INTRODUCTION:

Chronic pancreatitis (CP) remains a significant problem nowadays: its prevalence is estimated as 13.5–52.4 cases per 100,000 population, and about 5 new cases per 100,000 population are registered every year. In the USA, pancreatitis is among the three most common diseases of the gastrointestinal tract, not associated with malignant neoplasms¹. The pathogenesis of CP is driven by both genetic and environmental factors, and alcohol abuse is one of the most relevant among the latter, being a widespread problem around the globe². Alcohol abuse targets both exocrine and endocrine functions of the pancreas leading to permanent structural and functional damage³.

The directions of conservative treatment in chronic pancreatitis include adequate analgesia, replacement therapy and dietary modifications to eliminate exocrine insufficiency, correction of metabolic disorders in endocrine pancreatic insufficiency (antihyperglycemic agents)^1,4,5. There is a possibility to influence the pathological processes in the pancreas in the early stages to prevent the development of the disease with a typical clinical manifestation⁶. In the literature available, there are results of antioxidants studies for this purpose^7,8.

Besides, a combined chronic pathology of the liver and pancreas is often seen in clinic^1-3. Therefore, it may be expedient to investigate the substances possessing a multitargeted cytoprotective effect, namely those combining hepatoprotective and pancreatoprotective activity, able to normalize metabolic processes and to counteract the hazardous effects of alcohol. And among such substances, there is the innovative molecule –
a coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid (CCAA). It exerts a significant hepatoprotective effect and, under conditions of an acute or chronic inflammatory process, demonstrates an anti-inflammatory, analgesic, and antipyretic effect. It has a positive effect on indicators of mesenchymal inflammatory syndrome partially connected to the antioxidative properties, as well as the ability to decrease hepatotoxins influence⁹. Notably, on the model of tetrachloromethane and ethanol-induced hepatitis CCAA displayed high efficacy contributing to the maintenance of NADP(+)/NADPH ratio and supporting the activity of alcohol dehydrogenase¹⁰. At the same time, inhibition of hepatic alcohol dehydrogenase during chronic alcohol abuse is considered to be the key metabolic event in the pathogenesis of CP (due to the further formation of fatty acid ethyl esters via nonoxidative metabolism)³.

The indications for CCAA use in adults and children include acute and chronic hepatitis of various genesis (viral, alcoholic, medicinal, toxic); fatty dystrophy and liver cirrhosis; inflammatory diseases of the gallbladder; postcholecystectomy syndrome. It is used for the prevention of liver injury caused by food toxins, hepatotoxic drugs including chemotherapy, and radiation therapy⁹. Mefenamic acid in the structure of CCAA as a coordinative compound represents a widely used nonsteroidal anti-inflammatory drug, nonselectively blocking cyclooxygenase isoforms COX-1 and COX-2¹¹. The interest in mefenamic acid is still significant, and new medicinal forms are being developed^12-14.

The use of coordination compounds is considered to be a promising way to decrease toxicity and a manifold decrease in acute toxicity values, the changes in toxicodynamic characteristics were registered for such compounds compared with the correspondent inorganic metal salts as well as with the organic ligand toxicity¹⁵. Therefore, such compounds can be successfully used as therapeutic agents. This substantiates CCAA further studies providing the answer to the possible question concerning potential aluminium toxicity.

The interest in this compound is reflected in several studies. Thus, manganese complexes with mefenamic acid were screened for antiproliferative activity and its mechanisms were investigated¹⁶. Mefenamic acid and its metal complexes with manganese, cobalt, nickel, copper, and zinc were evaluated for anti-oxidant, anti-inflammatory, and antiproliferative action¹⁷. High free radical scavenging activity as well as inhibitory activity against lipoxygenase was shown for zinc complexes of mefenamic acid¹⁸.

MATERIALS AND METHODS:

Animals and treatment:

The experiments were conducted on 40 random-bred male albino rats weighing 220 ± 20 g in accordance with the principles and requirements of the EU Council Directive (2010) on the protection of animals used for scientific purposes (the study protocol was approved by the bioethics committee of the National University of Pharmacy, Kharkiv, Ukraine).

