Prediction Of The Active Ingredients Of Kudingcha
To determine the chemical ingredients of kudingcha, we searched the Traditional
Chinese Medicine Systems Pharmacology (TCMSP) (http://tcmspw.com/tcmsp.php) and the Encyclopedia of Traditional Chinese Medicine (ETCM) databases (http://www.tcmip.cn/ETCM/). In this study, molecules with an oral bioavailability (OB) value ≥ 30% and drug-likeness (DL) index ≥ 0.18 were identified as meaningful active ingredients[11].
Prediction Of Targets Of Kudingcha
The active ingredients were analyzed using the TCMSP database to identify the known drug targets. The two-dimensional structure of the compound obtained from PubChem was imported into the SwissTargetPrecision database, and a threshold (probability > 0.6) was set to obtain a possible target for each combination[11].
Prediction Of Endometriosis Targets
The keyword “obesity” was searched in three different disease gene databases: (1) GeneCards Human Gene database (https://www.genecards.org/); (2) DisGeNET database (https://www.disgenet.org/home/); (3) Online Mendelian Inheritance in Man (OMIM) database (https://omim.org/). UniProt (http://www.UniProt.org) was used to obtain the gene symbols of all targets (obesity and kudingcha), and the information was used for subsequent network pharmacological data analysis[12].
Screening For Key Targets (Dup: Abstract ?)
The Venny R package was employed to map the targets of kudingcha and the known therapeutic targets of obesity to build a Venny diagram. We combined the crossed targets of kudingcha and obesity after deleting the duplicate targets of obesity. These cross targets were defined as critical targets for the treatment of obesity.
Drug-ingredient-target Interaction Network
We used the Cytoscape software (version 3.7.2, Boston, MA, USA) to construct the drug-ingredient-target interaction network.
Protein-protein Interaction (Ppi) Network Construction
The key targets were subjected to PPI analysis using the STRING database (https://string-db.org/), and the species "Homo sapiens" was selected to generate a PPIbnetwork. After processing the data by R software (R version 4.1.2), we obtained the top 30 hub genes. Subsequently, we imported the hub gene data into the Cy software and obtained the HUB gene PPI network diagram[12].
Gene Ontology (Go) And Kyoto Encyclopedia Of Geld Genomes (Kegg) Analyses Of Hub Genes
We imported 30 critical genes into the Bioconductor library of R software to perform GO and KEGG pathway enrichment analyses. The screening criteria were set at P < 0.05, Q < 0.05, and the restricted species was Homo sapiens. GO and KEGG enrichment analyses revealed the top 15 results. Bar and bubble graphs represent the visualization, and a pathway atlas is presented for related pathways that meet clinical significance.
Cell Culture
The human liver cancer cell line HepG2 was used in this study. HepG2 is a hepatoma cell line, which has the fat storage capacity of hepatocytes and can form fat droplets that are easy to observe. The HepG2 cells were obtained from Dr Ying Zhang (Qilu Hospital). HepG2 cells were cultured in DMEM (Life Technologies, USA) containing 1% penicillin/streptomycin (Gibco, USA) and 10% FBS (Gibco, USA) in a humidified atmosphere containing 5% CO2 at 37°C. Cells were passaged when they reached 85% confluence and stored in liquid nitrogen for long-term preservation.
Cell Viability Assay (Dup: Abstract ?)
HepG2 cells were seeded in 96-well plates at a density of 5000 cells per well. When the confluence of cells was 80%, the culture medium was changed to DMEM medium containing 150µM palmitic acid (without FBS). Eight hours later, kaempferol or quercetin was added to the culture medium to make the concentration of quercetin or kaempferol 0, 2.5, 5, 10, 20 and 40 µmol /L, and the culture medium was cultured for 48 h. Then 10 µL solution of sterile Cell Counting Kit (CCK)8 (Bimake, USA) was added to each well and incubated for another 1.5h at 37°C. Absorbance was measured at 450 nm using universal microplate reader (ELx 800, BioTek Instruments, Winooski, VT). In order to exclude the influence of drug-induced cell death on the experiment, the concentration of two drugs with cell viability greater than 90% (IC10) was selected for further study.
Oil Red Staining
HepG2 cells were cultured for 8h in DMEM complete medium containing palmitic acid, and either kaempferol or quercetin. After 24h, discard the original culture medium, wash it with PBS for 3 times, put it into the fume hood to dry. And then, add 500µl of 4% paraformaldehyde to each hole, fix it for 20min, dye it with oil red O staining solution for 10-15min, cover the 24 hole plate during dyeing to prevent dye precipitation caused by solvent volatilization. After that, wash the excess dye with 60% isopropanol, next wash it with PBS for 3 times. Take photos under 20 times magnification of the microscope. Add 300µl DMSO to each hole to make the oil red dye completely dissolved in DMSO, 100µl of the above solution was added to 96 well plate. Colorimetric absorbance at 450nm was measured by microplate reader.
Quantitativepcr(Qpcr)
Total RNA was extracted from HepG2 cells by using RNeasy mini Kit (RNA Fast 200, China). An amount of 1 µg RNA was reverse-transcribed into cDNA by using SureScript First-Strand cDNA Synthesis Kit (GeneCopoeia, USA). PCR amplification was performed by using the SYBR PCR mix (Takara Bio. Inc., Kyoto, Japan). The primers for APOE were 5′-CGAGGTGTAGGTTATGTTC − 3′ (forward) and 5′-TACGCAACTTACGCAAAT-3′ (reverse); primers for Slc2a4 were: 5′-CCTGCCCGAAAGAGTCTAAAGC-3′(forward); and 5′- CTAAGAGCACCGAG
ACCAACG-3′(reverse); primers for INSR were: 5′-ACGGTCAATGAGTCAGCCAG
TC-3′ (forward) and 5′-ATCTCCATGAGCCATCAGTTCCA-3′(reverse). Primers for GADPH were: 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAA
GACGCCAGTGGA-3′(reverse).
Statistical Analysis
SPSS 22.0 was used to analyze the data. Quantitative data are presented as the mean ± SD of at least three independent experiments. One-way ANOV A were employed to compare the means. Differences were considered statistically significant if the p-value was < 0.05 or < 0.01.