Functional analysis of docosahexaenoic acid metabolism in skeletal muscle injury and regeneration

Functional analysis of docosahexaenoic acid metabolism in skeletal muscle injury and regeneration

好中球は 19,20-EpDPE 産生細胞である

0

1

2

3

4

5

0 5

10 15 20 25 30

n = 4 mean ± s.e.m

0*500*

1000*1500*2000*2500*3000*3500*

4+HD

oHE*

7+HD

oHE*

8+HD

oHE*

10+HDo

HE*

11+HDo

HE*

13+HDo

HE*

14+HDo

HE*

16+HDo

HE*

17+HDo

HE*

16,17+Ep

DPE*

20+HDo

HE*

19,20+Ep

DPE*

19,20+diHD

oPE*

21+HDo

HE*

22+HDo

HE*

WT*CYP4F18*KO*

19,20&Ep

DPE

n = 3 mean ± s.e.m

n = 4 mean ± s.e.m

2 A Gr-1 B Gr-1 19,20-EpDPE

1 CTX 12 DHA 19,20-EpDPE

n=4, mean sem

0"

2000"

4000"

6000"

20:4"AA" 20:5"EPA"22:6"DHA"

0"

5000"

10000"

15000"

20000"

25000"

30000"

PGE2"

PGD2

"PG

F2a"

TXB2

"125HHT

rE"

LTB4

"55HE

TE"

85HE

TE"

95HE

TE"

115HETE"

125HETE"

155HETE"

165HETE"

175HETE"

185HETE"

195HETE"

205HETE"

5,65EET"

8,95EET"

11,125EET"

14,155EET"

45HD

oHE"

75HD

oHE"

85HD

oHE"

105HDo

HE"

115HDo

HE"

135HDo

HE"

145HDo

HE"

165HDo

HE"

175HDo

HE"

205HDo

HE"

215HDo

HE"

225HDo

HE"

16,175Ep

DPE"

19,205Ep

DPE"

No"injury"

CTX"12h"

AA ω6 DHA ω3

19,20-EpDPE!

OH

O

O

0 5000

10000 15000 20000 25000 30000 35000 40000 45000

N.D.

454035302520151050 0"

5"10"15"20"25"30"

1" 3" 10" 1" 3" 10"

DHA μM 19,205EpDPE"μM

AP#TGF

α(release((%

)

GPR120(

RNA-Seq による損傷筋の網羅的遺伝子発現解析

Extract*significant*genes*Intensity1based*Z1score*>*2 *

Up*−*166*genes*Down*regulated*−*181*genes

DAVID*1*Func\onal*annota\on*GO*analysis* GOTERM_BP_FAT *

TOP10*of*p1value*

GOTERM_BP

In*CYP4F18*KO*mice,**Genes*involved*in*immune(system(and*inflammatory(response(are*signifiicantly*up1regulated*in*Day1.

0* 2* 4* 6* 8* 10* 12*

immune*response*

inflammatory*response*

response*to*wounding*

posi\ve*regula\on*of*immune*system*process*

bone*development*

defense*response*

regula\on*of*cell*prolifera\on*

ossifica\on*

posi\ve*regula\on*of*response*to*s\mulus*

an\gen*processing*and*presenta\on*

#LOG10((p#value)

Immune(response

Inflammatory(response

PosiIve(regulaIon(of((immune(system(process

Reponse*to*wounding

bone*development

A 4 A CTX Day1 CYP-X KO GO B

CYP-X KO n = 5, mean sem

CYP4F18 KO マウスにおける炎症性サイトカインの発現上昇は 19,20-EpDPE の投与によって回復する

0.00"0.50"1.00"1.50"2.00"2.50"3.00"3.50"4.00"

DMSO

"

DMSO

"

19,205ED

P"

WT" 4F18"KO"

mRN

A/18

s(rRN

A

0.00"0.50"1.00"1.50"2.00"2.50"3.00"3.50"

DMSO

"

DMSO

"

19,205ED

P"

WT" 4F18"KO"

mRN

A/18

s(rRN

A

0.00"

0.50"

1.00"

1.50"

2.00"

DMSO

"

DMSO

"

19,205ED

P"

WT" 4F18"KO"

mRN

A/18

s(rRN

A

B

0.00"0.50"1.00"1.50"2.00"2.50"3.00"3.50"

