Clinical Score Body Weight€¦ · Inhaled delivery of molecules provides direct access to the CNS via the ... EAE group + Ang -(1 7) Control group + saline Control group + liposomes

Evaluation of the anti-inflammatory response of Angiotensin 1-7 associated with liposomes administered via inhalation in mice with experimental autoimmune encephalomyelitis

Bárbara Fernandes Pinto1; Onésia Cristina Oliveira Lima (UFG) 2; Cláudia Martins Carneiro3; Robson Augusto Souza dos Santos1; Frédéric Jean Georges Frézard1; Juliana Carvalho Tavares1.

1. Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte - MG– Brasil.

2. Department of Pharmacology, Federal University of Goiás, Goias - GO - Brasil.

3. Department of Cytopathology, Federal University of Ouro Preto, Ouro Preto - MG - Brasil.

INTRODUCTION

DISCUSSION

Multiple sclerosis is a chronic, progressive and autoimmune diseasecharacterized by inflammatory infiltrates, gliosis, demyelination and axonaldegeneration in the central nervous system. Currently used therapies aregenerally associated with inconvenient methods of administration and severeside effects impairing patient safety and adherence to therapy.

So, the objective of this research was evaluate the anti-inflammatory responseof encapsulated-Ang-(1-7) in liposomes inhaled by mice with experimentalautoimmune encephalomyelitis.

Animals were divided in 5 groups

EAE induction

Clinical signs Score

No clinical signs 0

Partially limp tail 1

Paralyzed tail 2

Hind limb paresis, uncoordinated movement 3

One hind limb paralyzed 4

Both hind limbs paralyzed 5

Hind limbs paralyzed, weakness in forelimbs 6

Hind limbs paralyzed, one forelimb paralyzed 7

Hind limbs paralyzed, both forelimbs paralyzed 8

Moribund 9

Death 10

LITERATURE

Numerous studies have reported the association between cerebral RAS and thedevelopment of neurodegenerative diseases (Ang II/AT1 receptor axis).

Liposomes act as a drug targeting system and are able to prevent the rapiddegradation of the incorporated substance, promote penetration through theBBB and also exhibit high biocompatibility.

Inhaled delivery of molecules provides direct access to the CNS via theolfactory or trigeminal pathway without necessarily having to transpose the BBB.

Brain (IL -10)

0

500

1000

1500

2000

2500CTRL

CTRL + lipo

CTRL + Ang-(1-7)

CTRL + lipo + Ang-(1-7)

EAE

EAE + lipo

EAE + Ang-(1-7)

EAE + lipo + Ang-(1-7)

___*

Concentr

atio

n (

pg/m

L)

Spinal Cord (IL-10)

0

500

1000

1500

2000

2500CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)

EAE + lipo + Ang-(1-7)Concentr

atio

n (

pg/m

L)

Brain (TNF-a)

0

200

400

600

800CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


______________________________*

*

Concentr

atio

n (

pg/m

L)

Spinal Cord (TNF-a)

0

200

400

600

800CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


___*

Concentr

atio

n (

pg/m

L)

Brain (IL-1B)

0

200

400

600

800CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


atio

n (

pg/m

L)

Spinal Cord (IL-1B)

0

500

1000

1500CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


atio

n (

pg/m

L)

Brain (IL-6)

0

300

600

900

1200

1500CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


_________________

__________***

_____________**

*

Concentr

atio

n (

pg/m

L)

Spinal Cord (IL-6)

0

200

400

600

800

1000CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


atio

n (

pg/m

L)

Bearing post-capillary venules

0

2

4

6

***

CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Leukocyte

s r

olli

ng/

vessel/

20s

Bearing central venules

0

5

10

15

20

25

30

35

***

**CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Leukocyte

s r

olli

ng/

vessel/

20s

Adhesion post-capillary venules

0

2

4

6

8

***

CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Adhere

d leukocyte

s /

100µm

2

Adhesion central venules

0

10

20

30

***

CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Adhere

d leukocyte

s /

100µm

2

0

10

20

30

40

50

60

70

80EAE

EAE + lipo

EAE + Ang-(1-7)


