| Sažetak (engleski) | Introduction.Enterovesical fistula present an important clinical problem caused by
divericulosis, IBD, colorectal cancer.Enterovesical fistula represents an abnormal
communication between the intestine and the bladder.
Although EVF are uncommon, they cause significant morbidity and may markedly affect
patient’s quality of life. While colovesical fistula is the most common form of
vesicointestinal fistula and is most frequently located between the sigmoid colon and the
dome of the bladder, we hypothesized that colovesical fistulas may be healed by the
therapy with stable gastric pentadecapeptide BPC 157 , all consistent with its initial
clinical application effective (BPC 157, GEPPPGKPADDAGLV, M.W. 1419, is
originally an anti-ulcer peptide, stable in human gastric juice, designated to be a novel
mediator of cytoprotection LD1 not achieved, implemented in inflammatory bowel
disease trial with particular effect in fistulas healing (and now in multiple sclerosis trial)
recently reviewed.
Of note, current nonsurgical management of colovesical fistulae (medical therapy
including bowel rest, total parenteral nutrition, antibiotics, steroids, immunomodulatory
drugs, and urethral catheter drainage) is generally reserved for patients, but most patients
will require a diverting stoma in due course of a disease unfit for major intervention or
with extensive unresectable neoplastic process.
Thereby, an additional background might be important for these fistulas healing. BPC
157 has particular effects on the wound-healing (including blood vessels likely due to the
stimulation of the early growth response-1 (egr-1) gene and its co-repressor nerve growth
factor 1-A binding protein-2 (naB2) also responsible for cytokine and growth factor
generation and thereby, early extracellular matrix (collagen) and blood vessel formation
Also, BPC 157 might improve intestinal anastomosis healing while in the remaining
intestine, in 1-4 week-periods after massive small bowel resection, adaptation function of
all intestinal layers was consistently improved and weight gain of normal non-operated
rats achieved.
Recently, BPC 157 beneficial effect, using the same peroral regimen, might link the
model of complicated ulcerative colitis (colon-colon anastomosis healing in the after
highly damaging cysteamine enema application) as a peripheral disturbance, and central
disturbance model, cuprisone neurotoxin diet and application that fairly mimics multiple
sclerosis presentation.
Previously, for the fistulas healing background with stable gastric pentadecapeptide BPC
157, we reintroduced “wound healing-therapy” for (gastro)duodenal ulcer (an issue
recently reviewed in gastroduodenal ulcers theories, and revealed the general idea that
gastrointestinal ulcers are essentially internal wounds equivalent to external wounds,
chronic gastroduodenal ulcers and chronic skin ulcers as corresponding disturbances that
both equally fail to heal. Consequently, the wound /growth factors (for a review see theory
of the analogous nonhealing of wounds and persistent gastric ulcers was approved with
the evidence that BPC 157 indeed heals gastrocutaneous fistulas, both stomach and skin
defect, better than standard anti-ulcer agents, providing the fastest and simultaneous
beneficial effect.Subsequently, this BPC 157’s effect, accordingly with its indicated
particular wound healing potential ), was extended to colocutaneous and
esophagocutaneous fistulas healing , beneficial effects related to an interaction with NOsystem
while BPC 157 interacts with NO-system in different models and species .
And finally, specifically for colovesical fistulas healing, BPC 157 rescued urethral
sphincter function in rats.
Consequently, we focused on BPC 157 and on the colovesical fistulas healing,
macro/microscopically, and biomechanically, no passage obstruction and on adhesion
attenuation and on possible benefits which could further usefully applied in other internal
fistulas healing. We also focused on the possible particular therapy, stable gastric
pentadecapeptide BPC 157 given daily, intraperitoneally or perorally, in drinking water.
Materials and methods
Animals
Wistar Albino male rats (200 g b.w.) were randomly assigned to the experiments (10
animals at least per each experimental group and interval), all of which were approved by
the Local Ethic Committee. Furthermore, all experiments were carried out under blind
protocol and the effect was assessed by examiners who were completely unaware of the
given protocol.
Drugs
Pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, M.W. 1419), (Diagen, Ljubljana,
Slovenia) dissolved in saline, was used in all experiments. The peptide BPC 157 is part of
the sequence of human gastric juice protein BPC and is freely soluble in water at pH 7.0
and saline. It was prepared as described previously with 99% high pressure liquid
chromatography (HPLC) purity, expressing 1-des-Gly peptide as an impurity
Procedure
In deeply anaesthetized rats, peritoneal cavity was entered through 3 cm midline incision
in the rectal and bladder area, and then we created colovesical fistulas 5 cm from the anus,
with colon and bladder defect of 3 mm, where a precise caliper was used to verify the
initial size of the defect.