After the adaptation period (7 days), the animals were examined by a qualified veterinarian, weighed and marked, the experimental groups were formed by randomization using the method of minimizing the difference in body weight.

The rats were kept at constant humidity of 45–65 % and air temperature of 20–24 °С, 12/12 light cycle in the vivarium of the Central Research Laboratory of the Educational and Scientific Institute of Applied Pharmacy of the National University of Pharmacy. A standard diet for rodents and free access to water was provided.

The experimental groups were as follows (n = 8 in each group):

1. The intact control group (IC).

2. The untreated control group, in which chronic pancreatitis was induced (CP–UC).

3. The group with chronic pancreatitis induction receiving CCAA at a dose of 30 mg/kg (CP+30).

4. The group with chronic pancreatitis induction receiving CCAA at a dose of 60 mg/kg (CP+60).

5. The group with chronic pancreatitis induction receiving CCAA at a dose of 120 mg/kg (CP+120).

The test sample of CCAA tablets (one tablet containing 200 mg of the substance) was used for the study. The route of administration was equivalent to that used in the clinical practice, namely the intragastrical one. In accordance with the general FDA recommendations, the doses were extrapolated from those used in the clinical practice (namely, 300, 600, 1200 mg per day for humans for different indications⁹) taking into account the differences in body weight and body surface area. The calculated doses equaled 30, 60, and 120 mg/kg.

The animals of all groups except for the intact control (IC) received the standard Lieber–DeCarli isocaloric alcohol liquid diet for 10 weeks²². The rats of the IC group received the corresponding isocaloric liquid diet without ethanol. LPS solution was injected once a week through the tail vein for the three last weeks of the diet modification at a dose of 3 mg/kg. The animals of the IC group received 0.9% sodium chloride solution according to a similar scheme.

After the first injection of LPS, CCAA intragastric administration was started and lasted for 21 days. The test sample was suspended in purified water; the suspension (in the permissible volume for the chosen species) was administered each day at 9.00–10.00. The choice of the terms of treatment is substantiated by the data in the literature and aimed at the beginning of the treatment after the development of the irreversible changes of the pancreas^19-22.

The body weight of the animals was registered during the study using the scales WLC 0/6/C/1 (Radwag), and general physical appearance was estimated daily.

24 hours after the last administration of LPS CO₂-induced euthanasia was done as the generally accepted method from the point of view of bioethics. Blood samples were taken from the inferior vena cava and serum was obtained. The small intestine was harvested and the duodenal contents were used to prepare 10 % homogenate in the standard PBS, the supernatant was used for further analysis. The pancreas was harvested, macroscopically evaluated, weighed, and divided into parts for the preparation of homogenate as described above and for the fixation in a 10 % neutral formalin solution.

Histological studies:

The fixed samples were dehydrated in increasing concentrations of ethanol and embedded in paraffin. The sections were stained with hematoxylin and eosin (for general histological evaluation) and with picrofuxin by Van Gieson (for the evaluation of fibrotic changes)²³. The microscope “Granum L3003” (“Laboratory Granum Ltd,” Ukraine) and the camera ToupCam 310 UCMOS 3.1MP were used, and the photographs were processed through a Levenhuk Toup View program.

Biochemical methods:

The supernatant of the homogenates of the duodenal contents and the pancreas were analyzed immediately to avoid proteolysis. Since the standardized quantity of the biological material was used for homogenate preparation, the volume concentrations of the determined values were calculated.

Non-specific inflammatory markers were determined in the blood samples, namely the leucocyte content and erythrocyte sedimentation rate²⁴. Serum samples were kept at –20 °C until analyzed. Basal glycemia as an indirect indicator of the pancreas endocrine function was determined using the glucosooxidase method. Total lipids level in blood serum and lipids concentration in the duodenal contents were measured through the reaction with phospho-vanillin reagent, α-amylase activity in these samples was estimated through starch-iodine assay method, and commercially-available standard kits from NVP Filisit-Diagnostika Ltd. (Ukraine) were used. Rat pancreatic elastase activity was measured using ELISA “Rat Elastase 1, Pancreatic Elisa kit” (BT Lab, the People’s Republic of China). α-amylase and pancreatic elastase were considered as the markers of the pancreas exocrine function.

