Preparation of GJG extract and chemical compounds
GJG extract powder was purchased from Tsumura & Co. (Tokyo, Japan). GJG extract powder was suspended in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) and autoclaved (121°C, 15 min). After autoclaving, fetal bovine serum (FBS) (Sigma, Marlborough, MA, USA) was added so that the final concentration was 10%, and the mixture was passed through a 0.22-µm filter. Chemical compounds were selected and purchased based on the three-dimensional high-performance liquid chromatography profile and LC/MS analysis of GJG10 as below: benzoylpaeoniflorin (Cheminstock Ltd., Shanghai, China), Paeoniflorin, Genipin, 2'-hydroxy-4'-methyoxyacetophenone, morroniside, cinnamaldehyde (AK Scientific, Inc., Union City, CA, USA), Loganin, Isoacteoside (BioBioPha Co., Ltd., Kunming, China), Geniposidic acid, Benzoylmesaconine, Chikusetsusaponin V, Mesaconitine, Catalpol, Alisol A, Neoline, Benzoylaconine, Aucubin (Chemexpress Co., Ltd., Shanghai, China), Benzoylhypaconine (JR MediChem LLC, Princeton, NJ, USA), Azomycin (Selleck Chemical, Houston, TX, USA), 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (Toronto Research Chemicals, Toronto, Canada), and Inokosterone (ChemFaces Biochemical Co., Ltd., Wuhan, China).
SAMP8 and SAMR1 mice (8 weeks old), purchased from SLC, Inc. (Shizuoka, Japan), were divided into two groups of 5 each one week after they were acclimated based on the diet that they were fed: a normal diet (powdered mouse food; Oriental Yeast Co. Ltd. (Tokyo, Japan; P8 + N group); and a normal diet supplemented with 4% (w/w) GJG (P8 + GJG group). The control group, which consisted of 8-week-old male SAMR1 mice also purchased from SLCm was also divided into two groups of 10 each based on the diet that they were fed: a normal diet (R + N group); and a normal diet supplemented with 4% (w/w) GJG (R + GJG group). The general conditions and body weight of all mice were recorded. Housing care and the experimental protocol were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of the Animal Care and Use Committee of Osaka University. We confirmed the study was carried out in compliance with the ARRIVE guidelines
Cell Culture And Immunofluorescence Staining
Mouse skeletal muscle cell line C2C12 cells were obtained from DS Pharma Biomedical (Osaka, Japan). C2C12 cells were seeded in a 6-well plate at 5×104 cells/ml and cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 ug/mL streptomycin (Nacalai, Kyoto, Japan) for 48 h. After 100% confluence, the medium was replaced with DMEM containing 2% horse serum (Gibco BRL, Grand Island, NY, USA) and cultured for 96 h. Differentiation of C2C12 cells from myoblasts to myotubes was confirmed using immunofluorescence staining of sarcomeric α actinin13. C2C12 cells were fixed with 4% paraformaldehyde solution in PBS at room temperature for 10 min and permeabilized with 1 mL of 0.25% Triton/PBS for 20 min. After blocking with 5% skim milk/PBS at room temperature for 30 min, anti-Sarcomeric Alpha Actinin antibody (ab9465) (Abcam, Cambridge, UK) with 5% skim milk/PBS was added to the dilution and left overnight at 4°C. The secondary antibody was diluted with PBS, the diluted solution was added, shielded from light with aluminum foil, and allowed to stand at room temperature for 1 h. Nuclear staining was performed with DAPI-containing encapsulant (ProLong ™ Gold Antifade Mountant with DAPI: Invitrogen) on a glass slide13.
