound causing changes in the expression of anorexia-associated genes, we reconstructed a prescription-herb-compound-target network focusing on anorexia-associated genes that were differentially expressed. To build the network, information about the herb-component relationship was obtained from TM-MC, and compound-target information was gathered from curations by Hwang et al., which included data from ChEMBL, BindingDB, STITCH, Herbal Ingredients’ Targets Database, and the Traditional Chinese Medicine Integrated Database.
The data were performed using the GraphPad software (GraphPad Prism 5, USA). The results were analyzed using one-way analysis of variance to determine differences between the treatment and control groups. All data are presented as mean ± standard error of the mean. Values of *p < 0.05 were statistically significant.
We first investigated cytotoxicity under varying concentrations (25, 50, 100, and 200 µg/mL) of SDT and HYT in the GT 1–7 cell line. As shown in Fig.[1], both samples showed no cellular toxicity, even at high concentrations. SDT showed viability in 100 ± 0.28, 100.50 ± 0.06, 100.34 ± 0.48, 100.45 ± 0.49, 100.17 ± 0.20% (25, 50, 100, and 200 µg/mL) and HYT in 100 ± 0.28, 98.04 ± 0.51, 99.55 ± 0.19, 99.94 ± 0.50, 100.56 ± 0.49%, 25, 50, 100, and 200 µg/mL, respectively. Therefore, 25 and 200 µg/mL concentrations were selected for further study (Fig.[1]).
We conducted a pathway enrichment analysis based on the KEGG pathway.
