DUODENOCOLIC FISTULA HEALING BY PENTADECAPEPTIDE BPC 157 IN RATS. A CYTOPROTECTION VIEWPOINT

This study attempts to highlight the particular cytoprotection aspects of the healing of fistulas (i.e. upgrading minor vessels) using duodenocolic fistula in rats. This can be the stable gastric pentadecapeptide BPC 157 (toxicology studies showed no lethal dose (LD1), used in ulcerative colitis clinical trials, without adverse effects) (1-9), supposed to act as a cytoprotection mediator (10, 11); in particular, known to cure both external (12-15) and internal fistulas (16-18) in rats, largely reviewed before (19, 20). Based on the obtained beneficial evidence, we claimed that with BPC 157 therapy, cytoprotection (10, 11, 21-25) as a particular vascular effect (10, 11, 21-25), wound healing (1, 19), and neuroprotection (6, 21, 26) combine the principle of upgrading minor vessels (10, 11).

Note, that the current approach of duodenocolic fistula requires complex surgery as the only solution (27, 28) when occurred as a rare complication of malignant and inflammatory bowel disease, perforated duodenal diverticulum, perforated duodenal ulcer, and inadvertent injury to duodenum and colon during surgery (27, 28). Possibly, this may be opposed in terms of the long-standing cytoprotection concept (29-34). Admittedly, seminal Robert’s and Szabo’s cytoprotection studies among the various noxious agents and procedures (i.e. intragastric absolute alcohol, strong base, strong acid, boiling water) (29-34) have not considered the issues of the perforated defect (i.e. in the stomach (35, 36) or colon (37)). However, the axiomatic statement (direct damage to cell), pointed already for standard cytoprotective agents, prostaglandins, somatostatin, and sulfhydryls (29-34), verifies such prototype of cytoprotection lesion (surgical perforation as a direct harmful effect) (35-37). Consequently, the healing of perforated defect by a cytoprotective agent (intended reversal of damaged tissue to normal structure (29) that BPC 157 application consistently exerts (10, 11, 21-25, 38), unlike standard cytoprotective agents, effective administration before but not after noxious agent) (29-34, 39-45)) conceptually goes as a prototype of the direct beneficial effect (35-37), and cytoprotection agent’s efficacy in fistulas healing (1, 19, 20).

Thereby, with duodenocolic fistula, the cytoprotection equation (direct injury - direct cell protection/recovery) (29) bears double trouble of innate perforated defect healing (two perforated defects, duodenal and colic, combined, as a duodenocolic fistula). Given the wound healing as a part of the cytoprotection effect (1, 19, 20), duodenal and colic defects, simultaneously presented as a duodenocolic fistula, posit the problem of inability to follow simultaneous healing of two distinctive tissues, and thereby unable to cure duodenocolic fistula. The healing by stable gastric pentadecapeptide BPC 157 may have particular effectiveness as native and stable in human gastric juice and as a cytoprotective mediator translating gastrointestinal mucosal integrity to other tissue healing (cytoprotection › organoprotection) (1-11, 19-26). The particular peptide’s background (no sequence homology with known gut peptides) easily combined with cytoprotection pleiotropic beneficial effect (1-11, 19-26), can provide consistent fistula healing (1, 19, 20). There is the easy application of BPC 157 using whatever way of application (parenteral or per-oral), BPC 157 as stable gastric pentadecapeptide has an undisputed therapy practical application (1-11, 19-26). Thus, wide interconnected activity (including fistula healing) (12-18, 20) can be regularly seen and consistently ascribed to its own activity (1-11, 19-26). Contrarily, standard growth peptidergic factors are easily destroyed because incubated in human gastric juice or water, this pentadecapeptide was not subjected to any degradation during 24 hours, unlike human epidermal growth facor (EGF) and human transforming groth factor alpha (h-TGF-a), which are stable in water but degraded in human gastric juice after only 15 minutes (46). Consequently, standard growth peptidergic factors while endogenously present commonly request the addition of various carriers (i.e. peptide + carrier(s) complex) to show the therapeutic effect when applied (1). Thereby, they can have uncertain activity attribution providing the given complex, peptide + carrier(s) (peptide, carrier, or peptide + carrier(s)) (1), essential limiting activity point, which is largely underestimated (i.e. and thereby rarely used in fistulas issue, basic fibroblast growth factor (bFGF) incorporated in gelatin hydrogel microspheres, fibrin glue with growth hormone) (47, 48).

