Preparation of Curcumae Radix Extract
Curcumae radix was obtained from Beneherb Agricultural Co. Ltd. (Jeju Island, South Korea). The material was washed and rinsed with distilled water. These materials were prepared using a grinder-mixer. The origin plant samples were deposited in the Korea Institute of Oriental Medicine (KIOM) in South Korea (voucher specimen KIOM M 130110). Dried Curcumae Radix power was extracted in 70% (v/v) ethanol for 120 min with sonication twice. The extracted 70% ethanol solution was filtered (Whatman No. 2), and then concentrated by a vacuum rotary evaporator (Büchi; Flawil, Switzerland) at 40 °C. To obtain Curcumae radix powder, the final extract was lyophilized (IlShin; South Korea) using a freeze-dryer (final yield of 11.4%, Supplementary Figure S1). Compounds of Curcumae Radix quantitative analysis was shown in previous study [12].
Animals and treatment
Mice on a C57BL/6N background were obtained from Orient Bio (Daejeon, Korea) and housed in a pathogen-free facility at Chungnam National University under a standard 12 h light:12 h dark cycle and fed standard chow with water provided ad libitum. All mouse experiments were approved and performed in accordance with the Chungnam Facility Animal Care Committee. Mice used 12 weeks old and were administered Curcumae Radix extract was orally 5times in a week (50 mg/kg body weight). vehicle group was treated with same capacity distilled water same as Curcumae Radix extract treatment. Mice were sacrificed at 5 weeks and isolated organs. Mice used for experiment were 6 for each vehicle and Curcumae Radix group.
C57BL/6-Tg(NSE-hTau23)Korl mice were obtained from National Institute of Food and Drug Safety Evaluation (NIFDS, Cheongju, Korea). and housed in a pathogen-free facility at Chungnam National University under a standard 12 h light:12 h dark cycle and fed standard chow with water provided ad libitum. All mouse experiments were approved and performed in accordance with the Chungnam Facility Animal Care Committee. Mice used 24 weeks old and were administered Curcumae radix extract was orally for 2 weeks (50 mg/kg body weight). vehicle group was treated with same capacity distilled water same as Curcumae Radix extract treatment. Mice were sacrificed at 2 weeks and isolated organs. Mice used for experiment were 3 for each vehicle and Curcumae Radix group.
Cell culture
All cell culture reagents were purchased from Welgene (Gyungsan, Korea). Murine delayed brain tumor (DBT) cell were maintained at 37 °C in a 5% CO2 atmosphere in DMEM (Welgene, LM001-05) supplemented with 5% (vol/vol) fetal bovine serum and 1% penicillin-streptomycin (vol/vol) in six-well tissue culture plates. For experiments, Curcumae Radix extract group were supplemented with concentration of 2.5ug/ml and 4ug/ml that dissolved in distilled water and vehicle group were supplemented distilled water that be same volume for 24h, Curcumae Radix extract treated volume. All cell experiments were repeated at least 3 times.
Western blotting
Both protein samples of cerebrums and DBT cell was extracted by using protein lysis buffer, called T-PER reagent (78510, Thermo Fisher scientific) and quantified by Bradford assay with PRO-Measure solution (#21011, Intron). The samples were run SDS-PAGE electrophoresis on 10, 12% polyacrylamide gels and transferred to membrane. And the membranes were blocked with 30mg/ml BSA100 (9048-46-8, LPS solution), diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies were operated during overnight in 4℃. Following the step, the membranes were washed with TBS-T and secondary antibodies were operated in identical way. Results were detected with ECL solution (XLS025-0000, Cyanagen) and Chemi Doc (Fu-sion Solo, VilberLourmat). The primary and secondary antibody information are as for Table.1.
