Professional Documents
Culture Documents
Grado en Bioqumica
Famacologa Molecular
doi:10.1016/j.phrs.2010.12.011
INDEX
Introduction
Methods
o
In vitro studies
Histological studies
Microbiological studies
Statistics
Reagents
Results
o
Discussion
INTRODUCTION
Inflammatory
bowel
disease (IBD)
Crohns
disease
Ulcerative
colitis
Pathogenesis
elusive
Chronic activation of
immune and
inflammatory cascade
Genetically susceptible
individuals
Imbalance in the
intestinal microflora
Manipulation of
enteric flora, an
important approach in
controlling the disease
Antibiotics, a
rational strategy in
the treatment of IBD
Minocycline
Anti-apoptotic, immunosuppressive and anti-inflammatory
properties
Increases the mRNA of IL-10 and reduces TNF-, inducible
nitric oxide synthase (iNOS) and cyclooxygenase-2
Hypothesis
Immunomodulatory and antimicrobial properties of
minocycline point out as a new therapeutic approach for IBD
treatment.
General Objetive
To study the role of its immunomodulatory properties and its
antibiotic activity in different models of colitis.
Specific Objetive
To evaluate the direct immunomodulatory properties of
minocycline in two cell types actively involved in the intestinal
immune response.
To test the intestinal antiinflammatory effect of orally
administered minocycline in different models of colitis, as a
prophylactic or curative treatment.
Caco-2
cells
RAW 264.7
cells
Minocycline,
tetracycline or
metronidazole
Negative control
Positive control
Untreated cells
Range: 2
to 100 m
Minocycline,
tetracycline or
metronidazole
Range: 2
to 100 m
Negative control
Positive control
Untreated cells
Laboratory Animal
Service Of the UGR
6 GROUPS
Female
Wistar rats
4 groups
Tetracycline
(80 mg/kg)
Untreated
TNBS control
group
Metronidazole
(40 mg/kg)
Non-colitic
group
(control)
10mg TNBS
dissolved in 0,25
ml of 50% ethanol
2 ml of
distilled
water and
administered
daily by oral
gavage
SACRIFICE
Overdose of
halothane
7 days
Antibiotic
+ TNBS
2 days
Sacrifice
- Antibiotic
7 days
Antibiotic
+TNBS
Sacrifice
- Antibiotic
DAILY EVALUATION
Macroscopically
visible
damage (010 scale)
Remaining
colon samples
4% buffered
formaldehyde
Biochemical
determinations
Histological
studies
RNA isolation
Female
C57BL/6J mice
Non-colitic (n=10)
Concentration
DSS of 3%, 5
days
Minocycine treated
group
Control mice
Full-thickness sections of 5 m
Haematoxylin and eosin stain
Evaluation histological damage
MPO activity
Marker of neutrophil infiltration.
Glutathione
Tripeptide with antioxidant power.
iNOS
Inducible enzyme in inflammatory proccesses.
Produces NO, which reacts with superoxide to
form nitrite.
Determined by Western blot.
Cell viability
Not affected by antibiotics
** P < 0.01
Conclusions
Cell viability
Not affected by antibiotics
* P < 0.05
** P < 0.01
Conclusions
Macroscopic damage
** P < 0.01
Conclusion
2)
Microscopic damage
Non-colitic
TNBS control
2)
Microscopic damage
Non-colitic
TNBS control
2)
Microscopic damage
Non-colitic
TNBS control
Microscopic damage
* P < 0.05
Conclusions
* P < 0.05
** P < 0.01
Conclusions
* P < 0.05
Conclusions
* P < 0.05
** P < 0.01
Conclusion
6)
Conclusions
MNC: iNOS
MNC: IL-17 (TNBS-induced colitis)
MNC and TTC: pro-inflammatory markers.
Only MNC: colonic barrier function (mucus production).
7)
* P < 0.05
Conclusions
MNC: non-pathogenic/potentially
pathogenic ratio.
MNC effect = immunomodulatory +
microbiota modulation.
* P < 0.05
** P < 0.01
Conclusions
Reduction in BWL
DAI diminished
Macroscopic evaluation
Conclusion
3)
Non-colitic
DSS-control
Conclusions
IL-1
4)
Biochemical analysis
TNF
* P < 0.05
** P < 0.01
IL-6
Conclusion
expression of proinflammatory
cytokines
Macroscopic score
* P < 0.05
Conclusions
Bacterial ratio
* P < 0.05
Conclusion
* P < 0.05
Conclusion
DISCUSSION
TNBS-induced
colitis in rats
Histological studies
Reduction in the
inflammatory infiltrate
Minocycline
showed
anti-inflammatory
effect
DSS-induced
colitis in mice
Biochemical
determinations
Reduction of main
pro-inflammatory
cytokines
Only exerted
following a
curative
treatment
protocol.
No preventive
effect was
observed
Tetracycline
Similar antimicrobial spectrum
Did not show the same efficacy
Metronidazole
Prevented intestinal inflammation
Could not ameliorate colitis once it was
established
Minocycline
Immunodulatory properties definitively
participate in its ability to reduce colonic
inflammation
Minocycline
Reduces the release of IL-8 by
Caco-2
Inhibits NO production, in vivo
and in vitro
Down-regulates IL-17
Down-regulated the
counts of enterobacteria
Increased the counts
of lactobacilli and
bifidobacteria
Restoration of beneficial bacteria
levels
CONCLUSION