Therapy Effect of the Stable Gastric Pentadecapeptide BPC 157 on Acute Pancreatitis as Vascular Failure-Induced Severe Peripheral and Central Syndrome in Rats

We revealed the therapy effect of the stable gastric pentadecapeptide BPC 157 (10 μg/kg, 10 ng/kg ig or po) with specific activation of the collateral rescuing pathways, the azygos vein, on bile duct ligation in particular, and acute pancreatitis as local disturbances (i.e., improved gross and microscopy presentation, decreased amylase level). Additionally, we revealed the therapy’s effect on the acute pancreatitis as vascular failure and multiorgan failure, both peripherally and centrally following “occlusion-like” syndrome, major intoxication (alcohol, lithium), maintained severe intra-abdominal hypertension, and myocardial infarction, or occlusion syndrome, and major vessel occlusion. The application-sacrifice periods were ligation times of 0–30 min, 0–5 h, 0–24 h (cured periods, early regimen) and 4.30 h–5 h, 5 h–24 h (cured periods, delayed regimen). Otherwise, bile duct-ligated rats commonly presented intracranial (superior sagittal sinus), portal and caval hypertension and aortal hypotension, gross brain swelling, hemorrhage and lesions, heart dysfunction, lung lesions, liver and kidney failure, gastrointestinal lesions, and severe arterial and venous thrombosis, peripherally and centrally. Unless antagonized with the key effect of BPC 157 regimens, reversal of the inferior caval and superior mesenteric vein congestion and reversal of the failed azygos vein activated azygos vein-recruited direct delivery to rescue the inferior-superior caval vein pathway; these were all antecedent to acute pancreatitis major lesions (i.e., acinar, fat necrosis, hemorrhage). These lesions appeared in the later period, but were markedly attenuated/eliminated (i.e., hemorrhage) in BPC 157-treated rats. To summarize, while the innate vicious cycle may be peripheral (bile duct ligation), or central (rapidly developed brain disturbances), or peripheral and central, BPC 157 resolved acute pancreatitis and its adjacent syndrome.

Being stable and native in human gastric juice for more than 24 h, an easy application (i.e., in ulcerative colitis trial, it was safe without adverse effects, and a lethal dose (LD1) was not achieved in toxicology studies) (for review, see i.e., [1][2][3][4][5][6][7]) may indicate the particular therapy potential of this stable gastric pentadecapeptide BPC 157 in acute pancreatitis. Furthermore, due to its particular vascular recovering potential (for review, see i.e., [6]), BPC 157 could be fully capable at resolving the complete noxious course, acute pancreatitis, general vascular failure and occlusion-like syndrome. As emphasized before, the particular resolution of the activation of the collateral pathways, relayed on the given injury, would mean that the stable gastric pentadecapeptide BPC 157 therapy may realize an effective upgrade of the cytoprotection maxim endothelium maintenance → epithelium maintenance (for review, see i.e., [6]) as a powerful cytoprotective agent. The recognition of the cytoprotection maxim endothelium maintenance→ epithelium maintenance is potentially an essential but unresolved active key of Robert's and Szabo's cytoprotection concept, although they were overwhelmingly focused on stomach cytoprotection [32][33][34]. Recently, in these terms (activation of the collateral pathways, relayed on the given injury) [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], we reviewed the greater pleiotropic cytoprotection potential of the stable gastric pentadecapeptide BPC 157 as a novel cytoprotection mediator largely involved in healing (for review, see i.e., [1][2][3][4][5][6]), inside and outside the gastrointestinal tract (i.e., BPC 157 counteracted leaky gut (for review, see i.e., [4]). Special areas of therapy could be the bile duct ligation-induced disturbances [29][30][31]. As a specific, novel, rapid therapy effect (for review, see i.e., [6]), competing with the Virchow triad, commonly present, vascular recovery was consistently noted when confronted with multiorgan failure due to severe vascular failure [6]. As counteraction was generally seen peripherally and centrally [14][15][16]20,[22][23][24][25], it might also be possible in rats with an occluded bile duct. Intracranial (superior sagittal sinus) hypertension, portal and caval hypertension, aortal hypotension, progressing venous and arterial thrombosis peripherally and centrally, and ECG disturbances [14][15][16]20,[22][23][24][25] were commonly counteracted. Congested (i.e., inferior caval vein and superior mesenteric vein) and failed (azygos vein) blood vessels, multiple organs lesions, heart, lung, liver, Male Albino Wistar rats, 12 weeks old, 200 g body weight, bred in-house at the Animal Pharmacology Facility, School of Medicine, Zagreb, Croatia (registered with the Veterinary Directorate (Reg. No: HR-POK-007)), randomly assigned as 6 rats/group/interval were used in all experiments. Rats were acclimated for five days and randomly assigned to their respective treatment groups, housed in polycarbonate (PC) cages (identified with dates, number of study, group, dose, number and sex of each animal) at 20-24 • C, with a relative humidity of 40-70% and noise level of 60 dB, illumination 12 h per day (fluorescent lighting), a standard good laboratory practice (GLP) diet and fresh water ad libitum. Procedures were in line with the standard operating procedures (SOPs) of the Animal Pharmacology Facility, and the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123). This study was approved by the local Ethics Committee. The ethical principles of the study complied with the European Directive 010/63/E, the Law on Amendments to the Animal Protection Act (Official Gazette 37/13), the Animal Protection Act (Official Gazette 135/06), the Ordinance on the protection of animals used for scientific purposes (Official Gazette 55/13), Federation of European Laboratory Animal Science Associations (FELASA) recommendations and the recommendations of the Ethics Committee of the School of Medicine, University of Zagreb. The experiments were assessed by observers blinded as to the treatment.