Prooxidant-antioxidant balance was evaluated in the blood serum and the homogenate of the pancreas. The level of the lipid peroxidation products such as conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) was determined by the routine method after extraction with heptane and precipitation with trichloroacetic acid respectively²⁵. Antioxidative system components such as glutathione content (GSH), superoxide dismutase (SOD), and catalase (CAT) activities were measured using the commercially available standard kits “Total Glutathione (T-GSH)/Oxidized Glutathione (GSSG) Colorimetric Assay Kit,” “Total Superoxide Dismutase (T-SOD) Activity Assay Kit (WST-1 Method),” “Catalase (CAT) Activity Assay Kit” (Elabscience^®, the United States of America). Glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px) activities were estimated using the commercially-available standard kits “Glutathione Reductase (GR) Activity Assay Kit,” “Glutathione-S-Transferase (GST) Activity Assay Kit,” “Glutathione Peroxidase (GSH-Px) Activity Assay Kit” (Elabscience^®, the United States of America).

Semi-automatic biochemistry analyzer MapLab Plus (BSI, Italy) and ELISA microplate reader LabAnalyt M201 (Shenzhen Emperor Electronic Technology Co., the People’s Republic of China) were used for these studies.

Statistical Analysis:

Initially, the Shapiro-Wilk test was used to check if the variables are normally distributed. The results were expressed as mean (M) ± standard error of the mean (SEM). Statistical differences between groups were analyzed using the one-way analysis of variance (ANOVA), and Tukey’s HSD test was used post-hoc to determine the differences between the certain groups. The level of statistical significance was considered as
p < 0.05 ²⁶.

The results of the calibration of the enzyme immunoassay data were pre-processed using the 4Pl statistical-logistic method using the My Assays^® data analysis tools ²⁷. The standard package of IBM SPSS 22 and MS Excel 2013 programs was used.

RESULTS AND DISCUSSION:

Against the background of ethanol consumption during the first 7 weeks, the weight of the animals did not change significantly (p ˃ 0.05, one-way ANOVA). A significant decrease in this value was seen in the groups CP–UC, CP+30, CP+60 (Table 1) compared to the IC values. This was probably associated with a gradual decrease in the exocrine function of the pancreas caused by LPS, which led to a moderate disorder in nutrient assimilation. At the same time, no changes were observed in the CP+120 group at week 8 compared to the IC group value. On the 9^th and 10^th week, a moderate albeit statistically significant decline in body weight was registered in all groups with chronic pancreatitis induction (Table 1).

The induced pathological process led to the development of a moderate inflammatory process as seen from the slight but statistically significant increase in the non-specific markers indicating chronic low grade inflammation²⁸. Thus, erythrocyte sedimentation rate increased to 6.63 ± 0.60 mm/h; 5.25 ± 0.65 mm/h;
5.25 ± 0.49 mm/h in the groups CP–UC, CP+30, CP+60 respectively, while in the IC group, it equaled
3.00 ± 0.27 mm/h (p = 0.001; p < 0.05; p < 0.05 respectively). The increment in leucocytes content was seen and the values equaled 14.3 ± 1.51 × 10⁹/L;
12.5 ± 1.59 × 10⁹/L; 13.4 ± 0.72 × 10⁹/L in the groups CP–UC, CP+30, CP+60 respectively, while in the IC group it equaled 8.88 ± 0.51 × 10⁹/L (p < 0.02; p > 0.05; p = 0.06 respectively). At the same time, CCAA at the highest dose used led to the normalization of the mentioned markers: erythrocyte sedimentation rate equaled 3.50 ± 0.42 mm/h (p > 0.05 vs the IC group value, p = 0.001 vs the value of CP–UC group). Leucocyte content equaled 9.66 ± 1.10 × 10⁹/L (p > 0.05 vs the IC group value, p = 0.05 vs the value of CP–UC group).