Further, given updating original Robert’s and Szabo’s cytoprotection concept (i.e. rapid defensive response) by BPC 157 therapy (10, 11, 29-34), rapid upgrading minor vessels by prompt activation of the vascular collaterals (i.e. azygos vein direct blood flow delivery) as a common counteracted the occlusion/occlusion-like syndrome, as a whole, major vascular and multiorgan failure peripherally and centrally induced in rats (10, 11). These events were induced with permanent major vessel occlusions, central (49, 50) and peripheral (51-57), similar noxious procedures (35, 58-60), and agents’ applications (61-66). These all severely affect endothelium function. On the other hand, with an ulcer (perforated defect) there is also rapid ‘running’ (i.e., filled vessels ‘appear’; empty, ‘disappear’) toward the defect along with defect shrinking (35-37). With severe ischemia/reperfusion-induced colitis or arcade vessels, there is rapid collateralization ‘running’ over the left colic artery and vein occlusion along with recovering large pale areas without mucosal folds to the preserved mucosal folds (67). With duodenal mucosal and serosal lesions following occlusion of the superior anterior pancreaticoduodenal vein, rapid collateralization ‘runs’ over the superior anterior pancreaticoduodenal vein, along with attenuation of mucosal lesions and serosal congestion (68).

Thus, it may be hypothetized that in the particular case of the duodenum and colon defect, combined as duodenocolic fistula, treatment with BPC 157 would rapidly initiate such particular vascular response, simultaneously from both sides, duodenal and colonic, simultaneous collateralization and ‘running’ toward defects, at both, the duodenum and colon. Thereby, this vascular effect (35-37, 67, 68) can be the prime effect, and thereby, it attenuated/eliminated the persistent lesions, leaking, failure biomechanical, functional, diarrhea, weight loss, a long-standing perilous course leading to cachexia. To support this very early point, a particular gene expression i.e., cyclooxygenase-2 (Cox2), vascular endothelial growth factor A (VEGF-A), nitric oxide synthase 1 (Nos1), Nos2, Nos3, nuclear factor-kappa-B-activating protein (Nkap) (15, 50), was carried out as a likely special point to explain how the dysfunction and its counteraction is causal to, or result of. Note, that this can be due to this particular endothelium effect (10, 11), BPC 157 interaction with many molecular pathways (8, 9, 69-77), especially nitric oxide (NO)-system (73-82). It acts as a stabilizer of cellular junctions (8). There, it significantly mitigated indomethacin-induced leaky gut syndrome via increasing tight junction protein ZO-1 expression, and transepithelial resistance (8), inhibited the mRNA of inflammatory mediators: inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNF-α), increased expression of heat shock protein 70 (HSP70) and HSP 90, and antioxidant proteins, such as heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone-1 (NQO-1), glutathione reductase, glutathione peroxidase 2 and glutathione S-transferase pi (GST-π) (8). In particular, the interaction with the NO-system is a large one, in the various models and species, and BPC 157 which releases NO by itself, counteracts the adverse effect of NO-synthase (NOS)-blocker much like that of NOS-substrate (78-82) that maintains vascular tone (73-82) and thrombocytes function (82-85). Thus, BPC 157 may have a pleiotropic beneficial effect (1-11, 19-26). There, in the vessel wall, the rapid change in the lipid contents and protein secondary structure conformation, produced instantly via BPC 157 therapy (86), supported the vessel function under pathological conditions.

Consequently, this study investigated how BPC 157 can promptly restore vessels at both organs, improve healing of both duodenal and colon lesions, and mediate fistula closing (macro-/microscopically, and functionally, with no fistula leakage; diarrhea and weight loss rescued).

For two distinctive organs, the duodenum, and colon, which are normally separated, but now with fistula pathology interconnected, vascular rescue provides a common trigger to adjust distinctive tissues for appropriate healing. In theory, in internal fistulas, between the two organs, i.e., the duodenum and the colon, a simultaneous beneficial effect (healing of the duodenal defect, healing of the colon defect, closing of the duodenocolic fistula, healing process(es) in both of them) would match as an orchestrated action the internal and external fistulas healing as noted with BPC 157 therapy (12-18). Such orchestrated action, perceived in the described cytoprotection terms (10, 11), would signify a new quality in the combined tissues healing along with a beneficial effect in duodenal lesions (68, 87-92), ulcerative colitis (92-98), and complications (92-98). Hereby, this study emphasizes that BPC 157 given per-orally, or parenterally, given in the same dose range can be effective in the duodenal lesions, ulcerative colitis, and complications (68, 87-98) and internal and external fistulas healing (12-18), thus, a common therapeutic background of this compound should be further elaborated.

