EGF and L-Glutamine Accelerates the Restoration of Epithelial Barrier Function From Oxidative Stress-

Induced Injury

Nichole Barnhart

• Oxidative stress occurs when there is an accumulation of free radicals, such as superoxide, hydrogen peroxide and hydroxyl radical, which have one or more unpaired electrons, and can damage cells, by oxidizing proteins, fats and DNA (Del Maestro 1980).

• Oxidative stress is seen is patients with many gastrointestinal diseases (IBD, NEC, Colon cancer).

• A significant body of evidence indicates that tight junctions become weak or loose during oxidative stress-induced injury. Thus, injurious factors are able to penetrate the epithelium and induce inflammatory reactions in the tissue. (Farber 1994).

• So, the healing process during the treatment of diseases the oxidative stress-induced injury is expected to be restored.

Oxidative Stress

Epidermal Growth Factor (EGF)

• EGF, a polypeptide of 53 amino acids, is a GI mucosal protective factor.

• It is secreted in saliva at very high concentration.

• It stimulates the growth and repair of epithelial tissues.

L-Glutamine

•Glutamine, an important amino acid, is essential for the maintenance of intestinal metabolism, structure, and function.

•Glutamine has been shown to protect the GI mucosa from burn injury, infectious enterocolitis and in surgery patients with total parenteral nutrition.

It is hard to prevent oxidative stress, especially if damage has already occurred (such as a heart attack or stroke). It may be possible, however, to

accelerate the restoration of cells after oxidative stress has occurred.

GI Mucosal Protective Factors

Hypothesis

EGF and L-glutamine accelerate the restoration of epithelial barrier function from oxidative stress-induced injury.

1. Epithelial model

• Caco-2 cells, a colon cancer cell line that grow as a normal intestinal epithelium when cultured on Transwell inserts.

2. Induction of oxidative stress

• Caco-2 cell monolayers were exposed to hydrogen peroxide (100 M) for 60 min to induce oxidative stress.

• Restoration of barrier function was monitored after removing the hydrogen peroxide and continued incubation in the absence or presence of EGF (30 nM) or glutamine (2 mM)

• Dr. Rao’s Lab Protocol as found in Rao (1997).

Experimental Strategy

Data SheetData SheetData Form

0

20

40

60

80

100

120

0 30 60 90 120 150 240 270

C

HP+EGF

HP

Effect of EGF on Restoration of Caco-2 Epithelial Barrier Function

Time (min)

TE

R (

% o

f ba

selin

e va

lue)

Effect of EGF on Restoration of Caco-2 Epithelial Barrier Function

I

nulin

Flu

x (

% f

lux/

hr/c

m2)

0

0.5

1

1.5

2

2.5

3

C HP HP+EGF

40

50

60

70

80

90

100

110

0 30 60 90 120 150

C

HP

HP+GL

Effect of Glutamine on Restoration of Caco-2 Epithelial Barrier Function

Time (min)

TE

R (

% o

f ba

selin

e va

lue)

Effect of L-Glutamine on Restoration of Caco-2 Epithelial Barrier Function

I

nulin

Flu

x (

% f

lux/

hr/c

m2)

0

0.5

1

1.5

2

2.5

3

3.5

C HP HP+GL

Effect of L-Glutamine on Restoration of Occludin Distribution

ControlRestored for 3 h

without Glutamine

Restored for 3 h with Glutamine

Effect of L-Glutamine on Restoration of ZO-1 Distribution

ControlRestored for 3 h

without Glutamine

Restored for 3 h with Glutamine

Recovery of TER and reduced inulin flux indicates that EGF and glutamine accelerate the restoration of epithelial barrier function after the hydrogen peroxide-induced injury.

Confocal microscopy shows that glutamine accelerates the restoration of the integrity of tight junction after the damage induced by hydrogen peroxide.

Summary

Conclusion In conclusion EGF and glutamine may accelerate the

healing of gastrointestinal mucosa following oxidative stress injury by sealing the tight junctions and reducing the paracellular permeability.

Thanks to:

•Dr. R.K. Rao of the UTHSC Physiology dept.

•Parimal Sheth

•Ankur Seth