Table 1: Body weight dynamics in rats with chronic alcohol-induced pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid (n = 8; M ±SEM)

Group	Body weight, g
Group	Baseline	Day 56	Day 63	Day 70
IC	219±5.0	282±4.8	289±5.0	298±5.2
CP–UC	219±5.7	244±5.5^***	243±5.4^***	246±5.3^***
CP+30	219±4.6	261±3.8^*	263±3.0^{** #}	269±2.9^{*** ##}
CP+60	218±4.7	251±4.7^***	254±4.7^***	257±4.3^***
CP +120	220±4.6	264±4.7^#	269±4.5^{* ##}	275±4.2^{** ###}
ANOVA	p = 0,9995	p = 0,0000	p = 0,0224	p = 0,0000

Notes:

1. * – p < 0.05 compared to intact control; ** – p < 0.01 compared to intact control; *** – p = 0.001 compared to intact control; Tukey HSD test;

2. # – p < 0.05 compared to CP–UC; ## – p < 0.01 compared to CP–UC; ### – p = 0.001 compared to CP–UC; Tukey HSD test.

The results of the indirect assessment of the functional state of the pancreas according to biochemical indicators are shown in Table 2. Glycemia remained unchanged in all of the groups with pancreatitis induction. However, there was a significant reduction in the blood plasma total lipids level with the corresponding increase in total lipids concentration in the duodenal contents, indirectly indicating insufficient secretion of pancreatic juice enzymes, in particular lipolytic ones. In animals receiving the studied compound at doses of 60 and 120 mg/kg, blood plasma total lipids level did not statistically differ from the value of the IC group
(p ˃ 0.05 in both cases), and the lipid content in the duodenal contents was reduced almost by half compared to the value of the untreated rats (CP–UC group).

In all of the groups with pancreatitis induction, there were no significant changes in the activity of α-amylase and the content of pancreatic elastase in blood serum (Table 3) indicating the development of a chronic rather than an acute process. One of the main target points of this study was the assessment of α-amylase and pancreatic elastase in the duodenal contents as the markers of the exocrine function of the pancreas. Thus, against the background of the Lieber–DeCarli diet and repeated injections of LPS, there was a pronounced exocrine insufficiency of the pancreas evidenced by the decrease in α-amylase activity by 2.85 times in the CP–UC group, as well as in the content of pancreatic elastase – by 2.52 times compared to the intact animals value (p = 0.001 in both cases, Table 3). At the same time, a dose-dependent therapeutic effect of the studied compound was seen: the statistically significant normalization of the aforementioned values was registered in the group receiving it at a dose of
120 mg/kg, where amylase activity was increased by 1.98 times, and the elastase content – by 1.71 times
(p ˂ 0.02 vs the CP–UC group values in both cases). In the groups receiving the studied drug at lower doses, there was only a tendency to the normalization of the named markers of the exocrine function of the pancreas.

Table 2: Indirect indicators of the functional state of the pancreas in rats with chronic alcohol-induced pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid (n = 8; M ± SEM)

Group	Blood serum		Duodenal contents
Group	Glucose, mmol/l	Total lipids level, g/l	Total lipids level, g/l
IC	5.35±0.36	2.31±0.23	0.21±0.03
CP–UC	5.15±0.13	1.35±0.05 ^**	0.42±0.06 ^**
CP+30	5.29±0.19	1.52±0.17 ^*	0.31±0.04
CP+60	4.86±0.18	1.93±0.14	0.19±0.03 ^##
CP+120	5.39±0.30	1.91±0.21	0.19±0.03 ^##

Notes:

1.* – p < 0.05 compared to intact control; ** – p < 0.01 compared to intact control; Tukey HSD test;

2. ## – p < 0.01 compared to CP–UC; Tukey HSD test.