Drugs
Stable gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, molecular weight 1419; Diagen, Slovenia), a partial sequence of the human gastric juice protein BPC, which is freely soluble in water at pH 7.0 and in saline, was prepared as a peptide with 99% high-performance liquid chromatography (HPLC) purity, with 1-des-Gly peptide being the main impurity. The BPC 157 dose and application regimens (10 µg or 10 ng/kg given as an intragastric administration or continuously orally, in drinking water) were as described previously (i.e., without use of a carrier or peptidase inhibitor) (for review, see, i.e., [1][2][3][4][5][6]).

Experimental Protocol
In deeply anesthetized rats, intraperitoneal (ip)-injected 40 mg/kg thiopental (Rotexmedica, Germany) and 10 mg/kg diazepam (Apaurin; Krka, Slovenia) was injected to induce acute pancreatitis and concomitant vascular failure general syndrome. We induced ligation of the bile duct at its entry into the duodenum [34,35] with assessments at 30 min, 5 h and 24 h.
Rats received BPC 157 (10 µg or 10 ng/kg) therapy as an early regimen immediately upon bile duct ligation (i), or as a delayed regimen at time of the already advanced acute pancreatitis (ii). (i) As an early regimen, rats received BPC 157 or saline (5 mL/kg) as an intragastric administration immediately following ligation, for assessment at 30 min, 5 h and 24 h of ligation time. (ii) As a delayed regimen, the therapy (BPC 157 or saline (5 mL/kg)) was given as an intragastric administration at 4.30 h of ligation time for assessment at 5 h of ligation time, or BPC 157 as an oral administration, given continuously in drinking water from 5 h of ligation time until the sacrifice at 24 h of ligation time (0.16 µg/mL/rat, 0.16 ng/mL/rat) while controls received drinking water only (12 mL/day/rat).
After complete calvariectomy, recordings of brain swelling (before procedure, after ligation and therapy application, and before sacrifice) followed the procedure previously used in our vascular studies [14][15][16]20,[22][23][24]. The calvariectomy procedure included, medially to the superior temporal lines and temporalis muscle attachments, 6 burr holes drilled in three horizontal lines (just basal from the posterior interocular line (two rostral burr holes), just rostral to the lambdoid suture (and transverse sinuses) on both sides (two basal burr holes) and in line between the basal and rostral burr holes (two middle burr holes)).

Brain Volume and Vessel Volume Presentation
The procedure from our previous vascular studies [14][15][16]20,[22][23][24] was applied. Brain volume and vessel volume and heart volume and pancreas volume were proportional to the change in the brain, vessel, heart or pancreas surface area. The presentation of the brain and peripheral vessels (superior mesenteric vein, inferior caval vein and azygos vein) was recorded in deeply anaesthetized rats, with a camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, Dayton, OH, USA) [14][15][16][17][18][19][20][21][26][27][28]. The border of the brain (or veins, heart or pancreas) in the image was marked using ImageJ software and then the surface area of the brain (or veins, heart or pancreas) was measured. This was conducted with brain (or vein) images for healthy rats, and then for both the control (saline) group and the treated (BPC 157) group of rats at the same intervals after the application and at the time of sacrifice. The arithmetic mean of the surface areas was calculated for both groups. Then, the ratio of these two areas was calculated as ( A con A bpc ), where Acon is the arithmetic mean brain (or veins) area of the control group and Abpc is the arithmetic mean brain (or veins, heart or pancreas) area of the treated group. Starting from the square cube law, Equations (1) and (2), an equation for the change in brain (or veins, heart or pancreas) volume proportional to the change in brain surface area (6) was derived. In expressions (1)-(5), l is defined as any arbitrary one-dimensional length of the brain (for example, rostro-caudal length of the brain), used only for defining the one-dimensional proportion (l2/l1) between two observed brains (or veins) and as an inter-factor (thus, not measured (6)) for deriving the final expression (6). The procedure was as follows: square-cube law: square-cube law: from (1), after dividing both sides by A1: from (3), after taking the square root of both sides: from (2), after dividing both sides by V1: after incorporating expression (4) into Equation (5):

Gross Assessment of Gastrointestinal and Pancreas Lesions
For recording, we used a camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, Dayton, OH, USA). As described before, gross lesions in the gastrointestinal tract and in the stomach (sum of the longest diameters, mm) were assessed in deeply anaesthetized rats, laparatomized before sacrifice [12][13][14][15][16]20,[22][23][24]. The acute pancreatitis damage was assessed using the same macroscopic system as previously [29,30] (score 0: pancreas without changes; score 1: pancreas with edema only; score 2: separate hemorrhagic zones and/or foci of necrosis, largest diameters < 1 mm; score 3: separate hemorrhagic zones and/or foci of necrosis, largest diameters > 3 mm; score 4: confluent hemorrhagic zones and/or foci of necrosis, largest diameters > 6 mm; score 5: diffuse hemorrhagic zones and/or necrosis in whole pancreas).