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 along with study protocol were approved by the Local Ethics Committee at the School of Medicine (University of Zagreb, Zagreb, Croatia)(380-59-10106-17-100/273). 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 and experimental protocol

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 (12-18). After surgery, as a therapy regimen, pentadecapeptide BPC 157 (10 µg/kg/day, 10 ng/kg/day) was given per-orally, in drinking water (0.16 µg/ml, or 0.16 ng/ml, 12 ml/rat/day) till sacrifice, or alternatively, intraperitoneally (5 ml/kg), first application at 30 min after surgery, last at 24 h before sacrifice, using anesthesia overdose. Controls simultaneously received an equivolume of saline (5.0 ml/kg, intraperitoneally) or water only (12 ml/rat/day). To investigate an immediate effect on the blood vessels presentation upon fistula creation, we used two regimens of BPC 157 application: 10 µg, 10 ng/kg per 1 ml bath/rat or 10 µg/kg, 10 ng/kg instilled into the rat stomach, at 1 min fistula-time (that 10 ng/kg instilled into the rat stomach regimen was used also in mRNA expression studies). Controls received a saline bath of equal volume at 1 min of fistula-time or an equal volume of saline instilled into the stomach.

Procedures

In deeply anesthetized male Wistar Albino rats, 200 g body weight the peritoneal cavity was entered through a 3 cm midline incision. A fistula length of 4 mm was surgically created as a junction between the duodenum (1 cm aboral from pylorus) and colon (2 cm aboral from ileocecal valve). It was created using the single layer suture technique. A precise caliper was used to verify the initial size of the defect.

Immediate effect on the blood vessels presentation upon fistula creation was assessed considering the point immediately before therapy (point ‘0’ as 100%) and scoring the vessel presentation (recorded filled/appearance or emptied/ disappearance (camera attached to a USB microscope (Veho discovery VMS-004 deluxe, Veho, Southampton, UK))) at the 30 mm duodenum segment aboral and oral to the fistula, arcade vessels arising from the superior anterior pancreaticoduodenal vein and artery on the ventral duodenum side as described before (34-36), and a corresponding colon segment, after 3, 6, 9, 12, and 15 min.

Colon and duodenal defect, fistula leakage, weight, diarrhea, adhesions, and intestinal obstruction assessment was performed at days 1, 3, 7, 14, 21, and 28.

Colon defect, duodenal defect. Briefly, a precise caliper was used to verify the final size of the defect, and the largest diameter of the colon and duodenal defect was assessed (mm), photographed, and further verified using the program ISSA (VAMSTEC Software Company, Zagreb, Croatia) as described before (31, 33-37). The tissue was processed for further microscopic analysis (31, 33-37). The tissue specimens were fixed in buffered formalin (pH 7.4), for 24 h, dehydrated, and embedded in paraffin wax. The samples were stained with hematoxylin-eosin (H&E). Tissue injury was evaluated microscopically by a blinded examiner.

Fistula leakage. To assess fistula leakage (31, 33-37) and the closure of the fistula, we assessed 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 there was no leaking till the end of the fifth minute, the fistula was considered to be functionally closed.

Fecal observation. All rats were observed for normal feces or diarrhea. Stool consistency score (0–3) was defined as 0 - formed, 1 - mild-soft, 2 - very soft, and 3 - watery soft (diarrhea) (70).

Adhesions assessment. 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 anastomosis covered; 3 - exaggerated adhesions with whole anastomosis covered; 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’, as described before (36, 37, 53).

Intestinal passage obstruction. Likewise, as described before (36, 37, 53) intestinal passage obstruction was scored 0–3, according to the loop diameter ratio, close to the fistula formation. Briefly, if loop diameters at 2 cm orally/loop diameters at 2 cm aborally = 1, the 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 a moderate obstruction (score 2); and more than 1.66 is a severe obstruction (score 3).