Table 3: Markers of the exocrine function of the pancreas in rats with chronic alcohol-induced pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid (n = 8; M±SEM)

Group	Blood serum		Duodenal contents
Group	α-amylase, mg/ (sec × l)	Pancreatic elastase, ng/ml	α-amylase, mg/ (sec × l)	Pancrea-tic elastase, ng/ml
IC	468±17.1	8.46±0.55	1607±107	27.6±1.77
CP–UC	438±11.6	8.32±0.32	565±52.4 ^***	10.9±1.68 ^***
CP+30	458±11.7	8.14±0.47	760±70.0 ^***	12.2±1.81 ^***
CP+60	438±11.2	8.07±0.47	874±107 ^***	14.9±1.40 ^***
CP +120	442±13.0	8.18±0.54	1119±60.4 ^{ ###}**	18.7±1.67 ^{ #}**

Notes:

1. ** – p < 0.01 compared to intact control; *** – p = 0.001 compared to intact control; Tukey HSD test;

2. # – p < 0.05 compared to CP–UC; ### – p = 0.001 compared to CP–UC; Tukey HSD test.

The significant disorders of the prooxidant-antioxidant balance^29,30 were seen after pathological process induction, with a significant increment in lipid peroxidation markers and the corresponding decline in the activity of the antioxidative system, both in blood serum and in the pancreas homogenate (Table 4). Thus, in the CP–UC group compared with IC group values, the content of TBARS was statistically significantly increased by 2.96 times in the serum and by 1.85 times – in the pancreas; the content of CD was increased by 1.58 times in the serum and by 1.59 times in the supernatant. On the other hand, the activity of enzymes of the antioxidant system was statistically significantly reduced, namely SOD activity – by 22 % in the serum and by 25 % in the pancreas, and catalase activity – by 52 % and 46 %, respectively (Table 4).

In the group receiving the studied compound at a dose of 30 mg/kg, a tendency to a moderate decrease in TBARS and CD content as well as statistically significant maintenance of SOD activity (p = 0.001 vs the CP–UC value) was noted in blood serum. The normalization of TBARS level and SOD activity was reached in the pancreas (for both markers, p ˃ 0.05 vs. IC values,
Table 4). SOD activity normalization is considered to be an important marker of antioxidant activity used in in vivo studies³¹.

The studied drug at a dose of 30 mg/kg did not lead to significant improvements in the GSH system, it only restored the activity of GST in blood serum (p ˃ 0.05 vs IC group value).

Under the administration of CCAA at a dose of 60 mg/kg, T-GSH content in the pancreatic tissue was increased by 62 % (p ˂ 0.05 compared to the CP–UC value) and GST activity in this medium was normalized (p ˃ 0.05 vs. IC value). In addition, there was a tendency to the increment in GSH-Px activity (p ˂ 0.1 compared to the CP–UC value).

CCAA at a dose of 120 mg/kg allowed maintenance of GST activity in the blood serum (p ˃ 0.05 vs IC group value). GR activity was increased significantly compared to the data of the intact animals. An excessive increase in the level of glutathione reductase could be a consequence of the intensive involvement of the glutathione system in neutralization of oxidation products. At the same time, the reactive activation of the enzyme which reduces glutathione is necessary for the compensatory maintenance of the reduced/oxidized glutathione balance. In addition, under the influence of the studied drug at the mentioned dose, T-GSH content in the pancreatic tissue was significantly increased (by 49.6%; p˂0.05 compared to the CP–UC value). Together with an unchanged level of GSSG, it indicates an increase in the antioxidant active fraction of glutathione. Nevertheless, there was a normalization of GSH-Px level (p ˃ 0.05 vs IC group value) and a decrease in tissue GST activity (p < 0.05 vs IC group value). The latter can occur when the glutathione system is involved in the metabolism of xenobiotics³², and some drugs can reduce its activity, which is not considered to be a marker of a toxic action^33-35,moreover, the induction of this enzyme does not happen in such a way in the rat pancreas as in the rat liver²⁶.

The results of the histological studies:

The normal histological structure was observed in the pancreas of the intact male rats. In the exocrine component of the pancreatic tissue, the cells were of similar size and shape and formed densely packed acini with clear boundaries. The lumen of the acini was very limited. Two distinctive zones were identified within the cells, namely basophilic, with nuclei located on the periphery, and eosinophilic represented by the cytoplasm evenly filled with zymogen grains. The ratio of zones was within the range of 1:1.5 – 1:2. Small-sized excretory ducts were hardly visible among the acinar tissue, and medium and large ducts were surrounded by a limited amount of connective tissue. The epithelium of the ducts was normal, the lumen was sometimes widened. Blood vessels of various calibers were without any signs of pathologic changes (Figures 1, 2). Endocrine islets were clearly separated from the surrounding exocrine parenchyma. They were formed of strands of light-coloured polygonal cells. Between the strands, widespread sinusoidal capillaries were visible in some areas. β-cells were the predominant ones within the islet cells (the core of islets). Peripherally located cells, which could be classified as α-cells, were arranged in the form of chains.

