THE STUDY OF CELL HOMEOSTASIS STATE OF THE GASTRIC MUCOSA OF RATS ON MODEL OF RHEUMATOID ARTHRITIS, TREATMENT WITH IBUPROFEN AND ITS COMBINATION WITH VINBORON

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Recently, the attention of researchers has been payed to the problem of finding out new ways to prevent and treat the side effects of non-steroidal anti-inflammatory drugs (NSAIDs). This is primarily due to the fact that the diversity of the pathogenesis gastropathy induced by non-steroidal antiphlogistics, justifies the implementation of a comprehensive drug therapy or the use of drugs with polytropic pharmacological properties [1, 2, 3, 4]. Among the side effects of NSAIDs, the biggest part takes the toxic effect on the digestive system. At the same time, the attention of researchers is increasingly attracted by specific syndrome associated with taking this group of drugs - NSAIDs gastropathy, which morphological substrate may be necrosis of epithelial cells. However, as shown by recent studies, necrosis is not the single, but also is not the main cause of cell death. Apoptosis, the programmed cell death, is observed more often than necrosis. (Vladimirov V. Yu., 2002). One of the most important features of apoptosis is the activation of serine proteinases, called caspases, which trigger a cascade of biochemical reactions, ending the destruction of many proteins of the cytoplasm and nucleus, defragmentation of nuclear DNA and making a signal to phagocytes that a cell is to liquidate [5, 6]. Furthermore, ulcerogenesis problem is closely related to the ability of the epithelium of the gastric mucosa (GM) for adequate regeneration accompanied by increased aggressiveness of gastric juice induced by intake of NSAIDs [4]. Unfortunately, the known ways of reduction of gastrotoxicity NSAIDs, namely the use of proton pump inhibitors, H -histamine blockers, PG E synthetic analogues can`t satisfy the needs of clinicians fully [1, 7]. Among the existing NSAIDs we chose derivative of propionic acid - ibuprofen. Today, it is exhibited for treatment of children older than 6 months, as the over-the-counter medication (OTC) and of the age of 3 months - on the recommendation of a doctor, which means the lower toxicity of the drug and a minimum amount of adverse reactions [8]. However, with prolonged use (more than 7-14 days), even at low doses of ibuprofen (800 mg/day) lesions of the gastrointestinal tract may be caused [9]. In order to prevent the development of ibuprofen-induced gastropathy the new Ukrainian drug vinboron caught our attention, which favorably combines a wide range of pharmacological properties: antispasmodic, anti-inflammatory, analgesic (local and central), anti-ischemic, antioxidant, antiplatelet, immunomodulatory, antimicrobial, tocolytic, cardioprotective, cerebroprotective, stimulating effect on the microcirculation [2, 10]. Order of the Ukrainian Ministry of Health from September 1, 2014 no. 665 “About state registration (re-registration) of medicinal products (medical immunobiological preparations) and amendments to the registration materials” (Addition 2) approved vinboron for use in the clinic as a spasmolytic with the local anesthetic effect at a local dysfunction of the gallbladder and sphincter of Oddi (hypokinetic and hyperkinetic types), cholecystitis, gallstones, irritable bowel syndrome, gastric ulcer and duodenal ulcer with pain, spastic nature, as part of comprehensive treatment of disorders of cerebral circulation, as well as in chronic coronary insuffiand flow angina. The drug can be also administered to patients with impaired hepatic function, as it has its hepatoprotective properties [11]. In earlier studies we found out that 42.8% of the animals with adjuvant arthritis (AA) who used ibuprofen at 218 mg/kg, on day 28 of the experiment revealed erosive and ulcerative lesions of the coolant. In addition ulcerative index of (UI) ibuprofen monotherapy group (0.97) is almost 10 times higher than the combined pharmacotherapy group UI AA ibuprofen and vinboron (0.10) [2]. Objective: to characterize the proliferative activity (nuclear label Ki-67) and the process of apoptosis (cytoplasmic or perinuclear label CPP32) epithelial coolant rats with AA ibuprofen monotherapy and its combination with vinboron according to immunohistochemistry (IHC) studies. Materials and methods. The study was carried out using 28 adult male rats, divided into 4 groups: I - intact rats (n=7), II - rats with modeled AA untreated (control), III - rats with AA (n=7) treated with ibuprofen (218 mg/kg, intragastric), IV - rats with AA (n=7) treated intragastrically with ibuprofen (218 mg/kg) in combination with vinboron (11 mg/kg, intragastric). All experimental studies on laboratory animals were performed in accordance with the requirements of Good Laboratory Practice (GLP) and compliance with the guidelines of the Council of Europe Convention on the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of March 18, 1986, the Directive of the European Parliament and of the Council EU 2010/63/EC of September 22, 2010 on the protection of animals used for scientific purposes; order by Ukrainian Ministry of Health from December 14, 2009 no. 944 “On approval of pre-clinical studies of medicinal products and the expertise of pre-clinical study of medicinal products materials” and the Law of Ukraine on February 21, 2006 no. 3447- IV “About the protection of animals from cruelty” [12]. AA modeled by subgaleal administration of complete Freund’s adjuvant (Sigma, USA. Composition: 1 part of lanolin, 2 parts of paraffin oil and killed Bacillus Calmette-Guerin vaccine in dose of 5 mg/ ml) in the right hind paw of 0,1 ml per rat [12]. AA treatment was carried out from 14 to 28 days by intragastric administration of study drugs. The start of drugs administration meets the maximal inflammatory response [3, 12]. Euthanasia of the rats and fence material out of its fixation in 10% neutral formalin was performed on day 28 of the experiment by cervical dislocation under ether anesthesia. Conversion of drugs doses for human in rats was carried out using species sensitivity factor Yu. G. Rybolovlev [7, 12]. Immunohistochemical studies were performed on paraffin sections using streptavidin-biotin method (“DAKO”, Denmark; LSAB2 Systems, HRP). Antigen unmasking was performed in citrate buffer pH 6.0. Mice’s and rabbit’s monoclonal antibodies were used as the primary antibodies. The cell nuclei were tinted with Mayer’s hematoxylin for 15-60 seconds. [13, 14]. Proliferative activity of cells was assessed using murine monoclonal antibody to nuclear antigen Ki-67 (“DAKO”, MIB-1 clone, Denmark) as a sensitive marker of proliferation by T. Scholzen [15]. The proliferation index (nuclear tag Ki 67) and apoptotic index (or perinuclear cytoplasmic label CPP32 - caspase-3) were measured in the formulations at 400-fold magnification in 5 randomly selected visual fields (≥500 cells) as the percentage of positively stained nuclei of epithelial cells of the coolant in the three compartments (I - patching and surface epithelium; II - isthmus area, III of - iron-based, middle and lower third of the glands in the basal). To assess CPP32 expression (caspase-3) in the coolant in similar areas used semiquantitative rating scale color intensity of 0 (none) - no positive reactions in cells, 1 (weak) - up to 30% of the cells re- acted positively, 2 (moderate) - 31-60%, and 3 (strong) - 60% more stained cells and [16, 17, 18]. Immunohistochemical study was carried out on the basis Vinnitsa regional Office of Pathological anatomy (Vinnitsa, Ukraine). Microscopy and photography of histological preparations was performed using a light microscope OLIMPUS BX 41 at magnifications of 40, 100, 200 and 400 times. The photographs were processed, undergone morphometry and were statistically analyzed using Quick PHOTO MICRO 2.3 program. The contents of cellular elements were determined per unit of the convention- al area (1 mm2). Morphometric studies were guided by the basic principles laid down in the manual G. G. Avtandilov (2002) [1]. Results and discussion. To assess cell renewal coolant as a proliferation marker, Ki 67 nuclear antigen was selected because it was registered in all active phases of the cell cycle (G1, S, G2 and M), but not in the rest ing phase (G0) [5, 17]. Immunohistochemical analysis of expression showed that on 28th day of the experiment, proliferation index in the intact group (PI) was the highest, and the expression of Ki-67 was observed at the level of the zone of typical proliferative compartment coolant and dominated it in the germinal zone and sometimes extended to patching the epithelium (Fig. 1). PI in the control group was reduced by 5% relative to intact animals, indicating the systemic changes in rats with AA. Ibuprofen monotherapy was characterized by a low expression of Ki-67 (Fig. 2), which corresponded to statistically significant decrease in the PI by 36% compared to the same period of the control group animals. We