継続的運動はKupffer細胞の貪食能向上によりEndotoxinに対する生体クリアランス増大と炎症応答の低下を誘導する

継続的運動はKupffer細胞の貪食能向上により

Endotoxinに対する生体クリアランス増大と炎症応

答の低下を誘導する

著者

小峰 昇一

発行年

2018

学位授与大学

筑波大学 (University of Tsukuba)

学位授与年度

2017

報告番号

12102甲第8688号

URL

http://doi.org/10.15068/00152470

Kupffer

Endotoxin

1 2nonalcoholic fatty liver disease: NAFLD3 endotoxin

Kupffer 2KCs3 endotoxin NAFLD

ー ； KCs endotoxin i KCs i In vivo 8 2 5 8 , 10-18 m/ , 50 / 3 endotoxin20.01 µg/g body weight3 ・

endotoxin 2TNF-α IL-63 KCs

latex beads 2CD68, MARCO, SR-A3 2DHEA, testosterone, corticosterone, estradiol3 2leptin, adiponectin3

In vitro 1 2RAW264.73

latex beads endotoxin 2NFκB-p653

In vivo endotoxin 4 2area under the curve :

± 1.4 EU/ml, P < 0.053 Endotoxin 1.5 TNF-α IL-6 2TNF-α: 19.5 ± 5.2 vs. 11.0 ± 3.6, IL-6: 14.5 ± 0.9 vs. 10.6 ± 0.6 ng/ml, P < 0.053 endotoxin 1 KCs latex beads 2MFI: +20.8 ± 4.5%, P < 0.053 CD68, MARCO, SR-A 2CD68: +25.6 ± 1.1%, MARCO: +18.0 ± 1.1%, SR-A: +14.8 ± 3%, P < 0.053

DHEA 23.5 ± 1.1 vs. 6.8 ± 1.2 ng/ml, P < 0.053 In vitro RAW264.7 DHEA2 10 ng/ml3

latex beads 2MFI: +15.2 ± 1.7%, P < 0.053 endotoxin NFκB-p65

2-25.0 ± 4.1%, P < 0.053

DHEA KCs

endotoxin

1 2NAFLD3 Kupffer NAFLD NAFLD endotoxin 8 Kupffer

Kupffer Kupffer endotoxin Endotoxin DHEA 1 DHEA 1 9 ー

NAFLD

1

1 _{nonalcoholic fatty liver disease:}

NAFLD NAFLD 20-40%

12-30% 9-30% NAFLD

nonalcoholic steatohepatitis: NASH

NASH 3-5%

NAFLD Two hit hypothesis 1

1st hit

2nd hit

i i i NAFLD endotoxin 3 NASH i endotoxin ・ 々 endotoxin 4 endotoxin 1 Kupffer 2KCs3 NASH KCs endotoxin NAFLD

Kupffer

NAFLD NASH

NAFLD

NAFLD

16, 17 _{KCs latex beads} 16 i endotoxin KCs NAFLD KCs ． ； ー KCs endotoxin i KCs endotoxin

7 C57/BL6J Charles River Japan (Kanagawa, Japan) 12 ； 22.5 ± 1.4 , 55.6 ± 4.0% ） 6 8 23moR3 3 23moTr3 2 1 ー MK-6802Muromachi Kikai3 ー 3 5 m/min 10 10 m/min 10 2 10 m/min 10 15 m/min 10 20

10 m/min, 12 m/min, 14 m/min, 16

m/min 5 i 30 18 m/min

50 5 3moR 3moTr

1 Clophosome®-A

2FormuMax Scientific3 8 µL/g body weight 々 2 Lipo3moR Lipo3moTr3 3moTr

24 1 250 mg/kg body weight3 2

3

・ i 4000 rpm 15

1

ー X CT Latheta2LCT-200, Hitachi Aloka Medical3

i -80

KCs 1 i

Liver Perfusion Medium 2Gibco3 4 1 Type42Worthington Biochemical3

12Wako Chemical3 HEPES2Dojindo3 DMEM 10 − Hepatocyte Wash Medium2Gibco3

i 30 g 2 400 g 8

KCs 1 1

2Invitrogen, FluoSpheres®, 1.0 µm beads diameter, carboxylate-modified3 0.57 µl/g

body weight ・ 5 KCs KCs

1 1 F4/80 latex beads KCs latex beads

KCs 2CD68, macrophage receptor with collagenous structure; MARCO macrophage class A scavenger receptors; SR-A3