After chronic pancreatitis development, in the samples of the CP–UC group the majority of blood capillaries, venules and veins were dilated and a vascular plethora was seen. Erythrocytes in the lumen of vessels often formed aggregates. The excretory ducts of various calibers were dilated, proliferation of the ductal epithelium was visible (Figure 3).

Changes in the acinar tissue were observed, namely the zones of different sizes with disorganization of the cytoarchitectonics of the acini (Figure 4). Part of the acini was disorganized completely, with only sporadic changed pancreatocytes or their small groups seen. Part of the acini was destroyed partially. The cytoplasm of the zymogenic pole of pancreatocytes was unevenly stained, and areas of less intense staining were found within it. Destruction of the apical part of the cell membrane and the apical part of the cytoplasm was observed in some of these pancreatocytes. In numerous cells, the zymogenic zone of the cytoplasm was not determined at all. All of these changes could be attributed to a decrease in the synthesis of digestive enzymes. Pancreatic nuclei were often definitely enlarged, occupying a central position; perinuclear edema was registered (Figure 5).

In 50 % of the rats of the CP–UC group, the above-described changes in the cytoarchitectonics of the acini were complicated by signs of necrosis of the acinar tissue and focal edema of the inter-acinar stroma, its inflammatory infiltration and fibrosis (Figure 6). At the same time, no recognizable changes were observed within the incretory apparatus.

Against the background of the tested drug at a dose of 30mg/kg, the typical acinar structure of the pancreas was only partially maintained. Whole zones in different parts of the lobules were characterized by the destruction of the cytoarchitectonics of the acini and the dystrophic changes. At the same time, unlike the rats, which did not receive the drug, the beforenamed changes took the form of the cells dissociation, disorganization, uneven staining, or decrease/absence of the zymogenic zone of the cytoplasm, unclear vacuolization, rather than distinct destruction (that was seen in the samples of the animals from the CP–UC group). As a rule, the nuclei of pancreatic cells were characterized by the normal volume, nevertheless, the signs of perinuclear edema were sometimes visible (Figure 7). In one of the samples from this group, focal necrosis of acinar tissue with inflammatory infiltration and mild fibrotic changes was observed (Figure 8).

After the treatment with the studied drug at a dose of 60 mg/kg, the typical acinar structure of the exocrine component of the pancreas was, in general, maintained. Deformation and degradation of acini were not observed. Loss of zymogen granules or reduction of this part of the cytoplasmic zone was observed within certain groups of pancreatic cells in a minor part of the acini (Figure 9). In one sample, an increase in the intensity of the fibrotic changes in the periportal areas was detected after picrofuxin staining by Van Gieson (Figure 10).

Under the conditions of CCAA administration at the highest studied dose, the normal, typical acinar structure of the exocrine component of the pancreas of rats was practically restored in all samples. Only in the minor isolated areas, there were pancreatocytes with uneven staining or a certain reduction of the zymogenic zone, with very moderate vacuolization of the cytoplasm being present (Figure 11). In the samples stained with picrofuxin by Van Gieson, no changes were registered in the state of the interlobular/interacynar, periportal connective tissue (Figure 12).

Figure 1: Photomicrographs of the pancreas of the intact rat.

а – unchanged cells within the acini (× 250); b – the central eosinophilic zone of the acinar cells is evenly filled with zymogen grains (× 400). Hematoxylin and eosin staining.

Figure 2: Photomicrograph of the pancreas of the intact rat.
A small amount of connective tissue surrounding the interlobular blood vessel and bile duct. Picrofuxin staining by Van Gieson.
× 400.