Microscopy
As described in the previous studies [12][13][14][15][16]20,[22][23][24], evaluation was made by light microscopy using an Olympus 71 digital camera and an Olympus BX51 microscope (OLYM-PUS Europa SE & CO. KG). Digital images were saved as uncompressed 24-bit RGB TIFF files using the software program AnalySIS (Olympus Soft Imaging System GmbH, Munster, Germany). Representative tissue specimens (i.e., the brain, liver, kidney, pancreas, stomach, small and large intestine, lungs, and heart taken at the end of the experiment, fixed in 10% neutral buffered formalin (pH 7.4) at room temperature for 24 h) were embedded in paraffin, sectioned at 4 µm, and stained with hemalaun and eosin (H&E).
We also assessed the neuronal pathological changes in acquired digital images saved as uncompressed 24-bit RGB TIFF files in the software program AnalySIS (Olympus Soft Imaging System GmbH, Munster, Germany) by performing quantitative analysis of the neuronal damage in the karyopyknotic areas. The neurons of the cortical cerebral region, cerebellar region, hippocampus, and hypothalamus were counted in 10 different high powered fields (HPF, 400×) and 3 to 5 serial sections of each sample were used for the count, as described in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303860/ (accessed on 25 May 2022). The field size was 0.24 µm 2 .
We used four criteria for estimation of the edema: a pale myelin, sieve-like appearance of myelinated areas, dilation of perivascular and pericellular spaces and vacuolar appearance of the neuropil of gray matter. Edema was graded as heavy, moderate, slight or no edema (score 0-3). https://www.sciencedirect.com/science/article/pii/S0379073821001286 (accessed on 25 May 2022).
We estimated hemorrhage as a percentage of the affected brain area. Intraventricular hemorrhage was noted as present or absent.
The scoring criteria for hemorrhage were presented separately from the fat necrosis scoring criteria due to the morphological differences referring to hemorrhage between control and treated animals.
Quantitative Analysis of CD20, CD3, CD68 (PG-M1) and CD163 Protein Expression The expression of CD20, CD3, CD68 (PG-M1) and CD163 proteins in pancreatic tissue was independently assessed by two board-certified pathologists (A.S., E.L.). The score was obtained as the number of positive cells per one power field (magnification 200×). All the discordant results were resolved at the double-headed microscope evaluation. Immunohistochemically stained slides were analyzed using an Olympus 71 digital camera and an Olympus BX51 microscope. Complete pancreatic tissue section was scanned at 100× magnification to assess the area showing the densest area with positively stained cells, the 'hot spot'. Assessing the 100× magnification "hot spot", consecutive use of magnification 200× in the same area was used until the densest area with positive cells was included within the 200× field. The "hot spots" at 200× magnification were eligible for analysis.

Statistical Analysis
Statistical analysis was performed by parametric one-way analysis of variance (ANOVA), with the Newman-Keuls post hoc test or the non-parametric Kruskal-Wallis test, and subsequently the Mann-Whitney U test to compare groups. Values are presented as the mean ± standard deviation (SD) and as the minimum/median/maximum. To compare the frequency difference between groups, the chi-squared test or Fischer's exact test was used. p < 0.05 was considered statistically significant.
The stable gastric pentadecapeptide BPC 157 therapy application might have a prophylactic and curative effect on both acute pancreatitis (i.e., gross presentation, microscopy, serum amylase) and adjacent adverse syndrome (i.e., attenuated/counteracted intracranial (superior sagittal sinus) hypertension and aortal hypotension, major ECG disturbances, progressing arterial and vein thrombosis, lesions in the brain, heart, lungs, liver, kidneys, and gastrointestinal tract). The BPC 157 therapy effect was comparable to the previous BPC 157 therapy of the mentioned occlusion and occlusion-like syndromes [14][15][16]20,[22][23][24][25]. For this effect, the common key finding may be the prompt and sustained activation of the azygos vein in the BPC 157-treated bile duct-ligated rats, the direct blood delivery from the inferior caval vein to the superior caval vein instantly breaking the vicious cycle.

Blood Pressure Disturbances
Perceived as a cause-effect relation that should be essentially affected by the therapy application, BPC 157 rapidly reduced blood pressure disturbances that were induced by bile duct ligation, which was quite severe peripherally (portal and caval hypertension, aortal hypotension) as well as centrally (superior sagittal sinus hypertension) (Figure 1). With a rapid and then sustained effect, the portal and caval hypertension and intracranial (superior sagittal sinus) hypertension as well as the aortal hypotension were eliminated, or at least markedly attenuated, with BPC 157 application.

