Drug evaluation of LPS-induced acute lung injury (ALI)
mice models (2nd Report)○ Souta Ushijima, Hajime Wada, Masateru Furuta, Kimihide Okamoto, Kousuke Morizumi,Seiichi Katayama, Naoyuki Hironaka, Katsuhide Nishi

Summary in Japanese

Objective

急 性 肺 損 傷（acute lung injury：ALI），急 性 呼 吸 促 迫（窮 迫）症 候 群（acute respiratory distress syndrome：ARDS）は，呼 吸不全（肺 機 能不全）の総称で，新型コロナウイルス感染症の流行から注目を集めている．
急性 肺障害の動物モデルは，LPS の吸 入などにより作製し，短 期間，かつ，簡便に実施できることから薬物のスクリーニングに用いられている．
本研究では，マウスにα-GalCer および LPS を処置することで作製した急性肺炎モデルに対する薬物の作用をSpO2，BALF 中炎症細胞浸潤ならびにBALF上清中サイトカイン濃度により検討した．
その結果，薬物それぞれに特徴的な効果が認められ，薬物による肺炎治療に対するツールとして有用であることが確認された．

In the 94th Annual Meeting of the Japanese Pharmacological Society, we reported two kinds of pulmonary
inﬂammatory models using lipopolysaccharide (LPS): a LPS inhalation model and an α-GalCer-LPS instillation
model. In the previous report, we concluded that these LPS-induced ALI models could be useful to evaluate
therapeutic eﬃcacy of drugs used for treatment of pneumonia.
In the present study, we carried out further research on the LPS instillation model with a pretreatment ofα-garactosyl ceramide (α-GalCer).
We also examined eﬀects the eﬀect of exosome from the human adipose cell and anti-IL-6R antibody. We
evaluated inﬂammatory cell inﬁltration in the BALF (Broncho Alveolar Lavage Fluid), BALF supernatant cytokine
level and SpO2.

Materials and Methods

Discussion● Animal

BALB/cAnNCrlCrlj (Jackson Laboratory Japan, Inc.), ♂,7 weeks old (at Day 0)

Schedule● Reagents
LPS:

Lipopolysaccharides, from Escherichia coli
O111:B4, Sigma-Aldrich Inc.
α-GalCer (α-galactosyl-ceramide):
Kyowa Hakko Kirin Co., Ltd.
Dexamethasone:
Sigma-Aldrich Inc.
Exosome: Preadipocyte Exosomes (human), ZENbio
Anti-IL-6R antibody:
InVivoMAb anti-mouse IL-6R, BioXcell

Day 0Day 1

Day 3-30 min

After about
48 hours

0α-GalCer Instillation*
Anti-IL-6R Ab.
i.p.

● Measurement System
SpO2:
Cytokine:

Evaluation of the LPS-induced lung injury animal model wascarried out by administrating various reagents. Reagents
showed characteristic eﬀects on this animal model. Theresults indicate that characteristic pathophysiologicalchanges in the present model was identiﬁed and alsosuggested that this model is very useful for ALI study.

+4hr

・ Dexamethasone might be useful for medical treatment ofpneumonia at a high dose.
・ To explore the possibility that the Exosome may have atherapeutic eﬀect, it may be necessary to change thetiming of administration, etc.

SpO2, BALF,
Lung collection

LPS Instillation*
Exosome Instillation

・ Anti-IL-6R was partially eﬀective (cytokine), it may benecessary to change the timing of administration, etc forfurther conﬁrmation of eﬀectiveness.

Exosome Instillation

Dexamethasone
p.o.

Mouse OxPLUS (primetech)
Bio-Plex Multiplex Immunoassay System(Bio-Rad Laboratories, Inc.)+24hr*: Administration into lung by instillation to nasal
cavity under isoﬂurane inhalation anesthesia.

Results
Animal Model Conﬁrmation
Group ConﬁgurationGroup

α-GalCer

Sham
LPS
α-GalCer-LPS
Dexamethasone

1 µg

5 µg

10 mg/kg, p.o.

30 mg/kg, p.o.