1 1 ー

APC-conjugated anti-F4/80 2 17-4801-82, eBioscience 3 PerCP/Cy5.5-conjugated anti-CD682137010, BioLegend3, MARCO2MCA1849, Bio-Rad3 SR-A2AF1797, R&D Systems3 ー MARCO SR-A 2 Alexa Fluor 488

2Invitrogen3 ー Gallios 1 1 1

2Beckman Coulter3 ー 1 Kaluza 2ver. 1.2, Beckman Coulter3 ー

PBS 4% 24

70% 1 3

µm 1 ー

90 HistoVT One2Nacalai Tesque3 20

v 1 3% H2O2 with methanol ー

Tesque3 20 1 PBS F4/80 anti-body 2HM1066, Hycult Biotechnology3 4 v1 1

v 30 VECTASTAIN UNIVERSAL Elite ABC kit2Vector Laboratories Burlingame3 ー 1

2BZ-X710; Keyence Japan3 ー

endotoxin

24 ・ i endotoxin2E. coli O111: B4, Sigma- Aldrich3 0.01 µg/g body weight 々 Endotoxin 2Pre3

1.5, 3.0, 6.0, 12.0

0 4 4000 rpm 15 ー

-80 endotoxin LAL pyrochrome reagent in Glucashield buffer2Associates of Cape Cod3 ー i

PyroColor Diazo Reagents kit2Associates of Cape Cod3 ー 540 µm iMark Absorbance Microplate Reader2BioRad Laboratories3 ー

2TNF-α, IL-6, and IL-103 Multiplex bead analysis system2Bio-Rad Laboratories3 ー Testosterone2ADI-900-065, ENZO

Life Sciences3, estradiol2ES180S-100, Cal Biotech3, corticosterone2501320, Cayman Chemical Company3 DHEA2DI-900-093, ENZO Life Sciences3, leptin2M1305, Morinaga Institute of Biological Science3, adiponectin2MRP300, R&D Systems3

ー 1 2RAW264.73 10% 2FBS3 penicillin250 U/ml3 250 mg/ml3 1 DMEM2Nacalai Tesque3 5% CO2 95% 37 2.5 × 105 RAW264.7 6 well 1 24 1 DHEA2TCI3 0, 1.0, 5.0, 10.0 ng/ml DMSO 12 Latex beads 0.66 µl/ml

12 4% 1 1 ー 2 1 1 2 24000 rpm, 3 3 i latex beads 1 1 Endotoxin RAW264.7 24

24 endotoxin2100 ng/ml3 6 1

PBS protease inhibitor cocktail phosphatase inhibitor cocktail2Nacalai Tesque3 SDS buffer -80

1 BCA 2Thermo Scientific Pierce3

ー lysis buffer 1 well

i 10 µg

1 Blocking One P2Nacalai Tesque3 i 6 β-Actin2SC-1616, Santa Cruz3IκB2SC-371, Santa Cruz3 NFκB-p652C22B4, Cell Signaling Technology3 phospho-NFκB-p65293H1, Cell Signaling Technology3 1 Signal Enhancer HIKARI for Western Blotting and ELISA2Nacalai Tesque3 4

2 2 Chemi-Lumi One Super 2Nacalai Tesque3 ChemiDoc XRS + system chemiluminescence imager2Bio-Rad3 ー Image Lab software2Bio-Rad3

ー

1 IBM SPSS ver. 21 ー ±

5%． 2 1

Mann-Whitney U 2 1 1 Kruskal-

3 1 3moR 3moTr （ 2 23 ー CT i 2 33 2 3a3 3moR 3moTr 2 3b, c3

Kupffer

3moTr 7 2 4a3 Lipo3moR

F4/80

1 1 F4/80

3moR 3moTr 7 Lipo3moR

1.

2a3 2b3 2c3 n = 8

2.

2a3 2b3 2c3 2d3 n = 8

3.