Figure 3: Photomicrographs of the pancreas of the rat with alcohol-induced chronic pancreatitis (CP–UC group). Dilated blood capillaries, vascular plethora, proliferation of the ductal epithelium (a), dilation of the excretory duct (b). Hematoxylin and eosin staining. × 200.

Figure 4: Photomicrograph of the pancreas of the rat with alcohol-induced chronic pancreatitis (CP–UC group). Disorganization of the cytoarchitectonics of the acini of the entire lobe. Hematoxylin and eosin staining. × 250.

Figure 5: Photomicrographs of the pancreas of the rat with alcohol-induced chronic pancreatitis (CP–UC group). Destruction of the acini of different severity (a–b), significant in the volume of nuclei, perinuclear edema (c). Hematoxylin and eosin staining.
a–b – × 400, c –× 600.

Figure 6: Photomicrographs of the pancreas of the rat with alcohol-induced chronic pancreatitis (CP–UC group):
a – significant swelling and inflammatory infiltration of the interacinar stroma; b–d – signs of necrosis of different severity within the acinar tissue, with an inflammatory reaction and elements of fibrosis. a–b – hematoxylin and eosin staining,
c–d – picrofuxin staining by Van Gieson. × 200.

Figure 7: Photomicrographs of the pancreas of the rat with alcohol-induced chronic pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid at a dose of 30 mg/kg (CP+30 group). a – typical acinar structure partially maintained (× 250); b – dissociation, disorganization of pancreatic cells within the acini, uneven staining, decrease /absence of the zymogenic zone of the cytoplasm (× 400). Hematoxylin and eosin staining.

Figure 8: Photomicrograph of the pancreas of the rat with alcohol-induced chronic pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid at a dose of 30 mg/kg (CP+30 group). The signs of the focal necrosis of acinar tissue, inflammatory reaction and mild manifestations of fibrosis. Picrofuxin staining by Van Gieson.
× 250.

Figure 9: Photomicrographs of the pancreas of the rat with alcohol-induced chronic pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid at a dose of 60 mg/kg (CP+60 group).
a – restoration of the typical acinar structure in most areas observed within the glandular tissue (× 250); b – loss of zymogen granules or reduction of this cytoplasmic zone in certain groups of pancreatic cells of a minor part of the acini (× 400). Hematoxylin and eosin staining.

Figure 10: Photomicrograph of the pancreas of the rat with alcohol-induced chronic pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid at a dose of 60 mg/kg (CP+60 group). Intensified fibrosis signs in the periportal areas Picrofuxin staining by Van Gieson. × 250.

Figure 11: Photomicrograph of the pancreas of the rat with alcohol-induced chronic pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid at a dose of 120 mg/kg (CP+120 group).
a – restoration of the typical acinar structure in most areas observed within the glandular tissue (× 250); b – single pancreatocytes with uneven staining or a certain reduction of the zymogenic zone, with very moderate vacuolization of the cytoplasm (× 400). Hematoxylin and eosin staining.

Figure 12: Photomicrograph of the pancreas of the rat with alcohol-induced chronic pancreatitis receiving the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid at a dose of 120 mg/kg (CP+120 group). Normal structure of the interlobular connective tissue. Picrofuxin staining by Van Gieson. × 250

CONCLUSIONS:

1. The model of chronic alcohol-induced pancreatitis was induced in rats by a combination of long-term administration of alcohol and LPS (standard Lieber–DeCarli isocaloric alcohol liquid diet for 10 weeks + LPS administration once a week for the three last weeks), and there was clear evidence of the impairment of the cytoarchitectonics of the pancreas, disorders of the prooxidant-antioxidant balance, exocrine insufficiency.

2. The coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid administered during 21 days (beginning from the 8^th week of the diet modification, after the first injection of LPS) at doses of 30, 60, and 120 mg/kg intragastrically, was able to normalize the prooxidant-antioxidant balance markers in blood serum and pancreatic tissue. The normalizing influence on SOD activity was especially significant, and the values approached the level of the intact animals, being not dose-dependent.

3. The dose-dependent normalizing effect was registered in regard to the non-specific inflammatory markers, pancreatic exocrine function, and cytoarchitectonics of the pancreas, as well as on the sum of the changes of prooxidant-antioxidant balance markers, with the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid being the most active at a dose of 120 mg/kg intragastrically.

