Učinak pentadekapeptida BPC 157 na cijeljenje kolovezikalne fistule u štakora [Effect of the pentadecapeptide BPC 157 on the colovesical fistula healing in rat]

Grgić, Tihomir (2016) Učinak pentadekapeptida BPC 157 na cijeljenje kolovezikalne fistule u štakora [Effect of the pentadecapeptide BPC 157 on the colovesical fistula healing in rat]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

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.

Abstract in Croatian

Uvod.Enterovezikalna fistula je važan klinički problem najčešće uzrokovan divertikulozom kolona, kolorektalnim karcinomom ili upalnom bolesti crijeva. Pentadekapeptid BPC-157 pokazao se učinkovit u cijeljenu gastrokutanih i kolokutanih fistula kao modelima vanjskih fistula te akutnih i kroničnih upalnih stanja. Istraživanjem smo dokazali učinkovitost pentadekapeptida BPC 157 u cijeljenju enterovezikalnih fistula kao modelu unutarnjih fistula. Metode.Eksperiment smo proveli na mužjacima štakora soja Wistar , randomiziranih u pet grupa. Kontrolne grupe primali su vodu za piće, fiziološku otopinu( intraperitonealno ) , dok su terapijske grupe primale pentadekapeptid BPC-157 intraperitonealno ( mikrogrami, nanogrami ),jednom dnevno, te peroralno u mikrogramima preko vode za piće.Prva aplikacija BPC 157 bila je 30 minuta nakon operacije, zadnja 24 sata prije žrtvovanja. Na kraju svakog eksperimentalnog razdoblja životinje su se žrtvovale te je provedena funkcionalna, makroskopska i mikroskopska analiza.Za statističku analizu podataka koristili smo ne-parametrijski Kruskal-Wallis analizu ANOVA i Mann –Whitney U test radi usporedbe grupa.Za evaluaciju mortaliteta korišten je Fisherov test.Vrijednosti manje od P< 0,5 su smatrane statistički značajnima. Rezultati. Rezultati koje smo dobili pokazali su učinkovito djelovanje BPC 157 u cijeljenju enterovezikalnih fistula na eksperimentalnom modelu Wistar albino štakora.Došlo je do cijeljenja fistule te lezije na sluznici mokraćnog mjehura i sluznici kolona,što smo dokazali funkcionalnom, makroskopskom, biomehaničkom i histološkom analizom preparata.Zaključak. Pentadekapeptid BPC 157 dokazao je svoje pozitivno djelovanje u smislu cijeljenja unutarnje rane, tj. defekta dva različita tkiva koja ne cijele spontano. Dokazali smo da djeluje na sve tri komponente fistule istodobno i u relativno kratkom vremenu dovodi do saniranja fistule.

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