*** ***

******

Num

ber

of

cells

/M

icro

scopic

fie

ld

EAE group

+ Ang-(1-7)

Control group + saline

Control group +

liposomes

Grupo Controle

+ liposomes + Ang- (1-7)

Control group

+ Ang-(1-7)

EAE group + saline

EAE group

+ liposomes

EAE group

+ liposomes + Ang- (1-7)

Control Groups EAE Groups

Leukocyte

Recruitment

Intravital

microscopy

Cytokines

levels

ELISA

Vascular

permeability

Evans Blue

MOG35-55

CFA + Mycobacterium tuberculosis H37RA

Pertussis toxin (2 doses)

RESULTS0 3 6 9 12 15 18 200

1

2

3

4CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


*** ***###

###*###

Clinical Score

Days

Score

Body Weight

0 3 6 9 12 15 18 2080

85

90

95

100

105

110CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


*** *#########

Days

Rela

tive W

eig

ht (1

00%

)

0 3 6 9 12 15 18 200

1

2

3

4CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


*** ***###

###*###

Clinical Score

Days

Score

Body Weight

0 3 6 9 12 15 18 2080

85

90

95

100

105

110CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


*** *#########

Days

Rela

tive W

eig

ht (1

00%

)

Brain Permeability

0

10

20

30

40__________

___________

__________******

**

_____________**

**

CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Concentr

atio

n o

f

Eva

ns B

lue (

pg/m

L)

Spinal Cord Permeability

0

10

20

30_____________

_________________

____________

***

*________________

**

**CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Concentr

atio

n o

f

Eva

ns B

lue (

pg/m

L)

Lung Permeability

0

5

10

15

_________________

________________________

**

****

***

***

_____________***

*___

___****

CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Concentr

atio

n o

f

Eva

ns B

lue (

pg/m

L)

Spleen Permeability

0

5

10

15CTRL

CTRL + lipo

CTRL + Ang-(1-7)


EAE

EAE + lipo

EAE + Ang-(1-7)


Concentr

ation o

f

Eva

ns B

lue (

pg/m

L)

Ang- (1-7) is able to inhibit the NF-kB pathway, causing reduction ofinflammatory cytokine levels (BIHL et al., 2015). A study conducted by WU et al.(2015) has shown that administration of icv of Ang- (1-7) in models of middlecerebral artery occlusion (MCAO) reduced the permeability of BBB.

Recent study has demonstrated through the flow cytometry technique thatAng II induces the reduction of Treg cells in the kidneys and blood (CHEN X et al.,2018). Ang-(1-7) in turn, is able to neutralize the proinflammatory actiontriggered by Ang II (GIRONACCI et al., 2018).

According to ODOARDI (2012), before the entry of autoreactive T cells into theCNS, these establish in the lung, reactivate and acquire the capacity to enter inthe CNS.

Conclusion: The nebulization of liposomes containing Ang-(1-7) presents atherapeutic potential, since it was able to attenuate the clinical score and theweight loss in the EAE animals. These Ang-(1-7) treatment effects may beassociated with decrease of interleukin 6 (antiinflammatory cytokine) andlowered cerebral and lung vascular permeability.

Liposomes had a mean diameter of 133.0 nmand a PDI of 0.3 indicating a monodispersedsystem.

Histological

analysis

------------------------------------------------------

Rolling post-capillary venules Rolling central venules

Adhesion central venules Adhesion post-capillary venules

Histological analysis

EAE1


CTRL

Documents

Clinical Score Body Weight€¦ · Inhaled delivery of molecules provides direct access to the CNS via the ... EAE group + Ang -(1 7) Control group + saline Control group + liposomes