Blood Pressure Disturbances
Perceived as a cause-effect relation that should be essentially affected by the therapy application, BPC 157 rapidly reduced blood pressure disturbances that were induced by bile duct ligation, which was quite severe peripherally (portal and caval hypertension, aortal hypotension) as well as centrally (superior sagittal sinus hypertension) (Figure 1). With a rapid and then sustained effect, the portal and caval hypertension and intracranial (superior sagittal sinus) hypertension as well as the aortal hypotension were eliminated, or at least markedly attenuated, with BPC 157 application. application-sacrifice period. Therapy included BPC 157 (10 µg/kg (light gray bars) or 10 ng/kg (dark gray bars)) or saline (5 mL/kg) (white bars) as an intragastric administration either immediately following ligation, or as an early regimen, for assessment at 30 min, 5 h and 24 h-ligation time. As a delayed regimen, the therapy was given as an intragastric administration at 4.30 h ligation time for assessment at 5 h ligation time, or as oral administration, continuously in drinking water from 5 h ligation time until sacrifice at 24 h ligation time. Means ± SD, * p < 0.05, at least, vs. control.

Thrombosis
Likewise, the fact that BPC 157 reduced thrombosis, both peripherally and centrally (Figure 1), may indicate the effective cause-effect course of the therapy. Without therapy, from the very beginning, as noted already at 30 min of ligation time, thrombosis progressed along with ligation time, peripherally in veins (i.e., portal vein and inferior caval vein) as well as in arteries (i.e., superior mesenteric artery, and abdominal aorta) and centrally (i.e., superior sagittal sinus).

Collateral Pathways, Blood Vessels, and Brain Gross Presentation
As a follow-up to the attenuation of blood pressure disturbances, peripherally and centrally, activation of the collateral pathway may compensate for major vessel failure and blood stasis observable with progressing thrombosis, which was fully counteracted in all veins and arteries investigated, peripherally and centrally. Consequently, the activated defensive response may be seen indicatively with the particular effects of BPC 157 on the relative volume of the congested vessels (superior mesenteric vein and inferior caval vein, from the trapped volume, congested liver and lung), dilated heart and collapsed vessels (azygos vein not functioning), and swollen brain ( Figure 2). BPC 157 may decrease the increased relative volume of the superior mesenteric vein and inferior caval vein, increase the failed volume of the azygos vein, and bring these vessels and heart presentation close to normal vessel and heart presentation. Evidently, as a particular effect on blood vessels, congestion was reduced by the activation of the collateral bridging pathway, i.e., the azygos vein (Figures 2 and 3), as BPC 157 increased the azygos vein's relative volume (Figures 2 and 3), combining the inferior caval vein and left superior caval vein to reestablish blood flow. Finally, as additional proof, the brain swelling and increased volume (associated with considerable brain injuries) (Figures 2 and 4) was rapidly counteracted by BPC 157 administration, and induced a considerable decrease toward normal brain presentation. Interestingly, this effect was simultaneous with a comparable counteracting effect on the swollen pancreas ( Figures 2 and 5).

Heart and ECG Disturbances
Commonly, the ligation of the bile duct was continuously timely along with the prolonged QTc intervals. However, the evidence shows that despite continuously maintained ligation of the bile duct, in all BPC 157-treated rats, QTc intervals' prolongation was regularly absent. This occurred along with counteraction of the myocardial congestion ( Figure 2).

Serum Amylase Level
Without therapy, in all of the investigated ligation periods, the serum amylase levels were markedly increased. Likewise, BPC 157 therapy attenuated the increased serum amylase levels in all of the investigated ligation periods ( Figure 2).

Figure 2.
Timeline of relative volume (a-f) of the QTc intervals, ms (g), and serum amylase level, IU/L (h). Presentation of the relative volume of the pancreas (a), azygos vein (a), heart (c), inferior caval vein (d), superior mesenteric vein (e), and brain (f) to illustrate the effect on pancreas swelling, brain swelling, heart dilatation, superior mesenteric vein and inferior vein congestion, azygos vein failure, and counteraction as control/treated % or ligation/healthy % at the end of the ligation time period of 30 min, 5 h, and 24 h, as an early regimen at 0-30 min (e), 0-5 h (f), and 0-24 h (g) of application-sacrifice period, and as delayed regimen at 4.30 h-5 h (h) and 5 h-24 h applicationsacrifice period. Therapy included BPC 157 (10 μg/kg (light gray bars) or 10 ng/kg (dark gray bars)) or saline (5 mL/kg) (white bars) as an intragastric administration either immediately following ligation or as an early regimen, for assessment at 30 min, 5 h and 24 h ligation time. As a delayed regimen, the therapy was given as an intragastric administration at 4.30 h ligation time for assessment at 5 h ligation time, or as oral administration, continuously in drinking water from 5 h ligation time until sacrifice at 24 h ligation time. Means ± SD, * p ˂ 0.05, at least, vs. control, # p ˂ 0.05, at least, vs. healthy. , and brain (f) to illustrate the effect on pancreas swelling, brain swelling, heart dilatation, superior mesenteric vein and inferior vein congestion, azygos vein failure, and counteraction as control/treated % or ligation/healthy % at the end of the ligation time period of 30 min, 5 h, and 24 h, as an early regimen at 0-30 min (e), 0-5 h (f), and 0-24 h (g) of application-sacrifice period, and as delayed regimen at 4.30 h-5 h (h) and 5 h-24 h application-sacrifice period. Therapy included BPC 157 (10 µg/kg (light gray bars) or 10 ng/kg (dark gray bars)) or saline (5 mL/kg) (white bars) as an intragastric administration either immediately following ligation or as an early regimen, for assessment at 30 min, 5 h and 24 h ligation time. As a delayed regimen, the therapy was given as an intragastric administration at 4.30 h ligation time for assessment at 5 h ligation time, or as oral administration, continuously in drinking water from 5 h ligation time until sacrifice at 24 h ligation time. Means ± SD, * p < 0.05, at least, vs. control, # p < 0.05, at least, vs. healthy.