Table 1. Primary and secondary antibodies used for western blot
Primary antibodies
|
Type
|
Lot.
|
Inc.
|
Caspase3
|
Rabbit monoclonal
|
9665
|
Cell signaling technology
|
Cleaved caspase3
|
Rabbit monoclonal
|
9665
|
Cell signaling technology
|
Amyloid-beta
|
Mouse monoclonal
|
sc-28365
|
Santa Cruz biotechology
|
Tau
|
Rabbit monoclonal
|
A1103
|
Company ABclonal, Inc.
|
AMPKα
|
Rabbit monoclonal
|
5831
|
Cell signaling technology
|
Phospho-AMPKα
|
Rabbit monoclonal
|
2535
|
Cell signaling technology
|
Hexokinase ǀ
|
Rabbit monoclonal
|
2024
|
Cell signaling technology
|
PKM2
|
Rabbit monoclonal
|
4053
|
Cell signaling technology
|
Pyruvate Dehydrogenases
|
Rabbit monoclonal
|
3205
|
Cell signaling technology
|
LDHA
|
Rabbit monoclonal
|
3582
|
Cell signaling technology
|
Alpha-Tubulin
|
Mouse monoclonal
|
66031-1-Ig
|
Proteintech Group Inc
|
Secondary antibody
|
Type
|
Lot.
|
Inc.
|
Anti-Mouse IgG
|
Goat
|
121507
|
Jackonimmuno
|
Anti-Rabbit IgG
|
Mouse
|
123213
|
Jackonimmuno
|
Total RNA extraction and real-time quantitative PCR
Total RNA was extracted using TRIzol Reagent (15596-026, Life technologies) in both mouse cerebrum and DBT cells. Reverse transcription was performed with 1.5 µg of total RNA and Reverse transcriptase kit (SG-cDNAS100, Smartgene) following manufacturer’s protocol. Quantitative PCR (real-time PCR) was executed using Excel Taq Q-PCR Master Mix (SG-SYBR-500, Smartgene) and Stratagene Mx3000P (Agilent Technologies). Primers used in real time PCR were manufactured by Bionics Inc. (Seoul, Korea) or Genotech (Seoul, Korea). mRPLP0 was used as control in in vivo samples and in vitro respectively. All experiments were run more than triplicate, and mRNA values were calculated based on the cycle threshold and monitored for an amplification curve. The primers used for real-time PCR are as for Table.2.
Table 2. Primers used for real-time PCR
Gene Name
|
Upper Primer (5’-3’)
|
Lower Primer (5’-3’)
|
Species
|
Idh3a
|
TGC TTC GCC ACA TGG GAC TT
|
CGT TGC CTC CCA GAT CTT TT
|
Mouse
|
Sdhb
|
CTC TGT CTA CCG CTG CCA C
|
GGC ACA CTC AGC ACG GAC T
|
Mouse
|
Ndufb5
|
CTT CCT CAC TCG TGG CTT TC
|
CGC ACT TCC AGC TCC TTT AC
|
Mouse
|
Slc25a4
|
ATG GTC TGG GCG ACT GTA TC
|
TCA AAG GGG TAG GAC ACC AG
|
Mouse
|
RPLP0
|
GCA GCA GAT CCG CAT GTC GCT CCG
|
GAG CTG GCA CAG TGA CCT CAC ACG G
|
Mouse
|
Measurements of cellular glycolysis and mitochondrial respiration
DBT cells were grown in DMEM, 10% FBS, and 1% penicillin/streptomycin media and incubated for 24 hrs. And then After removing media, Curcumae Radix extract (4ug/ml) was treated for 24hrs. For only the glycolysis stress test, cells were further incubated in DMEM low-glucose medium [glucose 50 mg/dL without FBS]. Before the experiment, the cells were decarboxylated for 40 min to 1 h in XFp medium (103575-100, Agilent Technologies) containing the same amount of glutamine, sodium pyruvate, and glucose as that of the medium in which cells were grown. For the mitochondrial stress test (mitochondrial respiration and fatty acid oxidation), oligomycin (2 µM), FCCP (5 µM), and rotenone/antimycin (0.5 µM) were used, and oxygen consumption rate (OCR) was measured. For the glycolysis stress test, glucose (25 mM) was used, and extracellular acidification rate (ECAR) was measured. Seahorse XFp analyzer (Agilent Technologies), Seahorse XFp, XFp FluxPak (103022-100, Agilent Technologies), and XFp Cell Mito Stress Test kit (103010-100, Agilent Technologies) were used for analysis.
Statistical Analysis
Data are reported as mean ± SEM. Differences between means were obtained by Student’s t-test and the one-way ANOVA followed by a Dunnett post analysis was performed using Graph Pad Software (GraphPad Inc., San Diego, CA).