2a3 2b3 2c3 ー CT i

4. Kupffer

2a3 F4/80 1 1 50 µm 2b3F4/80

1 1 n = 8

Kupffer

3 KCs ；

KCs 1 1 i F4/80

latex beads i

latex beads 1

2 5a3 KCs latex beads i KCs 3moR

3moTr 2 5b3 KCs i

latex beads 2Mean fluorescence intensity: MFI3 3moR

3moTr 2 5c3

Kupffer

Kupffer KCs

1 1 KCs CD68

3moR 3moTr 2 6a3 KCs

MARCO SR-A 3moR

5. Kupffer

2a3F4/80 2Kupffer 3 latex beads 2b3Latex beads

2c3Latex beads 2MFI3

1 1 n = 8 ±

6. Kupffer

2a3CD68 2b3MARCO 2MFI3

2c3SR-A MFI n = 8 ±

5; P < 0.05

endotoxin

Kupffer 3

endotoxin

・ endotoxin 3

2 7a3 24 endotoxin

endotoxin 2 7b3 Endotoxin 3moR

1.5 1 3moTr 3moR

1.5 endotoxin 1 3moR

endotoxin 1 Lipo3moR Lipo3moTr endotoxin 1 3moR 3moTr

endotoxin 2AUC3 3moR 3moTr Lipo3moR Lipo3moTr 2 7c3

7. ・ endotoxin endotoxin

2a3 ・ endotoxin 2b3

endotoxin n = 8

Endotoxin

Endotoxin 8 3moR

endotoxin 1.5 TNF-α IL-6 1

3moTr 1.5 1

TNF-α IL-6 1 3moR

Lipo3moR Lipo3moTr 3moR 3moTr

TNF-α IL-6 AUC 3moR 3moTr Lipo3moR Lipo3moTr

i 3 endotoxin

IL-10 endotoxin 1.5 1 1

8. Endotoxin

2a3TNF-α 2b3IL-6 2c3IL-10 n = 8 ± 5; P < 0.05.

2 83 testosterone estradiol corticosterone

2 9a-c3 DHEA 3moR 3moTr 2 9d3

3b 3

leptin adiponectin 2 103 Leptin adiponectin 3moR 3moTr

9.

2a3Testosterone 2b3Estradiol 2c3Corticosterone 2d3DHEA

10.

2a3Leptin 2b3Adiponectin

DHEA

1

DHEA 2 9d3 DHEA

1 2RAW264.73

RAW264.7 DHEA i latex beads

2 11a3 1 1 latex

beads MFI DMSO 10 ng/ml

DHEA

1

11. DHEA 1 RAW264.7

2a3Latex beads latex beads 1 1 50 µm

2b3Latex beads 2MFI3 1 1

12. DHEA 1 2RAW264.73

Endotoxin Endotoxin (100µg/ml)

6 n = 3 ±

endotoxin 2 3a3 KCs latex beads i

endotoxin 2 3b3 KCs

Yano F344 15% 21 m/min 60 ・ endotoxin KCs

latex beads i 16 in vitro

endotoxin KCs 20

KCs ・

KCs latex beads i

2 5 min 3 2 53 KCs

CD68 1 々 2 63 KCs

CD68 latex beads i20

1 々 2MARCO SR-A3 endotoxin 21,22

i KCs KCs endotoxin endotoxin 1 1 21 ₀ KCs endotoxin Endotoxin i TNF-α IL-6 IL-10 1.5 1 2 73 endotoxin TNF-α IL-6 2 7a, b3 KCs i endotoxin endotoxin IL-10 2 7c3 40-60% endotoxin IL-10 23 Endotoxin KCs

24,25,26

i DHEA estradiol testosterone

corticosterone i 2 93 i 24 DHEA i DHEA/S 27 i DHEA/S 28 DHEA/S 7-22 i i 29 _{In vivo DHEA} endotoxin TNF-α 30 _i DHEA endotoxin KCs DHEA KCs in vitro 1 RAW264.7 ー

2 9d3 DHEA latex beads 2 113 endotoxin NFκB-p65 ( 12) i in vivo DHEA/S

KCs in vitro

2 3 31

RAW264.7 DHEA endotoxin TNF-α

32

1

33,34 _leptin

adiponectin 1

33,34 _{leptin adiponectin 3moTr 3moR}

2 103 35,36 3 KCs NAFLD NAFLD NAFLD endotoxin 4 KCs 9 37 _NAFLD DHEA （ 38 0 KCs NAFLD 15 NAFLD 1 ー ；

3 Kupffer i endotoxin

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i i i i i 1 . 1 1 i i i i