Palmitic acid로 지방 축적을 유도한 HepG2 cell에 …

1 Acid HepG2 cell , Original ArticleEffect of Samhwangsasim-tang and Daehwanghwangryunsasim-tang on Palmitate-induced Lipogenesis in HepG2 cellsEun sik Um, Young Chul KimDepartment of Clinical Korean Medicine, Graduate School, Kyung Hee UniversityObjectives: The goal of this study was to investigate the anti-lipogenic effects of Samhwangsasim-tang(SHT), Daehwanghwangryunsasim-tang(DHT) aqueous extract on HepG2 cells with palmitate. Materials and Methods: HepG2 cells treated with palmitate were used in this study as hepatic steatosis model. Cells were treated with different concentrations of SHT, DHT aqueous extract for 24 hours. Cell viability and cytotoxicity were analyzed by MTT assay.

2 Expressions of Bcl-2, Bax, Survivin, P21, TGF- 1, LXR- , ChREBP, ACC1, SCD1 mRNA were determined by Real-time PCR. Apoptosis of cells was detected by ELISA and FACS. Expression level of caspase-3 was studied by Western blot. Lipid accumulation was indicated by Oil Red O : SHT, DHT aqueous extract had no cytotoxicity, but decreased palmitate-induced lipid accumulation in HepG2 cells. SHT aqueous extract suppressed fatty acid synthesis by inhibiting LXR- , ChREBP, SCD1 activation and increasing TGF- 1 expression level. DHT aqueous extract also suppressed fatty acid synthesis by decreasing ChREBP expression and increasing TGF- 1 expression. Apoptosis of lipid accumulated cells was increased by enhanced activities of P21, caspase-3 and inhibited expressions of Bcl-2, : These results suggest that SHT and DHT have an anti-lipogenic effects on lipid accumulation of hepatic cell.

3 Also SHT and DHT have an efficacy to increase apoptosis of adipocyte without cytotoxicity. Therefore, SHT and DHT might have potential clinical applications for treatment of hepatic Words : Samhwangsasim-tang, Daehwanghwangryunsasim-tang, Hepatic steatosis, NAFLD, Lipogenesis, Apoptosis Received 27 November 2015 Revised 16 March 2016 Accepted 24 March 2016 Correspondence to (Young Chul Kim) 23 Tel +82-2-958-9118, Fax +82-2-958-9258, E-mail , 5% 1). , (Non-alcoholic fatty liver disease, NAFLD) 2). NAFLD , (hepatosteatosis) 3) (Non-alcoholic steatohepatitis, NASH) , 37 1 (2016 3 )J Korean Med.

4 2016;37(1):62-76 1010-0695 eISSN 2288-3339(63) 1 : Palmitic acid HepG2 cell of Samhwangsasimtang(SHT), Daehwanghwangryeonsasimtang(DHT)Scientif ic nameVolume(g)Herbs of DHT RheiRhizoma40 CoptidisRhizoma20 Scutellariae Radix20total amount80 Scientific nameVolume(g)Herbs of SHT RheiRhizoma48 Rehmanniae Radix32 CoptidisRhizomaScutellariae Radix1616total amount112 4). NAFLD 14~27% 5), 6). NAFLD 7,8), , 9). NAFLD , 10,11,12). , , 13), 14).

5 , , 15), NAFLD . , . MTT assay, Realtime-PCR, ELISA, FACS, Western Blotting, Oil Red O staining , .1. 1) , Prescription . 2) ( SHT) 4 (112 g) ( DHT) 5 (80 g) , 3 1000ml 2 . , 24 g( ) g( ) . (64) 37 1 (2016 3 ) ) Palmitic acid, Oil Red O, 3-[4,5-dimethylthiazol -2-yl]-2,5-diphenyltetrazolium bromide(MTT), Trizma base, Ammonium persulfate(APS), N,N,N',N tetramethylethylenediamine(TEMED), Phenylmeth- anesulfonyl fluoride(PMSF) Sigma Aldrich ( , MO, USA).

