Download Induction of oxidative stress

EGF and L-Glutamine Accelerates the
Restoration of Epithelial Barrier
Function From Oxidative StressInduced Injury
Nichole Barnhart
Oxidative Stress
• 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 stressinduced 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.
GI Mucosal Protective Factors
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.
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.
Hypothesis
EGF and L-glutamine accelerate the restoration of
epithelial barrier function from oxidative stress-induced
injury.
Experimental Strategy
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).
Data Sheet
Data Form
TER (% of baseline value)
Effect of EGF on Restoration of Caco-2
Epithelial Barrier Function
120
100
80
60
C
40
HP+EGF
20
HP
0
0
30
60
90
Time (min)
120
150
240
270
Effect of EGF on Restoration of Caco-2
Epithelial Barrier Function
3
Inulin Flux
(% flux/hr/cm2)
2.5
2
1.5
1
0.5
0
C
HP
HP+EGF
Effect of Glutamine on Restoration of Caco-2
Epithelial Barrier Function
TER (% of baseline value)
110
100
90
80
C
HP
HP+GL
70
60
50
40
0
30
60
90
Time (min)
120
150
Effect of L-Glutamine on Restoration of Caco-2
Epithelial Barrier Function
Inulin Flux
(% flux/hr/cm2)
3.5
3
2.5
2
1.5
1
0.5
0
C
HP
HP+GL
Effect of L-Glutamine on Restoration of
Occludin Distribution
Control
Restored for 3 h
with Glutamine
Restored for 3 h
without Glutamine
Effect of L-Glutamine on Restoration of ZO-1
Distribution
Control
Restored for 3 h
with Glutamine
Restored for 3 h
without Glutamine
Summary

Recovery of TER and reduced inulin flux indicates that
EGF and glutamine accelerate the restoration of
epithelial barrier function after the hydrogen peroxideinduced injury.

Confocal microscopy shows that glutamine accelerates
the restoration of the integrity of tight junction after the
damage induced by hydrogen peroxide.
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