Real-time Quantitative PCR And Droplet Digital PCR
The differentiated C2C12 myotubes underwent treatment with 100 µg/mL GJG, and then, 24 h later, the medium was replaced with a serum-free DMEM medium containing 100 µg/mL GJG and 10 or 100 ng/mL murine TNF-α (Peprotech, Rocky Hill, NJ). Then, 0, 3, and 6 h after the medium was changed, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Subsequently, SuperScript VILO Master Mix (Invitrogen, Carlsbad, CA) was used to synthesize cDNA from 200 ng of RNA. The ViiA 7 real-time PCR system (Life Technologies, Carlsbad, CA, USA) was used for real-time quantitative PCR, with the following cycling conditions: 50 ºC for 2 min, 95 ºC for 20 sec, followed by 40 cycles of 95 ºC for 1 sec and 60 ºC for 20 sec at annealing temperature. TaqMan Fast Advanced master mix (Applied Biosystems, Foster City, CA) was used to amplify the target genes from 10 ng of cDNA. Gene-specific primers and FAM-labeled probes (mouse MAFbx: Mm00499523_m1, mouse MuRF1: Mm01185221_m1, mouse PGC1-α: Mm01208835_m1, mouse B2M: Mm00437762_m1) were purchased from Applied Biosystems. Relative expression levels relative to beta2 macroglobulin (B2M) expression were then calculated.
A QX100 droplet digital PCR (ddPCR) system (Bio-Rad Laboratories, Hercules, CA) was used for ddPCR in a total volume of 20 µL, with 10 µL 2x ddPCR Supermix for the probes (Bio-Rad Laboratories), 1 µL primers and FAM-labeled probes (mouse B2M: Mm00437762_m1, mouse PGC1-α: Mm01208835_m1), and 5 µL sample cDNA (10 ng total RNA). After droplets were generated with a QX100 Droplet Generator (Bio-Rad Laboratories), a C1000 Touch Thermal Cycler (Bio-Rad Laboratories) was used to amplify each cDNA. The thermal cycling conditions were as follows: a pre-cycling hold at 95 ºC for 10 min, followed by 40 cycles of 94 ºC for 30 sec and 58 ºC for 1 min, with final heating at 98 ºC for 10 min. A QX100 Droplet Reader (Bio-Rad Laboratories) was used to measure droplets, and QuantaSoft software version 1.6.6 (Bio-Rad Laboratories) was used to analyze the target copy number, with normalization of the target copy number by B2M.
TNF-α ELISA and cell viability assay
A mouse macrophage cell line (RAW264.7), purchased from Dainippon Pharmaceutical (Osaka, Japan), was grown in DMEM with 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin at 37°C in a humidified 5% CO2 atmosphere. LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). The RAW264.7 cells were seeded in triplicate at 5 × 104 cells/mL in 96-well plates and left for 48 h. At that point, the cells were cultured for 24 h in DMEM with or without 10 or 100 µg/mL GJG (final 1% FBS). This was followed by stimulation of the cells by 10 ng/mL LPS for 16 h. The TNF-α concentration in the supernatant of the medium was then evaluated with a mouse TNF-α Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions 11. Cell viability, evaluated using Cell Counting kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol, was calculated by comparison to that of cells without GJG and LPS. Each of the above experiments was performed at least three times, and representative data are reported.
Western Blotting Analysis
SAMP8 and SAMR1 mice were sacrificed at 12, 16, 24, 36, and 48 weeks (each group, n = 3), and soleus muscles were obtained, with the muscle samples cut into small pieces and homogenized in RIPA buffer (Nacalai) with phosphatase inhibitor cocktail (Nacalai) and protease inhibitor cocktail (Thermo Fisher Scientific, Tokyo, Japan). After the lysates were centrifuged for 10 min at 14,000 g and 4°C, the supernatants were collected. The BCA Protein Assay Kit (Sigma) was then used to measure protein, followed by Western blot analysis according to previously described methods10,11.
After seeding of RAW264.7 cells in triplicate at 1 × 105 cells/mL in 6-well plates, they were grown in DMEM with 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin at 37°C in a humidified 5% CO2 atmosphere. Then, 48 h later, the cells were cultured for 24 h in DMEM with or without 10 or 100 µg/mL GJG (final 1% FBS). Dexamethasone(Dex) (Sigma, St Louis, MO, USA) was added for 24 h at 10–100 µM. To detect MyD88 and TLR4, cell stimulation was performed with 10 ng/mL LPS for 24 h, followed by homogenization of whole cell lysate in RIPA buffer with phosphatase inhibitor cocktail and protease inhibitor cocktail. To detect NF-κB p65, p-IκBα, and Ser32 IκBα, cell stimulation with 10 ng/mL LPS was performed for 30 min, followed by separation of nuclear extracts using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). The method described above was then used for Western blot analysis. Specific primary antibodies were then used for the following: TNF-α (Cell Signaling Technology #3707, Danvers, MA, USA); IL-6 (Cell Signaling Technology #3707); MyD88 (CST #4283); TLR4 (Santa Cruz Biotechnology sc-293072, Santa Cruz, CA, USA); IκBα (CST #9242); p-IκBα Ser32 (CST #2859); NF-κB p65 (CST #8242);GAPDH (CST); and PCNA (CST #13110).
