Geniposide

GCSB-5 was prepared by the Hanpoong Pharmaceutical Co., Ltd., Jeonju, Republic of Korea. The mixture of six crude drugs (Ledebouriellae Radix (4.444 g), Achyranthis Radix (4.444 g), Acanthopanacis Cortex (4.444 g), Cibotii Rhizoma (2.778 g), Glycine Semen (2.778 g), and Eucommiae Cortex (1.389 g)) was powdered and boiled for 3 h in distilled water (1 L). The resulting extract was subjected to ultrafiltration, and the components with molecular weight over 10,000 were excluded. The filtrate was lyophilized as powder and kept at 4°C until use. GCSB-5 was administered orally at a dose of 300 and 600 mg/kg in saline (1 kg/10 mL), and the same volume of saline was used as a vehicle control group. The validation of GSCB-5 was performed by high-performance liquid chromatography analysis of each ingredient extract using six indicator biological components: cimifugin for Ledebouriellae Radix, 20-hydroxyecdysone (0.311-0.312 mg/g) for Achyranthis Radix, acanthoside D (0.577-0.578 mg/g) for Acanthopanacis Cortex, onitin-4-O-β-D-glucopyranoside for Cibotii Rhizoma, genistin (0.0426-0.0427 mg/g) for Glycine Semen, and geniposide (0.431-0.432 mg/g) for Eucommiae Cortex. GCSB-5 was further standardized for quality control according to the regulations imposed by Korea Food and Drug Administration (KFDA).

HOK or HSC-3 cells were cultured in the dish which contained sterilized DMEM medium, and the concentration of cells was adjusted to 2 × 10⁴/dish. Then the cells solution was added into the 96-well plate with 50 μL per well, the cells were cultured with 5% CO₂ at 37 °C for 24 h. Sterile geniposide and LcS were added into the DMEM medium to prepare geniposide-L (25 μg/mL), geniposide-H (50 μg/mL), geniposide-L + LcS (1.0 × 10⁶ CFU/mL) and geniposide-H + LcS solutions, then these solutions were added into the 96-well plate with 50 μL per well. In the meantime, 50 μL of culture solution was added to the control group and cultured in a CO₂ incubator for 48 h. Subsequently, the untreated cells (control group) was added to MTT solution after the removal of clear solution and thereafter incubated for 4 h. In the blank control group, 100 μL of DMSO was added after the removal of the supernatant, followed by shocking for 30 min. The microplate reader was used for detection at 570 nm (680 Microplate Reader, Bio-Rad, Hercules, CA, USA) [13 (link)].

All the animal experiments were performed in accordance with the National Institutes of Health guidelines (NIH Publication, revised 2011) and the guidelines of the Animal Care and Use Committee of Renmin Hospital of Wuhan University. The animal studies also follow the ARRIVE guidelines. Male C57/B6J mice (age: 8–10 weeks; body weight: 25.5 ± 2 g) were purchased from the Institute of Laboratory Animal Science at the Chinese Academy of Medical Sciences (Beijing, China) and housed for more than 1 week before experimentation. After that, the mice were grouped according to a random number table and fed a high-fat diet (HFD, 45% kilocalories from fat, Institute of Laboratory Animal Science at the Chinese Academy of Medical Sciences, D12451, composition: protein 20 kcal%, carbohydrate 35 kcal%, and fat 45 kcal%) or a normal diet (ND, 10% kilocalories from fat) for 24 weeks, with only the last 3 weeks including a 21-day treatment with a previously used dose of geniposide (50 mg/kg, 0.2 ml, po) or an equal volume of saline (0.2 ml, po). The blood glucose levels were measured by mandibular puncture blood sampling. At the endpoint, all the mice were sacrificed with an overdose of sodium pentobarbital (200 mg/kg, i.p.) to harvest their heart and to calculate the following ratio: heart weight (HW)/tibia length (TL). To confirm the role of AMPKα in geniposide-mediated cardioprotection, Ampkα2 global knockout mice were used and subjected to HFD or ND for 24 weeks with treatment with geniposide for 3 weeks. The source of Ampkα2 global knockout mice has been described previously [16 (link), 17 (link)]. To verify the hypothesis that Sirt1 is involved in geniposide-mediated cardioprotection, siSirt1 and the siRNA control were delivered to the heart using a nanoparticle transfection reagent (Altogen Biosystems, NV, USA) via 3 injections (once every week) into the tail vein beginning from the initial geniposide treatment (21 weeks after HFD) [18 (link)].

To better understand the complex interactions among geniposide, AS, and the corresponding targets, we constructed a component-disease-target network based on geniposide, AS, and targets, and imported it into Cytoscape 3.8.0 for network drawing.

Chemicals for these experiments, including DMEM, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The treatment concentrations of geniposide (10~50 µM) were determined according to previous studies [18 (link)]. As in the previous study [20 (link)], baicalein, which is well known for its anti-inflammatory effects, was used as a positive control. Because the activation time of p38 MAPK in cells is due to stimulating factors, LPS and geniposide were administered simultaneously within a few hours.

First, the SMILES ID of geniposide was obtained through the PubChem database, and imported into the Swisstargets and similarity ensemble approach (SEA) databases to obtain the corresponding compound targets. Second, the compound targets of the drugs were searched using the Pharmmapper, Traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), and Herb databases. The targets obtained were corrected and deduplicated using the Universal Protein (UniProt) database. Then, “atherosclerosis” was used as the key word to search the human gene in GeneCards, NCBI, and DisGeNET databases. Targets obtained from the GeneCards database were filtered according to the median score to obtain more relevant targets. The selected drug targets and disease targets were input into Venny 2.1 diagram software, and common targets were obtained, which were used as the predicted targets of geniposide on AS for pathway enrichment analysis.