Gene expression analysis

Using the procedure as before (38, 45), after sacrificing at 5 min after intragastric application of the saline (5 ml/kg) or BPC 157 (10 ng/kg), tissue (duodenal and colon tissue, 1 mm around the defects) was rapidly dissected and frozen in liquid nitrogen. The tissue was disrupted using tissue homogenizer Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, CT, USA), in 1000 µl of TRIzol (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and the isolation itself was done using a TRIzol-based reagent method according to the manufacturer’s instructions.

After RNA isolation step, nucleic acid concentration was measured with Nano Drop ND-1000 spectrophotometer (Nano DropTechnologies, Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions and using a ProFlex PCR System machine (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).

TaqMan Gene Expression Assay (Applied Biosystems, Termo Fisher Scientific, Waltham, MA, USA) hydrolysis probes were used for gene expression analysis of selected genes (Table 1) with TaqMan Gene Expression Master Mix (Applied Biosystems, Termo Fisher Scientific, Waltham, MA, USA). Quantitative PCR was carried out in duplicate for every sample. Reactions were performed with Cobas z 480 instrument (Hoffmann-La Roche Ltd., Basel, Switzerland) according to the following protocol: 2 min at 50°C, 10 min at 95°C, 45 cycles of 15 s at 95°C and 1 min at 60°C.

We analyzed β-actin (Actb) as a reference gene to normalize the results of several genes of interest: Cox2, VEGF-A, NOS-1, NOS-2, NOS-3 and Nkap.

To determine the difference in gene expression between treated and non-treated samples, the formula 2–ΔΔCt was used, where the ΔΔCt is the difference between ΔCt of the treated sample and the ΔCt of the non-treated sample (Table 1).

Table 1. Selected gene details and TaqMan assays specifications.

An experimental design diagram is presented in Fig. 1.

Fig. 1. Experimental design diagram. Period in minutes (early events, upper) or in days (full experiment, low). Yellow indicate specific time points of assessment and sacrificing. Red box indicates surgery, and time of surgery (0). Black box indicates regimen and time of application (blue line indicate period of per-oral application given in drinking water; orange circles indicate time of once daily intraperitoneal application). Blue box indicates assessed parameters, and time point of assessment and sacrificing.

Statistical analysis

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 diarrhea rate assessment was used. Values of P<0.05 were considered statistically significant.

RESULTS

We focused on the rat duodenocolic fistula, healing therapy and the stable gastric pentadecapeptide BPC 157 (Figs. 2-9).

As may be expected in analogy with the known injuries in original cytoprotection studies (1, 3), in rats with duodenocolic fistulas, immediately after formation of the fistula, vascular failure progressed in controls quite rapidly, as seen with deteriorating vessels, losing collaterals, and branching was almost absent (Figs. 2-4). Then, they present a regular downhill course, resulting with the failure of the healing, and the persistence of the defects, duodenum defect much like colon defect, continuously considerable weight loss and diarrhea since first day, advanced adhesions formation and intestinal obstruction (Figs. 5-7).

Fig. 2. Bath application. Characteristic appearance of the rat duodenum and colon close to the created duodenocolic fistula (A, B, C, D, black letter) (arrows), presentation before therapy (white small italic letters), presentation after therapy (white capital italic letter). (A, C): at 1 minute after fistula formation, but before therapy (either BPC 157 (b, white small letter), or saline (control) (c, white small letter)). (B, D): Presentation at 5 minutes after direct application of the medication at the site of the fistula that mimics intraperitoneal application, BPC 157, 10 ng/kg, 2 ng/1 ml (B, white capital italic letter) or saline 1 ml-bath (C, white capital italic letter). B. BPC 157 rapidly induces vessels ‘recruitment’, ‘running’ toward the defect, simultaneously at duodenum and colon, providing numerous collaterals and branching (B). (D): deteriorating vessels, losing collaterals, and branching almost absent in controls (C). USB microscope camera.

Fig. 3. Intragastric application. Characteristic appearance of the rat duodenum and colon close to the created duodenocolic fistula (A, B, C, D, black letter) (arrows), presentation before therapy (white small italic letters), presentation after therapy (white capital italic letter). (A, C): at 1 minute after fistula formation, but before therapy (either BPC 157 (b, white small letter), or saline (control) (c, white small letter)). (B, D): presentation at 5 minutes after intragastric application of the medication, BPC 157, 10 ng/kg, 2 ng/1 ml (B, white capital italic letter) or saline 1 ml-bath (C, white capital italic letter). (B): BPC 157 rapidly induces vessels ‘recruitment’, ‘running’ toward the defect, simultaneously at duodenum and colon, providing numerous collaterals and branching (B). (D): Deteriorating vessels, losing collaterals, and branching almost absent in controls (C). USB microscope camera.