239521N

4444410000( 103 cells)

Total cell****

ShamLPS
α-GalCer-LPS
Dexamethasone 10 mg/kg
Dexamethasone 30 mg/kg
Day 0Day 1Day 2

Results
Body Wight (g)

TNF-α

4000

7515IFN-γ

250

140080*α-GalCer-LPS 48hr
Exsosome 4-48hr
Exsosome 24-48hr
Day 0Day 1Day 2***200

IL-435600
50010*†

5IL-9**

IL-1060

64030

42010030

*

**

20

#101050017Day 3

75015250

IFN-γ

IL-1βα-GalCer-LPS

Day 0Day 1Day 2

1600

MCP-1

8000

90

6000

854000802000Day 3750250

IFN-γ

35

IL-4

IL-91086420600

500

100

IL-1β35

IL-10305025402030152010105000IL-4300

15200

1020
0

IL-13

10IL-9

864
20

5100

0060

#IL-6400

2040600
500

2560

##

200

30

80

200

0400

0300

0600

50

0400

100

60

800
100

500

51000

1000

IL-6MCP-1

1200

150

01600
1400

200

200

150

IL-1030

50

2540

2030

1520

10

10

50

0

IL-13

#: p<0.05, ##: p<0.01, signiﬁcant diﬀerence compared to normal group (Student's -test)

Histopathological Study

1. Inﬂammatory cell inﬁltration and cytokine release
were observed in the airways and lungs due to theinduction of pulmonary inﬂammation.
2. Dexamethasone in excessive doses was eﬀective tothis reaction [cytokine level: IL-10, G-CSF (data notshown)].

TNF-α

1500

400

2020

Dex. 30 mg/kg

2000

600

3080

Histopathological Study
LPS95

Cytokine Concentration (pg/mL)*: p<0.05, **: p<0.01, signiﬁcant diﬀerence compared to normal group (Student's or Aspin-Welch's t-test)†: p<0.05, signiﬁcant diﬀerence compared to LPS group (Student's or Aspin-Welch's t-test)#: p<0.05, ##: p<0.01, signiﬁcant diﬀerence compared to normal group (Dunnett's or Steel multiple comparison test)

Normalα-GalCer-LPS
Anti-IL-6R

1000

10**( 103 cells)

Total cell

191200015202000

504025Inﬂammatory Cells
10000

100

2380

10060

IL-13

55SpO2 (%)

27

4000

250508*100*

Body Wight (g)
25800

0IL-6

1 mg/mLN

α-GalCer-LPS
Anti-IL-6R antibody

Results

85

150

5000

*

200

100

6000

0

1400

300

15090

200

10000

400

20208000

15000

025

40

TNF-α

400

306030000

20000

600

0IL-1β95Concentration

2125000

1000

500( 103 cells)

Total cell

1200100500100

MCP-1800*

α-GalCer-LPS
0Anti-IL-6R antibody 200 µg/body333333Inﬂammatory Cells
10000

DoseCytokine Concentration (pg/mL)

1600

200GroupN

Exsosome 4-24hr
Exsosome 4-48hr
Exsosome 24-48hr

100

170

150
1000

1×1010 particles/body,Instillation

19

2000

80

*†
1500

0

SpO2 (%)

27Cytokine Concentration (pg/mL)
2000

Dose and Route

2185

Day 3

48
2448
2448
48

Normalα-GalCer-LPS 24hr
α-GalCer-LPS 48hr

2390(hr)

444442425

8000
6000(hr)Shamα-GalCer-LPS 24hr
α-GalCer-LPS 48hr
Exsosome 4-24hr
Exsosome 4-48hr
Exsosome 24-48hr

Inﬂammatory Cells

Group Conﬁguration

Adm. time Sacriﬁce timeGroupDexamethasone 10 mg/kg
Dexamethasone 30 mg/kg

105
100

150 mg/kg, p.o.

SpO2 (%)

2517

Dose and RouteShamLPS
α-GalCer-LPS

2719LPSSaline
5 µg

Body Wight (g)Anti-IL-6R Antibody Study

Group Conﬁguration

PBSPBS

Results

Exosome Study

Normalα-GalCer-LPS

Exsosome 4-48hr Exsosome 24-48hr

1. Exosome was suppressed the Inﬂammatory cellinﬁltration, but were not suppressed theinﬂammatory cytokine levels.
2. Depending on the timing of Exosome
administration, lung inﬂammation was found toworsen.

Histopathological Study
α-GalCer-LPS

Anti-IL-6R Ab.

1. Anti-IL-6R antibody was not suppressed theInﬂammatory cell inﬁltration, but were partiallysuppressed the cytokine levels [MCP-1, IL-9,GM-CSF, IL-5, KC, and MIP-1α (data not shown)].