Facultad de Ciencias

Grado en Bioqumica
Famacologa Molecular

THE INTESTINAL ANTI-INFLAMMATORY EFFECT OF

MINOCYCLINE IN EXPERIMENTAL COLITIS INVOLVES BOTH
ITS IMMUNOMODULATORY AND ANTIMICROBIAL
PROPERTIES
Ral Albn Alba
lvaro Andrades Delgado
Paula Argente del Castillo Rodrguez
Jess Blanco Piar
Rosario Benavides Mndez

Granada, 10 Diciembre 2015

doi:10.1016/j.phrs.2010.12.011

INDEX
Introduction
Methods
o

In vitro studies

Trinitrobenzene sulfonic acid (TNBS) model of rat colitis

Dextran sodium sulfate (DSS) model of mouse colitis

Histological studies

Biochemical determinations in colonic tissue

Analysis of gene expression in colonic samples by RT-PCR

Microbiological studies

Statistics

Reagents

Results
o

In vitro effects of antibiotics in Caco-2 and RAW264.7cells

Curative effect of minocycline in different models of rodent colitis

Minocycline has no prophylactic effect in TNBS rat colitis

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

Beneficial effect attributed to their antimicrobial

properties
Modulation of the immune responses

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.

METHODS: in vitro studies

Caco-2 cells
Cell Culture
Unit of the
UGR

24-well plates at a density of 5 x 105 cells per well

and grown monolayer
Pre-incubation for 24 h with antibiotics

Caco-2
cells

RAW 264.7
cells

Stimulation with IL-1 for 20 h.

The supernatants were collected, centrifuged
at 10,000 g for 5 min

They were cultured in DMEM,

supplemented with 10% FBS, 2mM Lglutamine, in a humidified 5% CO2
atmosphere at 37C.

Minocycline,
tetracycline or
metronidazole

The supernatants were stored at 80 C until IL-8

determination by ELISA was performed.

Negative control

Positive control

Untreated unstimulated cells

Untreated cells

Range: 2
to 100 m

METHODS: in vitro studies

RAW 264.7 cells
24-well plates at a density of 5 x 105 cells per well and grown
confluence
They were cultured 1h with antibiotics

Minocycline,
tetracycline or
metronidazole

Range: 2
to 100 m

Stimulation with LPS (100ng/ml) for 20 h.

The supernatants were collected, centrifuged at
10,000 g for 5 min
Nitrite levels were measured using the Griess assay.

Cell viability was examined by the MTT-test.

Negative control
Positive control

Untreated unstimulated cells

Untreated cells

METHODS: trinitrobenzene sulfonic acid (TNBS)

model of rat colitis
Minocycline
(20 or 40
mg/kg)

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)

Colonic inflammation was

induced

10mg TNBS
dissolved in 0,25
ml of 50% ethanol

2 ml of
distilled
water and
administered
daily by oral
gavage

METHODS: TNBS model of rat colitis

SACRIFICE

Overdose of
halothane
7 days

Antibiotic

+ TNBS

2 days

Sacrifice

- Antibiotic

7 days

Antibiotic
+TNBS

Sacrifice

- Antibiotic

METHODS: TNBS model of rat colitis

The colon
was
removed
aseptically

DAILY EVALUATION

Macroscopically
visible
damage (010 scale)

Animal body weight

Occurrence of diarrhoea
Water and food intake
ndice de dao macroscpico (IDM) (Bell et al., 1995)

METHODS: TNBS model of rat colitis

Region of the
inflamed colon

Remaining
colon samples

4% buffered
formaldehyde

Biochemical
determinations

Histological
studies

RNA isolation

METHODS: Dextran sodium sulfate (DSS)

model of mouse colitis
2 GROUPS

Female
C57BL/6J mice
Non-colitic (n=10)

Concentration
DSS of 3%, 5
days

DSS colitic groups (n=20)

Minocycine treated
group

Control mice

30 mg/kg per day of

minocycline dissolved in
200 l of distilled water

METHODS: DSS model of mouse colitis

DAILY EVALUATION
Animal body weight
The presence of gross
blood
Stool consistency

METHODS: DSS model of mouse colitis

Selection of cross-sections
Embedding in paraffin

Full-thickness sections of 5 m
Haematoxylin and eosin stain
Evaluation histological damage

METHODS: Biochemical determinations in

colonic tissue
Biochemical determinations:

MPO activity
Marker of neutrophil infiltration.

Glutathione
Tripeptide with antioxidant power.

TNF-, IL-1 and IL-6

Pro-inflammatory citokynes.
Characterizaction of inflammatory status.
Determined by ELISA.

iNOS
Inducible enzyme in inflammatory proccesses.
Produces NO, which reacts with superoxide to
form nitrite.
Determined by Western blot.

METHODS: Analysis of gene expression in colonic samples

Expression of proteins by RT-PCR:
RNAs were extracted using TRIzol Reagent.
Semiquantitative PCR was performed using specific primers
for:
IL-17
IL-6
MCP-1
CINC-1
ICAM-1
MUC-2
TFF-3
-actin
PCR products were analysed by electrophoresis in 1%
agarose gel containing ethidium bromide.