4. The results substantiate broadening the use of the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid, namely the further inclusion of chronic pancreatitis to its indications list. The data also substantiate the dose choice for further clinical investigations and emphasize SOD activity as the possible target point for the studied drug activity.

Funding:

The research was conducted as a part of a scientific study of the coordinative compound of aluminium and N-(2,3-dimethylphenyl)-anthranilic (mefenamic) acid (No. SA/F-01/21, 2021/2022), carried out at the expense of the JSC “Farmak.”

Conflict of interest:

This funding source had no role in the design of this study, neither it had an influence during its execution, analyses, interpretation of the data, or decision to submit results.

Ethics approval:

Bioethics committee approval is reported in the Materials and Methods section.

REFERENCES:

1. Beyer G. Habtezion A, Werner J, Lerch MM, Mayerle J. Chronic pancreatitis. Lancet. 2020; 396(10249): 499-512. doi: 10.1016/S0140-6736(20)31318-0.

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Received on 31.10.2023 Modified on 30.12.2023

Research J. Pharm. and Tech 2024; 17(6):2531-2540.

DOI: 10.52711/0974-360X.2024.00396

Group	Thiobarbituric acid reactive substances, µmol/l	Conjugated dienes, µmol/l	Superoxide dismutase activity, IU/ml	Catalase, IU/ml
Blood serum
IC	0,133±0,024	0,222±0,009	11,3±0,39	138±3,49
CP–UC	0,394±0,043 ^***	0,351±0,012 ^***	8,83±0,47 ^***	65,5±11,6 ^***
CP+30	0,282±0,032 ^*	0,298±0,015 ^**	11,3±0,51 ^###	72,7±10,6 ^***
CP+60	0,226±0,027 ^##	0,291±0,012 ^** ^#	11,2±0,21 ^###	72,8±11,8 ^***
CP+120	0,207±0,020 ^###	0,269±0,016 ^###	11,0±0,24 ^##	80,1±6,22 ^***
Pancreatic tissue
IC	0,252±0,027	0,441±0,025	142±4,98	1703±63,3
CP–UC	0,465±0,050 ^***	0,699±0,020 ^***	107±5,03 ^***	912±47,8 ^***
CP+30	0,296±0,018 ^#	0,634±0,024 ^***	127±5,09 ^#	1135±54,6 ^***
CP+60	0,242±0,037 ^###	0,518±0,020 ^###	132±3,53 ^##	1160±47,9 ^***
CP+120	0,253±0,031 ^###	0,494±0,027 ^###	134±4,68 ^##	1310±58,1 ^*** ^#

Group	Total glutathione, µmol/l	Oxidized glutathione, µmol/l	Glutathione reductase, activity, IU/l	Glutathione-S-transferase, activity, IU/l	Glutation peroxidase, activity, IU/l
Blood serum
IC	19,2±0,73	2,44±0,07	9,43±0,90	0,084±0,004	336±3,84
CP–UC	13,0±1,01 ^***	1,98±0,29	8,19±0,74	0,060±0,002 ^***	304±15,7
CP+30	15,0±0,92 ^**	2,08±0,26	8,04±0,70	0,074±0,002 ^##	325±28,6
CP+60	14,1±0,51 ^**	1,74±0,21	13,4±2,25	0,067±0,003 ^**	295±24,3
CP+120	15,6±0,80 ^*	1,63±0,24	17,6±2,36^**##	0,074±0,002 ^##	320±23,2
Pancreatic tissue
IC	172±8,59	15,0±0,89	35,6±5,45	0,621±0,033	2507±336
CP–UC	77,6±5,45 ^***	15,7±0,82	26,3±4,92	0,552±0,019	674±114 ^***
CP+30	81,2±4,63 ^***	17,0±1,17	29,4±4,64	0,470±0,010 ^***	973±54,1^***
CP+60	126±7,27^***###	14,7±2,05	26,3±4,33	0,606±0,028	1647±336
CP+120	116±11,0 ^* ^##**	17,1±1,81	30,9±7,39	0,414±0,019^***##	1793±255 ^#