Heart and ECG Disturbances
Commonly, the ligation of the bile duct was continuously timely along with the prolonged QTc intervals. However, the evidence shows that despite continuously maintained ligation of the bile duct, in all BPC 157-treated rats, QTc intervals' prolongation was regularly absent. This occurred along with counteraction of the myocardial congestion ( Figure 2).  The lesions are commonly noted to be vigorously expressed in the untreated rats with bile duct ligation, with an increased severity with ligation time; the lesion evidence in the affected organs may indicate particularity in the lesion course (Table 1). Thereby, the reduced severity of lesions in the pancreas, gastrointestinal tract and in the other organs by BPC 157 therapy may be seen as part of the cause-effect therapeutic course along with the reduced intracranial (superior sagittal sinus), portal and caval hypertension, and reduced aortal hypotension (Figure 1, Section 3.1.1). Together, this may suggest an immediate impact of the activated collateral pathway (i.e., 0-30 min and 4.30 h-5 h ligation period) followed by a sustained beneficial effect (0-24 h and 5 h-24 h ligation period) (Figures 2-5). Table 1. Pancreatitis, heart, lung, liver, kidney, stomach, small and large intestine lesion assessment in the bile duct-ligated rats following ligation (time 0) at sequential periods (application sacrifice) to illustrate the effect of both early treatment and delayed treatment following ligation. * p < 0.05, at least, vs. control.

Regimens
Early  There were separate hemorrhagic zones and/or foci of necrosis, with the largest diameters in controls, while only edema was present in the pancreas of the BPC 157-treated rats. Microscopically, we noted in controls a diffuse edema of interlobar septe, interlobular septe, interacinal spaces, focal expansion of intercellular spaces, and perivascular infiltration of 6-10 leukocytes/HPF. Contrarily, BPC 157-treated rats with the early regimen presented only a focal edema of interlobar septe and no acinar necrosis, hemorrhage, fat necrosis, inflammation, or perivascular infiltrate ( Figure 6). Early regimen, period 0-5 h. There were separate hemorrhagic zones and/or foci of necrosis, with the largest diameters in controls while in the BPC 157-treated rats there were mostly only edema in the pancreas. Microscopically, in controls, there was a diffuse edema of interlobar septe and interacinal spaces, focal expansion of intercellular spaces, a diffuse occurrence of 5-10 necrotic acinar cells, a focal occurrence of 11-16 necrotic cells/HPF, up to 2 foci of hemorrhage, up to 4 foci of fat necrosis, and perivascular infiltration of 16-20 leukocytes/HPF. Contrarily, early BPC 157 treatment had a marked beneficial effect, with only a focal edema of interlobar septa, a focal occurrence of 1-4 necrotic acinar cells/HPF, no hemorrhage and fat necrosis, and perivascular infiltration of 2-5 leukocytes/HPF ( Figure 6).
Early regimen, period 0-24 h. There were confluent and diffuse hemorrhagic zones and/or foci of necrosis, with the largest diameters in controls while BPC 157 exhibited separate hemorrhagic zones and/or foci of necrosis. Microscopically, controls presented increased lesion severity. They expressed a diffuse edema of interlobar septe, and interlobular septae, and focal expansion of intercellular spaces, more than 16 necrotic acinar cells/HPF (extensive confluent necrosis), more than eight foci of hemorrhage, up to seven foci of fat necrosis, and perivascular infiltration of >30 leukocytes/HPF. Contrarily, an early BPC 157 treatment had a marked beneficial effect until the end of the experiment, presenting markedly less lesions. Only a focal edema of interlobar septe, a diffuse occurrence of 1-4 necrotic acinar cells, a focal occurrence of 15-10 necrotic cells/HPF, up to one focus of hemorrhage and fat necrosis, and perivascular infiltration of 6-10 leukocytes/HPF was presented ( Figure 6).
Delayed regimen, period 4.30 h-5 h. There were separate hemorrhagic zones and/or foci of necrosis, with the largest diameters in controls. Contrarily, in the BPC 157-treated rats, there were mostly only edema in the pancreas. Microscopically, controls presented a diffuse edema of interlobar septe, interlobular septe, interacinal spaces, focal expansion of intercellular spaces, a diffuse occurrence of 5-10 necrotic acinar cells and a focal occurrence of 11-16 necrotic cells/HPF, up to four foci of hemorrhage, up to six foci of fat necrosis, and perivascular infiltration of 6-10 leukocytes/HPF in the 4.30 h-5 h assessment period ( Figure 6).
Contrarily, in BPC 157-treated rats, we noted a focal edema of interlobar septe, a diffuse occurrence of 5-10 necrotic cells/HPF, a focal occurrence of 11-16 necrotic cells/HPF, up to one focus of hemorrhage, and up to seven foci of fat necrosis, mostly in the pancreatic tail, while the pancreatic head was not affected with fat necrosis. A perivascular infiltration of 16-20 leukocytes/HPF was found ( Figure 6). Delayed regimen, period 5 h-24 h. There were confluent and diffuse hemorrhagic zones and/or foci of necrosis, with the largest diameters in controls. Contrarily, BPC 157 treatment exhibited separate hemorrhagic zones and/or foci of necrosis. Microscopically, a diffuse edema of interlobar septe, interlobular septe, interacinal spaces, diffuse expansion of intercellular spaces, more than 16 necrotic acinar cells/HPF (extensive confluent necrosis), more than eight foci of hemorrhage and fat necrosis, perivascular infiltration >35 leukocytes/HPF, and confluent microabscesses were found in the 5 h-24 h assessment period in the control group ( Figure 6).
In BPC 157-treated rats, a focal edema of interlobar septe was noted along with diffuse occurrence of 5-10 necrotic cells/HPF, focal occurrence of 11-16 necrotic cells/HPF, up to one focus of hemorrhage, and up to seven foci of fat necrosis, mostly in the pancreatic tail, while the pancreatic head was not affected with fat necrosis. A perivascular infiltration >30 leukocytes/HPF was found in the 5 h-24 h assessment period ( Figure 6).