6 , Pageruler Plus Prestained Protein Ladder, Pierce BCA Protein Assay Kit, Supersignal West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific (Waltham, MA, USA) , 10 cell lysis buffer Cell Signaling Technology(Beverly, MA, USA) Western Blotting Detection Reagent GE Healthcare(Little Chalfont, UK) . detecting Enhanced Chemil- uminescence Kit Santa Cruz Biotechnology(CA, USA) , Davinch -Chemi Chemiluminescence Imaging System Davinch-K Co. Ltd(Seoul, Korea) . Dulbecco's Phosphate Buffered Saline(DPBS), Dulbecco's modification of Eagle's medium(DMEM) Welgene Inc.( , Korea) , Penicillin streptomycin, Trypsin-EDTA, no Glucose Gibco Invitrogen (Grand Island, NY, USA) , Fetal Bovine Serum(FBS) Atlas Biologicals(Fort Coliins, CO, USA).

7 2. 1) (Human hepatocellular carcinoma cell line) HepG2 cell . HepG2 cell FBS 10%, penicillin 10 U/mL, streptomycin 100 g/mL DMEM , 5% CO2, 37 C . 2) Palmitic acid Choi 16) . NaOH M PA 20 mM , 70 C 30 , 10% fatty acid free BSA solution 1:3 37 C conjugation . FA stock solution 5 mM Glucose DMEM mM, FBS 1:4 FA working solution 1 mM . 3) MTT assayCell viability MTT assay . HepG2 cell 24well cell culture plates 5 104 cells/well colony , 48 . 24 mM Glucose DMEM, FBS fasting PMS/MTS solution 490 nm .4) Real-time PCRT otal RNA total RNA Trizol reagent (Invitrogen).

8 Total RNA 1 g random hexamers reverse-transcription system(Promega, Madison, WI, USA) cDNA . primer sequences table1.. Real-timePCR Applied Biosystem (CA, USA) StepOnePlusReal_time PCR System , House keeping gene -actin . 2 x SYBR Green PCR Master Mix 10 l, cDNA 1 l, primer(sense) 1 l, primer(anti-sense) 1 l, Distilled water 7 l 20 l , (denaturation) cycle 95 C 3 , 40 cycle 95 C 10 , (annealing) 60 C 10 , (65) 1 : Palmitic acid HepG2 cell (extension) 72 C 15 .5) ELISA Cell death detection ELISA kit(Roche Molecular Biochemicals, Mannheim, Germany).

9 HepG2 6-well plate 24 PA 24 . ml (lysis buffer) 30 200 xg 10 405 nm .6) FACSA nnexin-V/PI assay kit , HepG2 . PA HepG2 ice-cold PBS buffer 2 , 500 l binding buffer , FITC Annexin-V (10 mg/ml) PI (50 mg/ml) 5 l , 5-15 , FACS Calibur (Becton Dickinson Immunocytometry System, San Jose, CA, USA) .FITC , FITC PI , . 7) Western Blotting100 mm cell culture dish 2 106 cells/well colony 48 mM Glucose DMEM, FBS 24 fasting.

10 PA SHT, DHT 24 1 cell lysis buffer+1mM PMSF , Pierce BCA Protein Assay Kit . Sample buffer 100mM DTT, 30% glycerol, bromophenol blue, 187 mM Tris-HCL 95 C 5 10~12% SDS-polyacrylamide gel . gel nitrocellulose membrane 1 5% nonfat dry milk blocking . 4 C overnight Cleaved Caspase-3, -actin , TBS-T 1:1000 . TBS-T 1:2500 horseradish peroxidase 2 1 , TBS-T 1 . Enhanced Chemiluminescence Kit detecting , Davinch-Chemi Chemiluminescence Imaging System . band intensity .8) Oil Red O stainingHepG2 cell , intracytoplasmic lipids Oil Red O17).