have identified, that the expression of Ki-67 in the depth of the coolant pits can be regarded as a sign of the formation of immunohistochemical NSAID-gastropathy, which coincides with the literature (Lauwers G.Y. et al., 2001). The above mentioned changes indicate that the coolant damages from the application of ibuprofen lead to changes in the life cycle of delay and epithelial differentiation, and thus to disruption of cell renewal coolant and its structural disorganization. In the group of combined use of ibuprofen and vinboron positive Ki-67 labeling was detected in the zone of the isthmus, and the basal gastric glands (Fig. 3). PI group combined pharmacotherapy (0.081±0.004) exceeded the value of ibuprofen monotherapy group by 62%, which is compared with virtually intact animals (0.082±0.004). This indicated the capacity to neutralize the antiproliferative properties of vinboron ibuprofen, which in turn led to the recovery of the regenerative epithelium coolant properties. These changes in proliferative activity of the combined use of ibuprofen and vinboron coincide with established in previous studies concerning the reduction of ulcer index ibuprofen mono- therapy group [2]. An important role in the maintenance of tissue homeostasis given to maintain the balance between the processes of proliferation and apoptosis. To examine the apoptosis of epithelial cells in the coolant as a marker has been selected caspase-3 (CPP32, Novocastra), in both the effector caspase cascade involved in apoptosis. Its activation means irreversible cell adherence by programmed death. We have found out that the in monotherapy ibuprofen group the caspase-3 expression was observed mainly in the fold and pit epithelium adjacent to the acute ulcers and erosions. The number of positively stained cells was more than 60% (Fig. 4) and was significantly higher in comparison with the group of combination drug therapy (p<0,05). Caspase-3 expression ranged from 10 to 30% in the group of combined use of ibuprofen and vinboron as in the group of intact animals (Fig. 5). Reduction of the caspase-3 expression in the combined use of drugs above indicates the ability to weaken vinboron ibuprofen-induced apoptosis of epithelial cells of the coolant. It should be noted that the decrease in the level of apoptotic cells does not carry the threat of carcinogenesis. Thus, according to the literature (Ivashkin V.T. et al., 2002), reduction of apoptosis in lesions of coolant is an indicator of the effectiveness of treatment. The reason for reduction of the number of apoptotic cells in combination therapy with ibuprofen with vinboron is probably a combination of several complementary factors, one of which is the presence of vinboron anti-inflammatory and immunomodulatory properties as inflammation regarded as an additional pathway of activation of apoptosis, besides the effects of directly stimulating programmed cell death (Kononov A.V. et al., 2005). Another possible reason for reduction of the level of apoptotic cells in the correction gastrotoxicity vinboron ibuprofen may be the antioxidant properties of the latter, since, according to the generally accepted ideas proapoptotic effect of NSAIDs can be realized through the activation of free radical oxidation (Fukumoto K. et al, 2011;. Tamaki K. et al, 2011). and antioxidants, having gastroprotective effect, reduce the level of apoptosis (Dursun H. et al, 2009;. Suleyman H. et al, 2009.). Conclusions. Analysis of the results of immunohistochemical studies indicates the ibuprofen property to suppress proliferation activity of gastric epithelial cells and induce apoptosis, as indicated by activation of caspase-3. Above mentioned changes lead to disruption of coolant capacity for adequate recovery. Gastroprotective effect of vinboron in terms of ibuprofen is conditioned by the increase in the proliferative capacity of the restoration of the regenerative properties of the gastric epithelium. Besides, vinboron is able to inhibit apoptosis induced by ibuprofen monotherapy epithelial cells of the stomach, which helps to maintain cellular homeostasis coolant.

About the authors

F. V Hladkykh

Vinnitsa National Pirogov Memorial Medical University

Email: fedir.hladkykh@gmail.com
Vinnitsa, Ukraine

N. H Stepaniuk

Vinnitsa National Pirogov Memorial Medical University

Vinnitsa, Ukraine

S. V Vernihorodskyi

Vinnitsa National Pirogov Memorial Medical University

Vinnitsa, Ukraine

References

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