海藻由来脂質における抗炎症 ... - saga-itc.jp

1 (1) - - LPS NO methanol , ethanol chloroform NO ethanol NO/ NO/ phospholipase LPS LPS toll TLR 1) LPS 2) LPS TLR NO 3-5) NO peroxinitrite 6) tumor necrosis factor- (TNF- ) interleukin-6 (IL-6) TLR 7) 8),9) 10) 11) 12) TLR TLR / 13)

2 Porphyra yezoensis 1 24 35 14) 15) 16) 17) 18) 19) 20) porphyra-334 21) in vitro LPS 2010 ZM-1; Retsch 125 m -18 C penicillin/streptomycin Life Technologies Gibco , (FBS; Thermo Scientific HyClone), Dulbecco's Modified Eagle Medium (DMEM; ) , LPS Escherichia coli 0111:B4 ; ), dimethyl sulfoxide (DMSO; ), mouse TNF- ELISA Kit (Mouse ELISA Ready-SET- Go!

3 ; Affymetrix eBioscience), Cell Counting Kit-8 ( ), (Wakogel C-300HG; ), 1-palmitoyl-2-oleoyl-phosphatidylglycero l (PO-PG), 1-palmitoyl-2-oleoyl-phosphatidylethanol amine (PO-PE), 1-palmitoyl-2-oleoyl-phosphatidic acid (PO-PA), 1-palmitoyl-2-oleoyl-phosphatidylserine (PO-PS), 1-palmitoyl-2-oleoyl-phosphatidylcholine (PO-PC), 1-palmitoyl-2-hydroxy-phosphatidylcholin e (P-LPC) ( 6 Avanti Polar Lipid), phenylmethylsulfonyl fluoride (Sigma-Aldrich Co), RIPA buffer (Cell Signaling Technology), Laemmli SDS-PAGE sample buffer (Bio-Rad), anti-mouse iNOS rabbit polyclonal antibody (Cell Signaling Technology), anti-mouse COX-2 rabbit polyclonal antibody (Enzo Life Sciences), anti-mouse GADPH rabbit polyclonal antibody (Gene Tex), alkaline phosphatase conjugated anti-rabbit IgG goat polyclonal antibody (Life Technologies Invitrogen). (Western-SuperStar; Life Technologies Invitrogen), phospholipase A1 (Thermomyces lanuginonus ; Sigma-Aldrich Co), phospholipase A2 (Naja mossambica mossambica ; Sigma-Aldrich Co) , phospholipase C (Clostridium perfringens.

4 Sigma-Aldrich Co) 7 methanol, ethanol, acetone, chloroform, ethyl acetate, petroleum ether, n-hexane 1 g 10 mL 1 1,500 x g, 10 10 mL 1 20 mg/mL -40 C 1% penicillin/streptomycin, 10% FBS DMEM 5%CO2 37 C LPS NO TNF- 22) 2 24 24 2 105 cells/mL 96 well 200 L 16 180 L 15 LPS 20 L 200 L n = 3 6 19 TNF- 6 NO 19 50 mg/mL DMSO DMSO DMSO TNF- ELISA DMSO LPS LPS TNF- TNF- (%)={1 (Sa Sb)/ (Sc Sb)} 100 Sa TNF- Sb TNF- Sc TNF- NO Griess 23)

5 NO2- 125 L 96 well Griess 125 L 10 Molecular devices M5 18 500 nm 650 nm A NO2- NO2- NO TNF- NO (%)={1 ( Aa Ab)/( Ac Ab)} 100 Aa Ab Ac Dunnett NO/ TNF- Cell Counting Kit-8 24) 2. 3 LPS 20 Cell Counting Kit-8 2 L reagent/98 L medium 6 450 nm 650 nm A A A NO/TNF- ethanol (SPE) 1g 80 mL chloroform/methanol/water = 1/2 (v/v) 20 mL chloroform 30 4 C 50% ethanol chloroform 25) chloroform acetone methanol 3 chloroform 20 mL 200 mg in 5 mL chloroform chloroform acetone methanol 200 mL -40 C ethanol (TLC) (150 g in 10 L solvent) TLC (Silica gel-60 TLC aluminum plates, Merck Germany) chloroform/methanol/water/triethylamine= 30:35:7.