Chemical Characteristic Analysis Of Chikusetsusaponin V
The solubility, membrane permeability, and metabolic stability of Chikusetsusaponin V were investigated. A 1% DMSO / PBS solution of Chikusetsusaponin V (final 500 µM) was created and filtered with a filtration plate (Multi Screen HTS-PCF, Merck-Millipore). Acetonitrile was added to the filtered samples, which were vortexed and centrifuged at 3500 rpm for 10 min. Supernatant was analyzed by LC/MS (Xevo TQ-S MS/MS, Milford, MA, USA). Solubility was calculated based on the area under the curve compared to 5% DMSO/PBS solution of Chikusetsusaponin V (final 50 µM or 500 µM). A caco-2 permeability assay was performed according to established methods24.
Briefly, 500 µM Chikusetsusaponin V/HBSS solution was added to the upper wells of cultured Caco-2 cells in a trans-well-plate, and HBSS with 1% BSA was added to the lower wells. After incubation for 2 h, the solution on the top plate and the bottom plate was collected. Acetonitrile was added to the solution, which was vortexed and centrifuged at 3500 rpm for 20 min. The supernatant was analyzed by LC/MS (Xevo TQ-S), and the apparent permeability coefficient (Papp) was calculated. For the metabolic stability assay, 500 µM Chikusetsusaponin V was mixed with liver microsomes (Sekisui XenoTech, LLC, Kansas City, KS, USA, final concentration 0.2 mg protein/mL), coenzyme group (NADPH, G-6-P : Sigma, Marlborough, MA, USA, MgCl2 : Wako, Osaka, Japan), and G6PDH (Oriental Yeast, Tokyo, Japan) Mix (200 µL) at 37 ˚C, for 0 min, 10 min, and 60 min. Acetonitrile was added to the solution, which was vortexed and centrifuged at 3500 rpm for 20 min. The supernatant was analyzed by LC/MS (Xevo TQ-S), and the ratio of unchanged chemical compound at 10 min and 60 min compared to the unchanged Chikusetsusaponin V at 0 min as 100% was calculated.
Plasma assay of Chikusetsusaponin V in mice administered GJG orally
After fasting for 16 h, extract powder of GJG 1 g/kg was administered orally to 8-week-old male CD1 (ICR) mice (Charles River Laboratories Japan, INC., Kanagawa, Japan). Plasma samples were collected from the caudal vena cava (heparin treatment) before and after administration at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h (n = 5 at each time point).
Eight-week-old, male SAMP8 mice were fed a normal diet (n = 3 from 1 week to 3 weeks, n = 6 at 4 weeks) or a normal diet supplemented with 4% (w/w) GJG for four weeks (n = 6 before and at each time point). Plasma samples were collected before and after administration at 1, 2, 3, and 4 weeks. Acetonitrile was added to the plasma samples, and the supernatants were collected after centrifugation. The supernatant was dried and dissolved in 100 µL of solution (10 mmol/L ammonium formate solution/acetonitrile (80:20)). The concentration of chikusetsusaponin V was measured by LC-MS / MS (MS / MS: QTRAP 5500; AB Sciex, Framingham, MA, HPLC: Agilent 1260; Agilent Technologies, Santa Clara, CA). The plasma concentration was calculated by adding a standard solution of chikusetsusaponin V (PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany) to plasma to prepare a calibration curve. For analysis of CD1(ICR) mice analysis, the range of the calibration curve was from 0.0200 to 10.0 ng/mL and for SAMP8 mice, the range of the calibration curve was from 0.0500 to 10.0 ng / mL.
The real-time PCR results and the TNF-α ELISA results were analyzed using Tukey-Kramer’s post hoc test, and the results of droplet digital PCR were examined by Dunnet’s test using JAMP Pro version 14. A p-value less than 0.05 was considered significant.