Fig. 4. Immediate effect on the blood vessels presentation upon fistula creation was assessed considering the point immediately before therapy (point ‘0’ as 100%) and scoring the vessel presentation (recorded filled/appearance or emptied/disappearance (camera attached to a USB microscope (Veho discovery VMS-004 deluxe))) at the 30 mm duodenum segment aboral and oral to the fistula, arcade vessels arising from the superior anterior pancreaticoduodenal vein and artery on the ventral duodenum side, and the corresponding colon segment, after 3, 6, 9, 12, and 15 minutes. To investigate an immediate effect on the blood vessels presentation upon fistula creation, we used two regimens of BPC 157 application: 10 µg, 10 ng/kg per 1 ml bath/rat (that mimics intraperitoneal application) or 10 µg/kg, 10 ng/kg instilled into the rat stomach, at 1 minute fistula-time. At 1 minute of fistula-time, controls received a saline bath of equal volume or an equal volume of saline instilled into the stomach. * P<0.05, at least versus control.

Unlike vascular failure in controls, upon duodenocolic fistula creation, BPC 157, given locally at the fistula (the application that actually mimics the intraperitoneal one), or intragastrically, rapidly induces vessels ‘recruitment’, ‘running’ toward the defect, simultaneously at duodenum and colon, providing numerous collaterals and branching (Figs. 2-4). As therapy, BPC 157, (10 µg/kg, 10 ng/kg, per-orally, in drinking water until sacrifice, or alternatively, intraperitoneally, first application at 30 min after surgery, last at 24 h before sacrifice), evidently rescues rats with the duodenocolic fistulas, since very beginning. BPC 157 therapy closed both defects, duodenal and colonic. Consequently, we evidenced no fistula leakage (finally, a maximal instilled volume much like in the healthy rats), no cachexia, the same weight as before surgery, no diarrhea (Fisher exact probability test P<0.05, at least versus control), markedly less adhesion formation and intestinal passage obstruction (Figs. 5-7).

Fig. 5. Duodenal (upper) and colon (low) defect healing. Fistula length of 4 mm was surgically created as a junction between the duodenum (1 cm aboral from pylorus) and colon (2 cm aboral from ileocecal valve). After surgery, pentadecapeptide BPC 157 10 µg/kg/day, 10 ng/kg/day was given per-orally, in drinking water (0.16 µg/ml, or 0.16 ng/ml, 12 ml/rat/day) till sacrifice, or alternatively, intraperitoneally, first application at 30 min after surgery, last at 24 h before sacrifice. Controls simultaneously received an equivolume of saline (5.0 ml/kg i.p.) or water only (12 ml/rat/day). Means ±SD, longest defect diameters, mm, *P<0.05, at least versus control.

Fig. 6. Leakage volume, ml H2O (upper), Δ weight, g, weight before surgery - weight after surgery (low). Fistula length of 4 mm was surgically created as a junction between the duodenum (1 cm aboral from pylorus) and colon (2 cm aboral from ileocecal valve). After surgery, pentadecapeptide BPC 157 10 µg/kg/day, 10 ng/kg/day was given per-orally, in drinking water (0.16 µg/ml, or 0.16 ng/ml, 12 ml/rat/day) till sacrifice, or alternatively, intraperitoneally, first application at 30 min after surgery, last at 24 h before sacrifice. Controls simultaneously received an equivolume of saline (5.0 ml/kg i.p.) or water only (12 ml/rat/day). Means ±SD, *PΔ0.05, at least versus control.

Generally, in duodenocolic fistula rats, the microscopic presentation followed the described macroscopical healing course (Fig. 8), thus internal fistula healing similar to the previously described healing course of rat other fistulas, external and internal (Fig. 8). Microscopically, the fistula wall in control rats is built partially of duodenum and partially of colon tissue separated by an area of granulation tissue with persistent fistula gap. Contrary to the poor healing of fistula in control rats, less granulation tissue, granulocytes, prominent regeneration of submucosal muscle fibers, and fistulous channel closure were noted in pentadecapeptide BPC 157-treated rats.