6 35 (v/v) 26) : (1) 50% sulfuric acid , (2) Dittmer-Lester 27 (3) diphenylamine (DPA) 28 . 6 PO-PG, PO-PE, PO-PA, PO-PS, PO-PC, P-LPC Rf LPS iNOS -2 3 24 24 COX-2 6 well 4 105 cells/mL 3 mL 16 15 LPS 10 ng/mL 3 mL 19 PBS(-) 1 mmol/L phenylmethylsulfonyl fluoride RIPA buffer mL RIPA buffer Sonifier 150, Branson 20,000 x g, 5 mol/L trichloroacetic acid Acetone 50 L Laemmli SDS-PAGE sample buffer (5% -mercaptoethanol ) 2 L 1 mol/L ammonium hydrogen carbonate 100 C5 SDS-PAGE 10 L Mini-Protean TGX, 4-20%, Bio-Rad SDS-PAGE iBlot 7-Minute Blotting System (Life Technologies ) PVDF anti-mouse iNOS rabbit anti-mouse COX-2 rabbit alkaline phosphatase conjugated anti-rabbit IgG anti-mouse GAPDH rabbit 29) Phospholipase methanol phospholipase A1, phospholipase A2 phospholipase C in vitro NO/ 4 mL methanol ( mg) 2 mL diethyl ether/methanol = 98/2 (v/v).

7 Acetate buffer (50 mmol/L, ) 6 acetate buffer DMSO 50 mg/mL 1 mg/mL 1% penicillin/streptomycin, 10% FBS DMEM in vitro 1 LPS NO TNF- NO TNF- chloroform methanol ethanol acetone NO TNF- n-hexane NO methanol ethanol WST 100 g/mL NO/TNF- ethanol chloroform/methanol/water SPE NO/TNF- DMSO , 100 g/mL 4 104cells/well, in 96 well microplate . LPS 10 ng/mL NO TNF- , DMSO.

8 *p< , **p< (Dunnett s test, vs control).Yield of extracts (%)Inhibition rate (%)Nitric ** ** ** *TNF- * ** ** viability (%) etherEthanolEthyl 4 24 24 2 ethanol SPE chloroform acetone methanol 100wt% 3 NO NO 3 acetone methanol 4 TNF- NO TNF- 3 acetone methanol TNF- NO NO/ TNF- iNOSCOX-2 GAPDHME ( g/mL) 0 0 1 10 100 LPS (10 ng/mL)

9 INOS COX-2 methanol Methanol (ME) DMSO , 1-100 g/mL 106cells/well, in 6 well plate . Cell lysate western blotting LPS iNOS COX-2 . ethanol Yield-1, 100 ; Yield-2, Ethanol 100 ; Yield-3, 100 .Yield-1 Yield-2 Yield-3 Ethanol layer (hydrophilic) layer (lipophilic) NO ethanol DMSO , g/mL 4 104cells/well, in 96 well microplate . LPS 10 ng/mL NO , DMSO . *p< , **p< (Dunnett s test, vs control). * ** ** ** ** ** ** ** ** **Methanol eluateLevel( g/mL)% Inhibition of nitric oxide productionEthanol extractSolvent partitionSPE fractionationHydrophilicLipophilicChloro form eluate Acetone eluate TNF- ethanol DMSO , g/mL 4 104cells/well, in 96 well microplate.

10 LPS 10 ng/mL TNF- , DMSO . *p< , **p< (Dunnett s test, vs control). * ** ** ** ** ** ** * ** ** **Methanol eluateLevel( g/mL)% Inhibition of TNF-alpha productionEthanol extractSolvent partitionSPE fractionationHydrophilicLipophilicChloro form eluate Acetone eluate 5 24 24 Methanol LPS 2 iNOS COX-2 1 LPS iNOS COX-2 10 g/mL methanol LPS GAPDH LPS TLC 2 ethanol TLC 50% sulfuric acid ethanol methanol Dittmer-Lester DPA acetone DPA Dittmer-Lester chloroform - NO/TNF- 020406080100 BufferPLA1 PLA2 PLC% Inhibition-80-4004080 BufferPLA1 PLA2 PLC% Inhibition1 g/mL10 g/mL1 g/mL10 g/mLA; NOB.