METHODS: Microbiological studies

Luminal content samples were weighted, homogenated and
diluted in peptone water.
Serial 10-fold dilutions were plated in specific media for
lactobacilli and bifidobacteria and incubated 24-48 hours at
37 C.
Enterobacteria were also determined by using specific
count plates Petrifilm.
Data was reported after incubation as log10 of colonyforming units per gram of material.

RESULTS: In vitro effects in Caco-2 cells

Minocycline
Concentration-dependent reduction
in IL-8 production.

Tetracycline and metronidazole

No significant effect in IL-8 production.

Cell viability
Not affected by antibiotics
** P < 0.01

Conclusions

MNC has immunomodulatory activity in intestinal

epithelial cells in vitro.
Direct effect: independent from antimicrobial
activity.

RESULTS: In vitro effects in RAW 264.7 cells

Minocycline
Concentration-dependent reduction
in nitrite production.

Tetracycline and metronidazole

No significant effect in nitrite
production.

Cell viability
Not affected by antibiotics
* P < 0.05
** P < 0.01

Conclusions

MNC has immunomodulatory activity in

macrophages in vitro.
Direct effect: independent from antimicrobial
activity.

RESULTS: Curative effect of minocycline in

TNBS-induced colitis in rats
1)

Macroscopic damage

** P < 0.01

Conclusion

MNC: significant reduction in the extension

of inflamed/necrotic colonic tissue.

2)

Microscopic damage

Non-colitic

TNBS control

Minocycline (40 mg/kg)

2)

Microscopic damage

Non-colitic

TNBS control

Tetracycline (80 mg/kg)

2)

Microscopic damage

Non-colitic

TNBS control

Metronidazole (40 mg/kg)

RESULTS: Curative effect of minocycline in

TNBS-induced colitis in rats
2)

Microscopic damage

* P < 0.05

Conclusions

MNC: improvement in colonic histology

(ulceration, goblet cell depletion).
MNC: reduction in the inflammatory infiltrate.

RESULTS: Curative effect of minocycline in

TNBS-induced colitis in rats
3)

Colonic MPO activity

* P < 0.05
** P < 0.01

Conclusions

MNC and TTC: neutrophil infiltration.

But, TTC showed no macroscopical effect.
MNZ: no significant effect.

RESULTS: Curative effect of minocycline in

TNBS-induced colitis in rats
4)

Colonic GSH content

* P < 0.05

Conclusions

MNC and TTC: GSH depletion.

MNZ: no significant effect.

RESULTS: Curative effect of minocycline in

TNBS-induced colitis in rats
5)

TNF and IL-1

* P < 0.05
** P < 0.01

Conclusion

MNC and TTC: proinflammatory cytokines.

6)

Other markers: WB and RT-PCR

Conclusions

MNC: iNOS
MNC: IL-17 (TNBS-induced colitis)
MNC and TTC: pro-inflammatory markers.
Only MNC: colonic barrier function (mucus production).

7)

Colonic bacterial profile

* P < 0.05

Conclusions

MNC: non-pathogenic/potentially
pathogenic ratio.
MNC effect = immunomodulatory +
microbiota modulation.

RESULTS: Effects of minocycline in

DSS-induced colitis in mice
1)

Disease Activity Index (DAI)

* P < 0.05
** P < 0.01

Conclusions

Reduction in BWL
DAI diminished

RESULTS :Effects of minocycline in DSSinduced colitis in mice

2)

Macroscopic evaluation

Conclusion

Beneficial effect: colonic W/L

ratio significantly diminished.

3)

Microscopic damage analysis

Non-colitic

Minocycline (50 mg/kg)

DSS-control

RESULTS: Effects of minocycline in

DSS-induced colitis in mice
3)

Microscopic damage analysis

Conclusions

MNC: ulceration, crypt hyperplasia

and goblet cell depletion
MNC treatment effectively reduces
inflammation.

IL-1
4)

Biochemical analysis
TNF

* P < 0.05
** P < 0.01

IL-6

Conclusion

expression of proinflammatory
cytokines

RESULTS: Prophylactic effect of

minocycline in TNBS-induced colitis in rats
1)

Macroscopic score

* P < 0.05

Conclusions

MNC: No prevention of colitis

development.
MNZ: decrease in macroscopic damage

RESULTS: Prophylactic effect of minocycline

in TNBS-induced colitis in rats
2)

Bacterial ratio

* P < 0.05

Conclusion

MNC & MNZ: Partial restoration of

non-pth/potentially pth ratio

RESULTS: Prophylactic effect of

minocycline in TNBS-induced colitis in rats
3)

MPO activity & GSH content

* P < 0.05

Conclusion

MNC: No anti-inflammatory effect.

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

Minocycline, only by modulating the

intestinal microbiota, is not able to prevent intestinal
inflammation.

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

Reverses the reduction of

MUC-2
Different effects than
tetracycline

Minocycline remarkable effect in restoring a

balanced intestinal microbiota

Down-regulated the
counts of enterobacteria
Increased the counts
of lactobacilli and
bifidobacteria
Restoration of beneficial bacteria
levels

CONCLUSION

Minocycline displays a better anti-inflammatory profile.

Derived from its ability to restore the luminal microbiota
imbalance and to inhibit iNOS and IL-17 expressions in
the inflamed tissue and promote intestinal membrane
integrity.
Both inmunomodulatory and antimicrobial properties of
minocycline constitute an advantage in the treatment
of intestinal inflammation in IBD experimental models.