A Perilous Syndrome Occurred Centrally
Brain Lesions, Cerebral and Cerebellar Cortex, Hypothalamus/Thalamus, and Hippocampus Without therapy, the consistently downhill course of ligation of the bile duct at its entry into the duodenum in rats led to multiple organ lesions, widespread thrombosis,

Brain Lesions, Cerebral and Cerebellar Cortex, Hypothalamus/Thalamus, and Hippocampus
Without therapy, the consistently downhill course of ligation of the bile duct at its entry into the duodenum in rats led to multiple organ lesions, widespread thrombosis, disturbed ECG and blood pressure, portal and caval hypertension, aortal hypotension, and in particular, intracranial (superior sagittal sinus) hypertension. From the immediate ligation period, the increased intracranial (superior sagittal sinus) hypertension coincided with a consistently swollen brain, severe brain edema and congestion, large intracerebral hemorrhage in the fronto-parietal area, and intraventricular hemorrhage in the central and lateral ventricles. Contrarily, reduced intracranial (superior sagittal sinus) hypertension (and eliminated portal and caval hypertension) with BPC 157 therapy counteracted the brain swelling with only mild brain edema and congestion, lesser brain hemorrhage, and no intraventricular hemorrhage.

Brain Damage
A pronounced edema and congestion were visible in the brain tissue in all four assessment time periods (i.e., 0-30 min, 0-5 h, 0-24 h, 4.30 h-5 h, 5 h-24 h) in the control group. A pronounced intracerebral hemorrhage involving larger areas of brain tissue was observed, affecting areas of the corpus callosum, amygdala, thalamus, neocortex, and striatum. Intraventricular hemorrhage involving the third and lateral ventricles was also seen ( Table 2, Figures 14-16). Table 2. Brain lesion assessment in the bile duct-ligated rats following ligation (time 0) at sequential periods (application sacrifice) to illustrate the effect of both early treatment and delayed treatment following ligation. * p < 0.05, at least, vs. control.
Biomedicines 2022, 10, x FOR PEER REVIEW 29 of 42 Figure 14. Gross presentation of the brain changes in rats with obstructed bile duct (a-H). Swollen brain (a,c-e,g) and brain swelling (saline application (d,g)), but counteracted by BPC 157 therapy (B,F,H) in rats with obstructed bile duct. I. Swollen brain before medication ((a,c) (immediate ligation-time)) and BPC 157 10 ng/kg intragastric administration, given immediately, ligation time 0, and effect assessed immediately thereafter; (B) the effect of the saline intragastric administration (d) assessed immediately (d) (worsened swelling due to uninterrupted progress of the injurious course). II. Swollen brain before medication (e) (4.30 h ligation time) and BPC 157 10 ng/kg intragastric administration, given at 4.30 h ligation time 0, and effect assessed immediately thereafter (F). III. Swollen brain (g) at 24 h ligation time, 24 h after saline (intragastric administration) given immediately after ligation. Swelling counteraction by BPC 157 therapy (H) at 24 h ligation time; BPC 157 10 ng/kg intragastric administration given at ligation time 0. Figure 14. Gross presentation of the brain changes in rats with obstructed bile duct (a-H). Swollen brain (a,c-e,g) and brain swelling (saline application (d,g)), but counteracted by BPC 157 therapy (B,F,H) in rats with obstructed bile duct. I. Swollen brain before medication ((a,c) (immediate ligationtime)) and BPC 157 10 ng/kg intragastric administration, given immediately, ligation time 0, and effect assessed immediately thereafter; (B) the effect of the saline intragastric administration (d) assessed immediately (d) (worsened swelling due to uninterrupted progress of the injurious course). II. Swollen brain before medication (e) (4.

Immunochemistry
There is a distinctive difference between the control and treated animals regarding inflammatory cells (Figures 17 and 18, Table 3). An increase in inflammatory infiltration, particularly CD3-positive lymphocytes and M1 CD68 (PG-M1)-positive macrophages, were observed in the control group, which follows the timeline of the inflammatory process. There was an increased number of M2 CD163-positive macrophages observed, lower than the number of M1 macrophages. This reflects the regular homeostasis of the inflammatory response to the tissue injury. Contrarily, in treated animals, there was none, or a distinctively mild nonspecific inflammation found, with only rare CD3-positive lymphocytes. There was no macrophage activation and barely one M2 CD163-positive macrophage in the "hot spot" area at 5 h.

