INFLUENCE OF PENTADECAPEPTIDE BPC 157 ON HEALING OF COLONCOLONIC ANASTOMOSIS UNDER VENOUS CONGESTION CONDITIONS IN RATS

Venska kongestija debelog crijeva je stanje zastoja venske cirkulacije debelog crijeva koje uzrokuje edem stijenke i smanjenje arterijske irigacije crijeva. U takvim je uvjetima cijeljenje kolokolične anastomoze rizično pa se na kongestiranom crijevu obično ne radi primarna anastomoza nego kolostoma. Pentadekapeptid BPC 157 je sintetski oblik aktivnog terminalnog dijela ljudskog želučanog proteina koji se u dosadašnjim istraživanjima pokazao kao važan promotor cijeljenja različitih tkiva, uključujući i različite dijelove probavne cijevi, te kao snažan aktivator kolateralne cirkulacije. U procesu cijeljenja stimulira stvaranje granulacijskog tkiva i organizaciju kolagena, smanjuje edem i stimulira angiogenezu. U ovom istraživanju je ispitano djelovanje pentadekapeptida BPC 157 na cijeljenje kolokolične anastomoze u uvjetima venske kongestije debelog crijeva izazvane podvezivanjem kaudalne mezenterične vene u štakora. Pokus je izveden na 105 mužjaka laboratorijskih životinja soja Wistar, tjelesne mase 200-220 g, u dobi 8-10 tjedana. Životinje su bile podijeljene u 15 skupina i 5 vremenskih intervala (3, 5, 7, 15 i 30 dana), a svaka se skupina sastojela od 7 životinja. Nakon postizanja kongestije silaznog dijela debelog crijeva učinjena je transekcija debelog crijeva i pripadajućeg mezokolona, a zatim je kreirana terminoterminalna kolokolična anastomoza. Tretirane životinje su nakon kreiranja anastomoze intraperitonealno dobile jednokratno pentadekapeptid BPC 157 u dozi 10 µg/kg ili 10 ng/kg, a kontrolne životinje su na isti način dobile fiziološke otopinu u dozi 5 mL/kg. U postoperacijskom periodu tretirane životinje su primale pentadekapeptid BPC 157 u dozi 10 µg/kg/dan ili 10 ng/kg/dan peroralno. Popuštanje kolokolične anastomoze je evidentirano kod 31,42 % kontrolnih životinja (11/35) i kod 2,85 % tretiranih životinja (2/70). Priraslice i smetnje pasaže su bile značajno manje izražene kod tretiranih životinja. Volumen venskih arkada debelog crijeva i broj venskih kolaterala između dviju susjednih venskih arkada uz oralnu i aboralnu stranu anastomoze su bili značajno veći kod tretiranih skupina. Edem i nekroza su bili značajno manje izraženi kod tretiranih životinja. Granulacijsko tkivo se javilo ranije kod tretiranih skupina uz vidljivu organizaciju kolagenih i retikulinskih vlakana dok kontrolne skupine životinja nisu imale organizaciju cijeljenja. Epitelizacija anastomoze se javila ranije i na kraju pokusa je bila značajno veća kod tretiranih životinja. Testiranjem anastomoze pokazana je veća čvrstoća anastomoze tretiranih životinja. Nije bilo statistički značajne razlike između skupina koje su dobivale pentadekapeptid BPC 157 10 µg/kg i skupina koje su dobivale pentadekapeptid BPC 157 10 ng/kg. Rezultati upućuju na značajno bolje cijeljenje kolokolične anastomoze kod tretiranih životinja, potvrđuju rezultate dosadašnjih istraživanja i sugeriraju mogućnost primjene pentadekapeptida BPC 157 u abdominalnoj kirurgiji.Background and objectives: Although great progress in colorectal surgery was achieved during the last few decades, the problem of colorectal anastomosis dehiscence, with resulting morbidity and mortality, remains to this day. Colonic anastomosis creation is an everyday procedure in abdominal surgery and basically does not differ from any other anastomosis creation. A particular type of colonic anastomosis is a colorectal-coloanal anastomosis, special due to rectum vascularization, absence of the serosa in the subperitoneal part of the rectum, and difficulty with the surgical procedure due to narrow space in the true pelvis. Thus, literature that focuses on colonic anastomoses usually refers to the colorectal anastomosis. Intestinal anastomosis healing is a complicated process that depends on the patient's general state, intestine state, technical conditions, and surgeon's knowledge and experience. Among many factors that affect the process of anastomosis healing, one of the most important is adequate arterial and venous circulation of the intestines. Colonic congestion is defined as colonic venous circulation stasis that causes intestinal wall edema and reduction of arterial irrigation, which is often observed in patients that suffer from colonic obstruction ileus caused by malignant tumors. Although ischemia and congestion both lead to tissue hypoxia, intestinal congestion is considered a more