Experimental protocol
BPC 157 was given perorally, in drinking water (10 μg/kg, 12 ml/rat/day) till
sacrifice, or alternatively, 10 μg/kg, 10 ng/kg intraperitoneally, first application at 30 min
after surgery, last at 24h before sacrifice.
Controls simultaneously received an equivolume of saline (5.0 ml/kg ip) or water only.
The assessment was at day 7, 14 and 28 as follows.
Colon defect, vesical defect, fistula assessment.
Briefly, a precise caliper was used to verify the final size of the defect and the largest
diameter of the colon and bladder defect was assessed (mm), photographed and further
verified using the program ISSA (VAMSTEC Software Company, Zagreb, Croatia) and
the tissue processed for further microscopic analysis.
To assess fistula leakage, a separate group of animals received a volume of water
(1ml/6sec), either to leaking induction, or to a maximal volume of 20 ml. In this case, if
leakage did not occur after 5 min, the fistula was considered functionally closed.
To assess fistula leakage, and the closure of the fistula were assessed in a separate group
of animals by studying the volume( mL) that was sustained before the initiation of the
leakage through the fistula. The volume of saline was infused through a syringe-perfusion
pump system ( Argus 600; Argus Medical A6, Heimberg,Switzerland ) at the rate of 1 mL
/10 s. The infusion was stopped at the point when the leakage through the external
aperture of the fistula started. If not leaking till the end of the fifth minute, the fistula was
considered to be functionally closed.
Defecation through fistula and fecaluria: all rats were observed for fecaluria and
defecation through fistula.
As we described before , adhesion presentation was scored 0–7: 0, no adhesion; 1, thin
adhesions covering less than one half of anastomosis; 2, more prominent adhesions with
more than half of anas- tomosis; 3, exaggerated adhesions with whole anasto- mosis; 4,
the mesenterial part of small bowel also included; 5 neighboring small intestine loop also
included; 6, many neighboring small intestine loops included; 7, neighboring loops,
stomach, liver “packed”.
Likewise, as described before Intestinal passage obstruction scored 0–3, according to the
loop diameters ratio close to anastomosis. In other words, if loop diameters at 2 cm
orally/loop diameters at 2 cm aborally = 1 passage is normal (score 0), between 1 and 1.33
is the sign of mild obstruction (score 1), between 1.33 and 1.66 is moderate obstruction
(score 2), and more than 1.66 is severe obstruction (score 3).
Statistical analyses
Statistical analysis was performed by a non-parametric Kruskal-Wallis ANOVA and
subsequent Mann-Whitney U-test to compare groups. Fisher’s exact probability test for
mortality rate assessment was used. Values of P<0.05 were considered statistically
significant
Results
In general, in rats with colovesical fistulas (i.e., colon and vesical defect, fistula
leaking, adhesions and intestinal obstruction as healing processes) we noted always
simultaneous effect, induced beneficial effect of therapy healing or spontaneous
worsening of colovesical fistulas aggravating course, the closure of defects and fistulas
accomplished (BPC 157) or otherwise definitively failed.
If not treated otherwise, colovesical fistulas exhibited poor healing persisting both
defects continuous fistula leakage, advanced adhesions formation and intestinal
obstruction .BPC 157 given perorally or intraperitoneally, in μg- and ng-regimens rapidly
improved complete presentation (maximal volume instilled continuously raised till the
values in healhty, counteracted both advanced adhesions formation and intestinal
obstruction . All control rats had fecaluria and all of them defecated through the fistula at
the end of 7, 14 and 28 day experimental period.
Contrary, all BPC 157 rats had no signs of fecaluria and defecation through the fistula
(Fisher exact probability test P<0.05, at least vs. control). Thus, BPC 157 effects appear to
be suited to induce full healing of colocutaneous fistulas in rats.
Generally, in colovesical fistula-rats the microscopic presentation followed the
described macroscopical healing and previous course of gastrocutaneous fistula-rats. In
control groups, transitional epithelium of bladder demonstrated pronounced infiltration
with inflammatory cells containing abundant mononuclear and polymorphonuclear cells,
while stroma showed edema with high number of neutrophils.
On the other hand, BPC157 treated animals showed only mild and mainly
mononuclear infiltration both in epithelium and in stroma, without pronounced edema.