Immunochemistry
There is a distinctive difference between the control and treated animals regarding inflammatory cells (Figures 17 and 18, Table 3). An increase in inflammatory infiltration, particularly CD3-positive lymphocytes and M1 CD68 (PG-M1)-positive macrophages, were observed in the control group, which follows the timeline of the inflammatory process. There was an increased number of M2 CD163-positive macrophages observed, lower than the number of M1 macrophages. This reflects the regular homeostasis of the inflammatory response to the tissue injury. Contrarily, in treated animals, there was none, or a distinctively mild nonspecific inflammation found, with only rare CD3-positive lymphocytes. There was no macrophage activation and barely one M2 CD163-positive macrophage in the "hot spot" area at 5 h.  In summary, after BPC 157 therapy, rats with a ligated bile duct exhibited no portal and caval hypertension, ameliorated aortal hypotension, markedly attenuated superior sagittal sinus hypertension, and no prolonged QTc interval. Additionally, venous and arterial thrombosis was attenuated, both peripherally and centrally, with reduced brain, heart, lung, liver, kidney, and gastrointestinal lesions, and counteracted acute pancreatitis, as either an initial or final result (severe intracerebral and intraventricular hemorrhage at the time when the pancreas exhibited only edema). The laboratory parameters improved and a particular inflammatory response was indicated. The key finding of an activated particular collateral pathway, i.e., the azygos vein, which combined the inferior caval vein and left superior vein to reorganize blood flow, might be responsible for the noted beneficial effects.  Table 3. Number of CD20-, CD3-, CD68-(PG-M1) and CD163-positive cells in the bile duct-ligated rats following ligation (time 0) at the sequential periods (application sacrifice) to illustrate the effect of both early treatment and delayed treatment following ligation. * p ˂ 0.05, at least, vs. control.
However, there might be an indication as to how the particular counteraction with BPC 157 therapy might advance during a noxious course. Namely, the evidence may be indicative that at an early time point, the pancreas exhibited mostly edema only; the major pancreatitis lesions (i.e., acinar, fat necrosis, hemorrhage) regularly appeared in the later period [55]. This might favor the prime central early course, the brain swelling, lesions and huge hemorrhage as the most prominent damage; increased (superior sagittal sinus) hypertension might have thereby a decisive role. Additionally, BPC 157's beneficial effects appear to be particularly appealing centrally. Illustratively, BPC 157 is very active in the rats with occluded superior sagittal sinus [20], as well as when given in the reperfusion in the rats with stroke, where BPC 157 therapy counteracted hippocampal ischemia/reperfusion injuries and functional and cognitive deficits [21]; when given in the rats with spinal cord compression, BPC 157 rapidly decreased spinal hematoma and increased function recovery [56,57]. In all of the vascular studies [14][15][16]20,[22][23][24][25], as also in the rats with ligated bile ducts, there was a prominent reversal of the brain swelling shortly after BPC 157 therapy application along with a decrease in intracranial (superior sagittal sinus) hypertension. It is possible that the BPC 157 therapy's effect might accommodate the particular central/peripheral and peripheral/central equation that may also take place in the bile duct-ligated rats. Namely, in analogy, we evidenced the occluded superior sagittal sinus-produced central disturbances (i.e., intracranial (superior sagittal sinus) hypertension) rapidly transmitted to the periphery (i.e., portal, caval hypertension, aortal hypotension, multiorgan lesions) [20]. Vice versa, direct occlusion of the superior mesenteric vessels, vein, artery, or vein and artery (i.e., portal, caval hypertension, aortal hypotension, peripheral lesions) rapidly induced the intracranial (superior sagittal sinus) hypertension, brain swelling, and lesions [14][15][16]. As this beneficial BPC 157 effect occurred in either circumstance, there was adequate resolution of the anatomical imbalance in venous drainage, which would thereby not occur [20]; centrally, a harmful inability to drain venous blood adequately for a given cerebral blood inflow without raising venous pressures was resolved, evidencing rapid counteraction of such venous and intracranial hypertension [20,[58][59][60][61][62]. Notably, in the timeline's causes and effects in the bile duct-ligated rats, the obtained frame (i.e., 0-30 min, 0-5 h, 4.30 h-5 h, 5 h-24 h, 0-24 h of the application sacrifice periods to verify consistency of the given therapy) might imply the timely advanced adverse effects of that shared syndrome, all and/or individual, into the essential presentation of the acute pancreatitis, and vice versa. This may indicate that the rapid initial presentation and subsequent injurious course is well presented. This may reveal the particular relationships (and therapy significance considering lesion counteraction in particular) and multidirectional network between the acute pancreatitis presentation and all other pathology targets. In particular relations, acute pancreatitis causing other organ lesions, we may characterize the brain swelling and lesions (i.e., intraventricular hemorrhage), heart dysfunction (i.e., prolonged QTc interval, congestion), lung lesions (i.e., hemorrhage), liver and kidney failure (i.e., severe congestion), and gastrointestinal lesions. Furthermore, there were widespread arterial