Fig. 7. Adhesions presentation, scored 0–7 (upper), intestine passage obstruction, scored 0–3 (low). Fistula length of 4 mm was surgically created as a junction between the duodenum (1 cm aboral from pylorus) and colon (2 cm aboral from ileocecal valve). After surgery, pentadecapeptide BPC 157 10 µg/kg/day, 10 ng/kg/day was given per-orally, in drinking water (0.16 µg/ml, or 0.16 ng/ml, 12 ml/rat/day) till sacrifice, or alternatively, intraperitoneally, first application at 30 min after surgery, last at 24 h before sacrifice. Controls simultaneously received an equivolume of saline (5.0 ml/kg i.p.) or water only (12 ml/rat/day). Min/Med/Max, *P<0.05, at least versus control.

Fig. 8. Gross presentation of duodenal and colonic defects at 14 day after surgery (white capital letters, A, B, C, D), microscopic presentation of fistula wall at 28 day after surgery (white capital letters, E, F). (A, B, C, D, upper): Gross presentation in controls (small letters) and BPC 157-rats (capital letters) of duodenal (d, D) and colonic (c, C) defects at 14 day after surgery. Arrows indicate open defects (controls) or practically closed defect (BPC 157 rats). (E, F, lower): Microscopically, at day 28, the fistula wall in control rats is built partially of duodenum (upper) and partially of colon tissue (low) separated by an area of granulation tissue with persistent fistula gap. Contrary to the poor healing of fistula in control rats (c), less granulation tissue, granulocytes, prominent regeneration of submucosal muscle fibers, and fistulous channel closure were noted in pentadecapeptide BPC 157 rats (B). Arrows indicate open defects (controls) or closed defects (BPC 157 rats), scale bars 200 µm, HE × 40.

The mRNA expression studies done at that very early fistula-time provided strongly elevated (NOS-2) and decreased (Cox2, VEGF-A, NOS-1, NOS-3, Nkap) gene expression, as a way how BPC 157 may act beneficially in both duodenum and colon lesions (Fig. 9).

Fig. 9. mRNA expression. Real-time PCR determination of mRNA expression of a set of targeted genes in three different tissue samples, duodenum (white bars) and colon (gray bars) time interval. Results are expressed as percentage changes (Means ±SD) of BPC 157 10 ng/kg (intragastric application) relative to control samples. P<0.05 is marked with an asterisk *. Results without a * have no biological difference to control samples.

DISCUSSION

The evidence that BPC 157 therapy heals duodenocolic fistula in rats as a whole course since the very beginning, can be likely indicative. Hereby, the evidence started with presented earliest findings (i.e. around defect (duodenum, colon), local blood vessels failure versus rapid particular vascular effect). This can elucidate the particular cytoprotection aspects of successful healing of fistulas versus failed healing and fistulas persistence (i.e. duodenocolic), and, clearly combine the principle of upgrading minor vessels and stable gastric pentadecapeptide BPC 157 cure of fistulas in rats, largely reviewed in earlier studies (1, 19, 20). Noteworthy, this can be important, given the previous evidence consistently demonstrating a particular effect depending on the injury (consistent with cytoprotection healing of different tissues (29)), as BPC 157 therapy heals gastrocutaneous, duodenocutaneous, esophagocutaneous, colocutaneous, colovesical, rectovaginal, and vesicovaginal fistulas (12-18). Thereby, BPC 157 has a pleiotropic beneficial effect (10, 11) and this effect equally involves external and internal fistulas (1, 12-20). Now, we may claim a particular background (particular gene expression, and relation with NO-system and prostaglandin-system) behind upgrading minor vessels. Accordingly, BPC 157 therapy produced instantly in the vessel wall, the rapid change in the lipid contents and protein secondary structure conformation (86), thus, supported the vessel function even under pathological circumstances. So, it may be that BPC 157 therapy confronted with the defects on two different sides, can have such vascular rescuing effect simultaneously initiated at two distinctive sides, realizing the simultaneous healing of the defects in different tissues (i.e. duodenum and colon), and thereby, resolving the false communication (i.e. fistula) and reestablish tissue integrity and function. Illustratively, in rats with duodenocolic fistulas, leaking, failure of biomechanical, functional, diarrhea, weight loss, and a long-standing perilous course with cachexia were all counteracted.