At 14 and 28 days BPC treated animals presented with only few inflammatory cells and
advanced epithelization, while in control groups there were still more inflammation,
mainly mononuclear cells, and mature granulation tissue with poor epithelization.
Discussion
For BPC 157’s fistulas healing we introduced the healing of rat colovesical fistulas as
internal fistulas healing as we previously addressed external fistulas healing with resolved
gastrocutaneous, colocutaneous and esophagocutaneous fistulas.
With colovesical fistulas known to have poor healing these colovesical lesions like
essentially resistant internal wounds,equivalent to resistant external wounds.
And thereby, we would suggest a commonality between external and internal fistulas
healing with BPC 157 therapy
This should be particularly claimed since both defects (internal and external internal and
internal in the present study) healed simultaneously. Thereby, we argue “wound healingtherapy
for complicated internal fistulas healing as we did previously for external fistulas
healing.
This may be particular for BPC 157 therapy, since, for instance, only BPC 157 in
gastrocutaneous fistulas healing (but not atropine, ranitidine, omeprazole (improvement),
or methylprednisolone (worsening) and in colocutaneous (but not sulphasalazine
(improvement) or methylprednisolone (worsening) might simultaneously affect both
defects.
Methodologically, colovesical fistula healing model resolves connected resistant colon
defect healing and vesical defect healing and requirement for reciprocal healing and
fistula closure as the end of suited healing process(es) requiring both defect healing. And
thereby, colovesical fistula healing model may accurately resolve the therapy success
(BPC 157) as a new therapy phenomenon.
Specifically for BPC 157 (i.e., in skin wound healing better than corresponding standard
agents (i.e. becaplermin) in wound and fistulas healing simultaneous combining and
initiation of the healing of esophageal, gastric, colonic, and skin defect colonic and vesical
defect in the present study), the final background might be its particular effect on collagen
as already claimed.
The consequence and prove is the largely improved biomechanical and functional
healing, maximal volume instilled already at 7th day, attenuated adhesions and passage
obstruction, lack of defecation through fistula and fecaluria in colovesical fistulas-rats in
the present study), alike previous advanced biomechanical healing findings
Accordingly, BPC 157 prominently stimulated expression of the egr-1 gene, which
induces cytokine and growth factor generation and early extracellular matrix (collagen)
formation, and its repressor naB2.
Also, for full intestine function recovery it is important that in the same range, BPC 157
recovers sphincters function (lower esophageal, pyloric and uretheral and has also a
particular angiogenic healing potential.
Of note, such angiogenic response is regularly augmented and shifted toward the left also
in hypocellular, hypovascular and hyponeural structures where BPC 157 induces higher
VEGF and CD34 positivity, preceding the increase in actual number of blood vessels.
Particular activity in angiogenesis and healing is concordant with its previous direct
endothelium protection influence on NO-system, counteraction of the effect of NOSinhibitor
and NO-precursor s well as counteracted over expression of endothelin.
Also important point for the rapid onset of action should be its peroral effectiveness,
probably due to its more advanced cytoprotective effect (i.e., stable in human gastric juice,
BPC 157 is claimed to be novel mediator of Robert’s cytoprotection.
Finally, pentadecapeptide BPC 157’s beneficial effect on both the wound and mucosa
healing , reduces the number of inflammatory cells and levels of leukotriene B4 (LTB4),
thromboxane B2 (TXB2), and myeloperoxidase (MPO) in the serum and inflamed tissues
and increases macrophages.
Conclusion.
The most common form of vesicointestinal fistula, colovesical fistulas mostly appear in a
disease unfit for major intervention or with extensive unresectable neoplastic process.
Logically, a simultaneous beneficial effect of a given agent will control healing of the
internal wound axis (healing of the colon defect, healing of the vesical defect, and suitable
closing of the colovesical fistula). This signifies a new quality in combined healing, and
not a simple repeat of separate defects healing effects, respecting all the healing
particularities of fistula healing. BPC 157 was the agent tested that promptly improved
both colon and vesical lesions healing and fistula closing (macro-/microscopically, and
functionally, with no fistula leakage upon application of the maximal volume water).
Thereby, the BPC 157 healing of colovesical , with other fistulas healing,
beneficial effect in ulcerative colitis , even when complicated , and rescue of urethral
sphincter function , should be the practical hallmark for further wound healing therapy in
fistulas healing, research and practical application in colovesical fistulas pathology. |