and venous thrombosis, vein congestion (inferior caval vein and superior mesenteric vein) or failure (i.e., azygos vein), and severe intracranial (superior sagittal sinus), portal and caval hypertension and aortal hypotension. Thus, in this mutually interacting large damaging circuit in acute pancreatitis syndrome, the evidence of effective therapy that did counteract pancreatitis may indicate that it rapidly travels to affects all of the mentioned points, as we demonstrated with the BPC 157 therapy regimens. Illustratively, as a common beneficial effect of BPC 157 therapy, the pancreatic head was not affected by fat necrosis, there were no changes or only mild myocardial, lung, liver, kidney, stomach and intestinal congestion, and limited brain lesions (i.e., unlike extensive intraventricular hemorrhage in controls, only mild intraventricular hemorrhage in the third ventricle) in the BPC 157 rats. Such a pleiotropic effect (i.e., in the bile duct ligation/liver cirrhosis studies, with BPC 157 therapy, portal hypertension was either not developed or rapidly annihilated, depending on the given BPC 157 s regimen, and BPC 157 therapy might have counteracted liver fibrosis and portal hypertension [31]) was constant. As in other studies, there was counteraction of the brain [21], heart [63][64][65][66][67][68], lung [69][70][71][72], and liver lesions [31,73,74], liver, gastrointestinal and brain lesions [75][76][77][78][79][80], and kidney [14][15][16]20,[22][23][24][25] and pancreas [29,30] lesions. In the original cytoprotection studies, pancreatitis was known as a particular target for the standard cytoprotective agent activity [81], combined with the stomach and duodenal lesions [29,30]. Thereby, the activated azygos vein [13][14][15][16]20,[22][23][24][25] (rat azygos vein partially resembles the atrial myocardium [82]) may indeed be the BPC 157 bypassing key. As a BPC 157 therapy effect, the combined inferior caval vein and superior caval vein via direct blood delivery initiated the defensive response that made rats capable of enduring the bile duct ligation at its entry into the duodenum with a more preserved pancreas. Thus, the consequent noxious "occlusion-like" syndrome, occlusion syndrome and multiple organ failure was peripherally and centrally avoided [13][14][15][16]20,[22][23][24][25], despite the permanent occlusion of the bile duct. Finally, with BPC 157 therapy Virchow was reversed, there was a reversal of arterial and venous thrombosis, a common effect that was rapid and sustained [11][12][13][14][15][16]20,[22][23][24][25]. This means that in rats with bile duct ligation, the therapy reversed the stasis peripherally (note also the counteracted arrhythmias) and centrally, and re-established blood flow and largely rescued rats with ligated bile duct at its entry into the duodenum, thereby counteracting the intracranial (superior sagittal sinus) hypertension, portal and caval hypertension and aortal hypotension [11][12][13][14][15][16]20,[22][23][24][25]. Thus, general multiorgan failure, in particular that of the acute pancreatitis, appears to be due to the constant lack of the activated collateral pathway and may be a special target for the therapy application.
Finally, this special issue (i.e., bile duct occlusion, acute pancreatitis, presented with vascular failure, antecedent and concurrent occlusion-like syndrome, with multidirectional relation between the acute pancreatitis and other organs' lesions) remains to be further elaborated. Likewise, the pentadecapeptide BPC 157 should be further investigated. The therapy involved an intragastric bolus, and continuous oral application in drinking water, activated azygos vein direct blood delivery, counteracted/attenuated the intracranial (superior sagittal sinus), portal and caval hypertension, aortal hypotension, multiorgan failure (brain, heart, lung, liver, kidney, stomach, and intestinal lesions), and progressing thrombosis. Thereby, we observed acute pancreatitis, improved gross and microscopy presentation, and decreased amylase levels that might be applied also to other acute pancreatitis-type induction. We used the bile duct occlusion, acute pancreatitis, occlusion-like syndrome, activated azygos vein collateral pathway, pentadecapeptide BPC 157, intragastric bolus, and continuous oral application in drinking water as a simple but useful key, and shared the same dose relation in all BPC 157/vascular studies (for review, see, i.e., [1][2][3][4][5][6]). This might be seen as a network of evidence for the physiological significance of the revealed BPC 157/vascular system interplay (i.e., BPC 157 was found in situ hybridization and immunostaining studies in humans to be largely distributed in tissues [84] and may have additional physiological regulatory roles [3,84]). Moreover, there is also a very safe BPC 157 profile (i.e., no adverse effects in clinical trials (ulcerative colitis, phase II); in toxicological studies, a lethal dose (LD1) could be not achieved) (for review see, i.e., [1][2][3][4][5][6]84]), a point recently confirmed in a large study conducted by Xu and collaborators [85]. Together, these findings (for review, see, i.e., [1][2][3][4][5][6]84]) might suggest BPC 157 therapy application for further vascular injuries, and appropriate use in acute pancreatitis therapy as well.
In addition, case reports and retrospective cohort studies have reported an association between acute pancreatitis and COVID-19 [86], while recently, due to the described effects, the stable gastric pentadecapeptide BPC 157 was suggested as a potential treatment for COVID-19 [7].

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.