Likely, such positive both early and late outcomes as consistent findings throughout the whole experiment course may determine the preceding significance of the early vascular rescue in the duodenum and the colon along with the same particular mRNA expression genes as the earliest event as manifested by i.e. strongly elevated (NOS-2) and decreased (Cox2, VEGF-A, NOS-1, NOS-3, Nkap) gene expression. This may be regardless of the limitation to the one point analyzed (Cox2, VEGF-A, NOS-1, NOS-2, NOS-3, Nkap pathway) and the common limitation as RT-qPCR has the limitation of results only reflecting mRNA expression levels, which may not correlate with protein expression (19, 50)). Possibly, this point may provide an early link (the complex interplay between NOS and COX pathways to the expression of inducible forms (NOS-2 ad COX-2) as a cellular response to stress (99, 100). We found strongly elevated NOS-2 (and decreased NOS-1, NOS-3, and Cox2) in the BPC 157 treated rats. Thus, while BPC 157 inhibited NSAIDs-induced lesions and leaky gut syndrome as well (4, 8, 37, 96, 101-106), at least, at the same time, there may be successfully resolving, rapidly recovered blood vessels, mediated likely by continuously high levels of NO through iNOS (99, 100). Note, once induced, iNOS produces continuously excessive levels of NO, until the enzyme is degraded (99, 100, 106). Thereby, benefits from iNOS acting in antimicrobial, antiviral, antiparasitic, and tumoricidal processes have been described (107, 108). Accordingly, this can be a notion of a particular BPC 157/NO-system self-controlling (99, 100) (i.e. commonly, the expression of iNOS is considered to be regulated by cytokines and determined primarily by the de novo synthesis and stability of iNOS mRNA and protein)) (78, 108-110). This may be especially the case, given the known BPC 157’s free radical scavenger effects (8, 9, 36, 37, 111-114) especially in vascular studies (51-53, 63, 65, 67, 68). BPC 157 may act as the stabilizer of the cellular junction and the adequate element of the oxidant defense system to counteract possible oxidative stress (8). Moreover, this may be a particular case knowing the BPC 157 relation with vascular endothelial growth factor (VEGF) (73, 74, 76). There is the increased expression and internalization of VEGFR2, the activation of the VEGFR2-Akt-eNOS signaling pathway without the need for other known ligands or shear stress (73), and Nkap-NO relations because nuclear factor kappaB (NFκB) plays a central role in the regulation of NOS expression (100). Also, BPC 157, while interacting with several molecular pathways (8, 9 ,69-77), exerts a particular interaction with the NO-system (73, 74, 77-82), a point consistently revealed in more than 80 targets in the body (78). BPC 157 has been shown to release of NO (73, 74, 80, 81) in various models and species (73, 74, 77-82), given that BPC 157 counteracted the adverse effect of NO-synthase (NOS)-blocker much like that of NOS-substrate, L-arginine (78, 79). Illustrative for a particular modulatory role should be the counteraction of hypertension, and anti-coagulant effect (L-arginine) as well as pro-coagulant effect (L-NAME) and maintaining thrombocyte function without affecting coagulation pathways (80, 82).

Very likely, this initial vascular rescue can be essential for the whole course of the fistula healing. First, such vascular rescue noted in the duodenum and colon is along with the principle of upgrading minor vessels by prompt activation of the vascular collaterals (i.e. azygos vein direct blood flow delivery) shown in the occlusion/occlusion-like syndrome, as a whole, major vascular and multiorgan failure peripherally-and centrally-induced in rats (51-66). Thereby, the complex fistula healing may correspond to the recovery of occlusion/occlusion-like syndromes induced with permanent major vessel occlusions both, central (49, 50) and peripheral (51-57), the similar noxious procedures (35, 58-60), and agents’ applications (61-66). BPC 157 therapy attenuated/eliminated vascular and multiorgan failure, major vessel failure, thrombosis, advanced Virchow triad circumstances, and blood pressure disturbances intracranial (superior sagittal sinus), caval and portal hypertension, and aortal hypotension (51-66). These effects of BPC 157 therapy resulted in, i.e. rapidly reestablished reorganized blood flow and attenuated/eliminated the lesions in the brain (intracerebral and intraventricular hemorrhage), heart (congestion, myocardial infarction, severe arrhythmias), lung (hemorrhage), and congestion in the liver, kidney, and gastrointestinal tract (51-66).