serious and complicated condition. By comparing ischemic and venous congestion intestinal injury, destruction of the intestinal mucosa induced by venous congestion arises earlier and is slower to normalize after etiological factor removal, which makes intestinal congestionreperfusion injury much more severe than ischemic-reperfusion injury. Colonic congestion is usually found in patients suffering from ileus caused by malignant tumors located in the colon, and in those cases, a colostomy is preferred over anastomosis creation. In case of venous congestion, meaning compromised venous circulation, primary anastomosis is usually not performed because congested intestinal anastomosishealing is risky and burdened with a high possibility of anastomosis dehiscence with accompanying peritonitis or intraabdominal abscess. Thus, colostomy is usually performed in those cases. Pentadecapeptide body protective compound 157 (BPC 157) is a terminal part of the BPC protein crucial for its activity. Pentadecapeptide BPC was isolated from human gastric juice and is found to need no additional carriers for absorption and to be stable in human gastric juice. The synthetic form of BPC 157 has been examined in great detail and scope via experimental research that has shown its positive effects on burn healing, gastric and duodenal ulcer healing, different fistula of the gastrointestinal tract, and intestinal anastomosis healing. The effectiveness of pentadecapeptide BPC 157 in healing promotion has been investigated in different animal models, proving its effect on connective tissue and blood vessels. In a burn animal model, it was shown that BPC 157 attenuates the inflammatory response, lessens the edema and necrosis of the burnt skin, and enhances collagen organization, angiogenesis, and tissue epithelization. In an animal model of esophagogastric anastomosis and colon-colonic anastomosis in cysteamine colitis caused by rectal cysteamine enema, greater mortality and anastomosis dehiscence was found in control groups, in contrast to treated groups. Pentadecapeptide BPC 157 helps the healing process by stimulating granulation tissue creation and collagen organization, as well as reducing the edema and stimulating angiogenesis. Recent studies have shown that pentadecapeptide BPC 157 activates anatomically present collateral circulation in arterial or venous obstruction conditions and thus lessens the negative impact of vascular obstruction.The exact mechanism of action of pentadecapeptide BPC 157 is still not clarified in full, but its effects on different tissues and organs are explained through many different interacting systems, the most important one being the NO system. It has been proven that pentadecapeptide BPC 157 induces NO formation and interferes with agonists (L-arginine, NO substrate) and antagonists (L-NAME) of the NO system. By inducing NO synthesis, pentadecapeptide BPC 157 stimulates vasodilatation in the early stages of the healing process. Additionaly, in early stages of the healing process, pentadecapeptide BPC 157 stimulates the production of EGR1, crucial for activation of genes that code for various cytokines and growth factors, thus stimulating collagen production and organization. It has been established that pentadecapeptide BPC 157 stimulates expression of EGR-1mRNA 15 minutes after application, and 30 minutes after application, BPC 157 stimulates NAB2 mRNA expression, which is a corepressor of EGR-1. Hsieh et al. have demonstrated that pentadecapeptide BPC 157 affects aortic tone via NO synthesis stimulation through the Src-Cav-1-eNOS signal pathway. The angiogenesis-modulating potential of pentadecapeptide BPC 157 manifests through stimulation of VEGF and VEGFR2 expression, which was examined in an animal model of limb ischemia. Pentadecapeptide BPC 157 increased VEGFR2 expression in blood vessel endothelium and thus enabled quick reestablishment of the circulation through the ischemic extremity. Angiomodulatory effects of pentadecapeptide BPC 157 were also examined in an animal model of duodenal lesions caused by ligation of superior anterior pancreaticoduodenal vein that revealed that pentadecapeptide BPC 157 causes collateral circulation activation through the NO system, and thus attenuates the congestion and duodenal mucosal lesions development. To this day, pentadecapeptide BPC 157 was administered in various different doses (µg/kg, ng/kg, or pg/kg) and applied in various different modes of drug delivery (locally: baths, cremes, eyedrops; systemically: intraperitoneally, intragastrically, perorally). Additionally, to this day no serious toxicity of pentadecapeptide BPC 157 has been noted. Based on described effects of pentadecapeptide BPC 157, we hypothesized that pentadecapeptide BPC 157 may have positive effects on colon-colonic anastomosis healing under venous congestion conditions. The aim of this study is to examine the effect of pentadecapeptide BPC 157 on colon-colonic anastomosis healing under venous congestion conditions caused by ligation of the caudal mesenteric vein in rats. Material and methods: The experiment was performed using 105 male albino Wistar rats, bodyweight 200-220 g, aged 8-10 weeks, divided into 15 groups and 5 time intervals (3, 5, 7, 15, or 30 days). Each group included 7 animals. For each time interval, there was one control group, one treated group that received pentadecapeptide BPC 157 dose of 10µg/ kg, and the second treated group that pentadecapeptide BPC 157 dose of 10 ng/kg. Two different doses of BPC 157 were used to examine the dosedependent effect of BPC 157. Animals were anesthetized via intraperitoneal injection containing diazepam 5 mg/kg (Apaurin, Krka, Slovenia), thiopental 50 mg/kg (Thiopental Injection BP, Rotexmedica, Germany), and buprenorphine 1 mg/kg (Bupredine Multidose, Dechra, Great Britain).In deeply anesthetized rats, median laparotomy was performed and caudal mesenteric vein (CMV) was ligated to achieve venous congestion in the descending colon. After venous congestion was achieved, transection of the colon descendens and adjacent mesocolon was done, after which terminoterminal colon-colonic anastomosis was created. After anastomosis creation, treated animals received pentadecapeptide BPC 157 intraperitoneally in doses 10 µg/ kg or 10 ng/kg and control animals were given 1 mL saline, also intraperitoneally. At each time interval (3, 5, 7, 15 or 30 days postoperatively) animals were again deeply anesthetized and relaparatomised to examine macroscopic changes: anastomosis dehiscence, adhesions, anastomosis passability, and venous arcade (vasa recta) presentation on the ventral and dorsal side of the colon, orally and aborally from the anastomosis. The anastomosis was tested as follows: intestinal clamps were used to occlude the descending colon 2.5 cm orally and 2.5 cm aborally from the anastomosis site and saline wasapplied intraluminally until volume that caused anastomosis leakage was reached. The colonic segment containing the anastomosis was explanted from euthanized animals (2 cm orally and 2 cm aborally from the anastomosis site). Surrounding mesocolon was removed from the resected part of the colon, the resected part was then rinsed in saline, placed over a styrofoam panel, and submerged into 4% formaldehyde solution for histopathological analysis. Mentioned analysis was performed by two independent pathologists who had no previous knowledge of tissue specimens. 5 fields of view were analyzed using 200x magnification. The morphological analysis included epithelisation, necrosis, edema, and granulation tissue formation evaluation. Histochemical coloring methods included hemalaun-eosine, Gommori for reticulin fibers and Masson for collagen fibers, and immunohistochemical coloring SMA for new smooth muscle cells visualization. All stages of the experiment were recorded using a Veho Discovery VMS-004 Deluxe USB camera with a microscope (Veho, Dayton, OH, SAD). Recordings took place before CMV ligation, after CMV ligation, after anastomosis creation and pentadecapeptide BPC 157 application, and at 3, 5, 7, 15 and 30 days postoperatively. Thus obtained films images were then saved as digital photographs to enable comparison between treated and control animals. Using digital photographs, two venous arcades (vasa recta) were analyzed on the ventral and dorsal side of the colon, orally and aborally from the anastomosis, as well as communicating branches between mentioned venous arcades.Program STATISTICA 12.1. (StatSoft, Inc, Tulsa, USA) was used for statistical processing. For the data distribution assessment, the Kolmogorov-Smirnoff test was used. Results are expressed as mean +/- standard deviation (SD) and as minimum/median/maximum. For parametric data, one way ANOVA test with NewmanKeuls post hoc test was used, and for nonparametric data Kruskal-Wallis test with Mann-Whitney post hoc test was used. Qualitative data in control and treated groups were analyzed using the Fisher test. p