WT" 4F18"KO"

mRN

A/18s(rRN

A

0.00"

0.50"

1.00"

1.50"

2.00"

WT" 4F18"KO"

mRN

A/18s(rRN

A

0.00"0.50"1.00"1.50"2.00"2.50"3.00"3.50"4.00"

WT" 4F18"KO"

mRN

A/18s(rRN

A

B

3 A CYP-X HEK293 19,20-EpDPE B WT CYP-X KO DHA C CTX 12h WT CYP-X KO 19,20-EpDPE n=3, mean sem

好中球は 19,20-EpDPE 産生細胞である

0

1

2

3

4

5

0 5

10 15 20 25 30

n = 4 mean ± s.e.m

0"500"

1000"1500"2000"2500"3000"3500"

45HD

oHE"

75HD

oHE"

85HD

oHE"

105HDo

HE"

115HDo

HE"

135HDo

HE"

145HDo

HE"

165HDo

HE"

175HDo

HE"

16,175Ep

DPE"

205HDo

HE"

19,205Ep

DPE"

19,205diHD

oPE"

215HDo

HE"

225HDo

HE"

WT"CYP4F18"KO"

19,20#Ep

DPE

n = 3 mean ± s.e.m

n = 4 mean ± s.e.m

CYP5X"KO

0"1000"2000"3000"4000"5000"6000"

Ctrl"

0"μM

"

1μM""

3μM""

10μM

""

30μM

""

100μ

M""

LPS"10"ng/mL"

pg/m

L

0"1000"2000"3000"4000"5000"6000"7000"

Ctrl"

0"μM

"

1μM""

3μM""

10μM

""

30μM

""

100μ

M""

LPS"10"ng/mL"

pg/m

L

DHA"

19,205EpDPE"

0"

5"

10"

15"

20"

25"

30"

1" 3" 10" 1" 3" 10"

DHA μM 19,205EpDPE"μM

A

B

0"

2000"

4000"

6000"

20:4"AA" 20:5"EPA"22:6"DHA"

0"

5000"

10000"

15000"

20000"

25000"

30000"

PGE2"

PGD2

"PG

F2a"

TXB2

"125HHT

rE"

LTB4

"55HE

TE"

85HE

TE"

95HE

TE"

115HETE"

125HETE"

155HETE"

165HETE"

175HETE"

185HETE"

195HETE"

205HETE"

5,65EET"

8,95EET"

11,125EET"

14,155EET"

45HD

oHE"

75HD

oHE"

85HD

oHE"

105HDo

HE"

115HDo

HE"

135HDo

HE"

145HDo

HE"

165HDo

HE"

175HDo

HE"

205HDo

HE"

215HDo

HE"

225HDo

HE"

16,175Ep

DPE"

19,205Ep

DPE"

No"injury"

CTX"12h"

AA ω6 DHA ω3

19,20-EpDPE!

OH

O

O

0 5000

10000 15000 20000 25000 30000 35000 40000 45000

N.D.

454035302520151050 0"

5"10"15"20"25"30"

1" 3" 10" 1" 3" 10"

DHA μM 19,205EpDPE"μM

AP#TGF

α(release((%

)

GPR120(

19,20-EpDPE 産生酵素の同定に向けた P450 酵素の活性スクリーニング

CYP-X

DHA!

19,20-EpDPE!

OH

O

OH

O

O

CYP4F18 は好中球に選択的に発現する P450 酵素である [JBC., 2006] → 損傷筋では CYP4F18 を介して好中球が 19,20-EpDPE を産生する？

5 Thioglycolate MΦ LPS IL-6, MCP-1

19,20-EpDPE DHA

0"1000"2000"3000"4000"5000"6000"

Ctrl"

0"μM

"

1μM""

3μM""

10μM

""

30μM

""

100μ

M""

LPS"10"ng/mL"

pg/m

L

0"1000"2000"3000"4000"5000"6000"7000"

Ctrl"

0"μM

"

1μM""

3μM""

10μM

""

30μM

""

100μ

M""

LPS"10"ng/mL"

pg/m

L

DHA"

19,205EpDPE"

0"

5"

10"

15"

20"

25"

30"

1" 3" 10" 1" 3" 10"

DHA μM 19,205EpDPE"μM

B