Thereby, the noted healing of these fistulas may have additional general significance. Illustrative can be the counteraction of cachexia (i.e. in rats with duodenocolic fistulas, short bowel (97), and tumors (9)). In duodenocolic fistulas rats as in other fistulas’ healing, this goes along with counteraction of leaking, failure of biomechanical and functional alterations such as the passage of the duodenal content into the colon, diarrhea, and weight loss, that would result in a long-standing perilous course with cachexia as in the patients with duodenocolic fistulas (27, 28). In short-bowel rats, the constant weight gain above preoperative values started immediately with peroral and parental BPC 157 therapy, and vigorous adaption of the whole intestinal wall occurred in the remaining short bowel after massive small bowel resection, all intestinal layers exhibited additional increase and adequate adaptation (95). In mice with colon adenocarcinoma C-26 application, BPC 157 prolonged survival, counteracted the muscle wasting, counteracted increase of the pro-cachectic and pro-inflammatory mediators, and changes in the expression of forkhead transcription factor O subfamily member 3a (FoxO3a), phospho-protein kinase B (p-AKT), phosphorylation of mammalian target of rapamycin (p-mTOR), and glycogen synthase kinase 3beta (P-GSK-3b) (8). Of note, BPC 157 was shown to inhibit growth of several tumor cell lines and counteracted the effect of VEGF (115).

Illustrative generalization in duodenocolic fistulas rats can be the counteraction of bowel adhesion, given that BPC 157 counteracts bowel adhesion formation related to different procedures (12-18, 54, 94-96). Likewise, this may resolve adhesions and fistulas as an aberrant wound-healing process (1, 19, 116). Namely, as a part of synchronizing healing (19), BPC 157 accelerates the healing of various wounds (i.e. skin (117-119), muscle (120-125), tendon (124-128), ligament (129), bone (130); corneal ulcers (131) and ulcers in the entire gastrointestinal tract (68, 87-98), along with external and internal fistulas (12-18) and anastomosis, intestinal perturbations (95-97, 132), nerve (133) and vascular damage (134). Also, once the peritoneum is damaged, the coagulation cascade is set in motion (135); BPC 157 counteracts the whole Virchow triad (51-66), venous and arterial thrombosis (51-66, 134) and attenuates prolonged bleeding and thrombocytopenia after perforation, amputation, and anticoagulant use (35-37, 82-84). Therefore, it is likely that BPC 157 limiting adhesion formation and reversing existing adhesions, interfering when two damaged peritoneal surfaces come into contact with each other, may additionally support vascular rescue as essential in fistulas’ healing and allows for cytoprotection concept implementation.

In summary, we could envisage prompt activation of the vascular collaterals (particular mRNA expression genes, same in duodenum and colon) as a particular target resolved by the BPC 157 therapy in fistulas healing compatible also with resolution of the multiorgan failure of even deadly occlusion/occlusion-like syndromes by BPC 157 therapy (51-66). The complexity essentially resolved can be interesting given the applied doses (intragastric, bath, peroral, intraperitoneal) of BPC 157 largely compatible with cytoprotective peptide native and stable in human gastric juice cytoprotective mediator. By holding stomach integrity maintenance this mediator should be translated to other organ healing, also mimicked by cytoprotective agents’ application (10, 11, 29). Thus, BPC 157 that can be released into the circulation, is sent to distant organs by complex biological processes to regulate physiology and behavior. A study (3H-PL-10.1.AK-15. Pharmacokinetics in the rat after single oral administration. Istituto di Ricerche Biomediche „A. Marxer“, RBM, May 03, 1996) revealed a BPC 157 half-life of 66 h in male rats and 69 h in female rats. Direct administration on the formed fistula and initiation of the whole chain of events can be seen as a direct cytoprotective effect (note, a direct cytoprotective effect was noted also in vitro studies (136, 137)). This goes with the evidence of the in situ hybridization and immunostaining of BPC 157 found in many tissues in both the human fetus and in adults (1). Possibly, BPC 157 may have a regulatory physiologic role in bodily functions, given its similar beneficial effects in other species as well (e.g., birds (138) and insects (139, 140)). The final advantage of BPC 157 therapy is also its remarkable safety (no adverse effects in clinical trials e.g. ulcerative colitis, phase II), and LD1 could be not achieved in toxicological studies) (1-11, 19-26), a point recently confirmed in a large study conducted by Xu and collaborators (141).

Conflict of interests: None declared.

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