Ability of Mexidol to Enhance Antiexudative Effect of Diclofenac Sodium and Etoricoxib in Rats and Mice with Carrageenan-Induced Edema

Nonsteroidal anti-inflammatory drugs (NSAIDs) are some of the most widely used drugs in existence. However, they can cause numerous side effects. Therefore, it is crucial to develop approaches to reducing the severity of their side effects. The purpose of this study is to investigate the effect of mexidol on the anti-inflammatory activity of NSAIDs, such as selective cyclooxygenase (COX)-2 inhibitor etoricoxib and nonselective COX inhibitor diclofenac sodium. Effect of a single oral dose of the drugs was evaluated in rats and mice with carrageenan-induced edema by measuring the severity of edema over a period of 4 and 6 h, respectively. Diclofenac sodium at doses of 1 and 10 mg/kg exhibits a dose-dependent antiexudative effect, the effect of etoricoxib at these doses is comparable, and mexidol at a dose of 25 mg/kg does not exhibit an antiexudative effect. In mice, the effect of a combination of diclofenac sodium at a dose of 1 mg/kg and mexidol at a dose of 25 mg/kg is noninferior to diclofenac sodium at a dose of 10 mg/kg and superior to the effect of diclofenac sodium at a dose of 1 mg/kg. In rats, the combination of diclofenac sodium and mexidol significantly reduces the severity of edema at 4 h after its induction compared with diclofenac sodium at a dose of 1 mg/kg only. In mice, a combination of etoricoxib at a dose of 1 mg/kg and mexidol at a dose of 25 mg/kg is more effective than etoricoxib at doses of 1 and 10 mg/kg. In rats, etoricoxib at a dose of 10 mg/kg and the combination of etoricoxib at a dose of 1 mg/kg and mexidol at a dose of 25 mg/kg have a comparable effect. Mexidol at a low dose (25 mg/kg) enhances the antiexudative effect of diclofenac sodium and etoricoxib. Therefore, a decrease in the dose of NSAIDs in combination with mexidol will help to reduce the side effects of these drugs.


Ability of Mexidol to Enhance Antiexudative Effect of Diclofenac Sodium and Etoricoxib in Rats and
INTRODUCTION Inflammation causes increase in vascular permeability, damage of vascular endothelial cells, intensification of lipid peroxidation (LPO), and activation of membrane phospholipases. Oxidation of low-density lipoproteins leads to the formation of oxidized lipids and lipoproteins with immunogenic and proinflammatory properties [1]; fragments characteristic of oxidized low-density lipoproteins are recognized by macrophage scavenger receptors and are involved in innate immune responses [2].
Under the action of phospholipase A2, fatty acids, in particular arachidonic, and lysophospholipids are formed from phospholipids [3], which, along with the products of free radical oxidation, increase the permeability of cell membranes for ions and water. Tissue damage leads to the induction of cyclooxygenase (COX), which catalyzes the formation of prostaglandins from arachidonic acid that provide the development of an inflammatory reaction [4,5].
COX inhibitors have established themselves as the most widely used anti-inflammatory and pain-relieving drugs. However, both selective inhibitors of COX-2, which plays a major role in the development of inflammation, and nonselective inhibitors of COX, including COX-1, which often initiates the inflammatory process, cause numerous side effects due to inhibition of the production of prostaglandins involved in various physiological functions [6][7][8][9][10][11]. In connection with the above, drugs with antioxidant, membrane stabilizing, and microcirculation normalizing properties are capable of affecting the development of the RESEARCH ARTICLE inflammatory process. One of these drugs is mexidol, developed at the Zakusov Institute of Pharmacology (ethylmethylhydroxypyridine succinate), which has an antioxidant and membrane-stabilizing effect, improves microcirculation and rheological properties of blood, including stabilizing the membrane structures of erythrocytes and platelets during hemolysis [12].
The aim of this work is to study the effect of mexidol on the severity of the anti-inflammatory effect of nonsteroidal, anti-inflammatory drugs (NSAIDs)-a selective COX-2 inhibitor etoricoxib and a nonselective COX inhibitor diclofenac sodium. To achieve this goal, we studied the effect of drugs on carrageenan paw edema in rats and mice, which is a model of acute exudative inflammation accompanied by a significant increase in the expression of COX-2, inducible nitric oxide synthase (iNOS) [13], and the concentration of free radical oxidation products of lipids in damaged tissues [14][15][16].

MATERIALS AND METHODS
We used sexually mature, outbred white male mice weighing 24-29 g and sexually mature, outbred white male rats weighing 245-280 g obtained from the laboratory animal nursery of the "Stolbovaya" branch of Carrageenan paw edema in rats was induced by subplantar injection of 0.1 mL of 1% carrageenan solution (Sigma-Aldrich, United States) into the hind paw of animals [17]. Paw edema was recorded by the difference in the diameter of the paw in the metatarsal area, measured with a caliper (millimeters), 1, 2, 3, and 4 h after the induction of inflammation relative to the diameter before the introduction of the carrageenan solution.
Carrageenan edema of the paw in mice was induced by subplantar injection of 0.05 mL of a 2.5% solution of carrageenan into the hind paw of the animals [13]. Paw edema was recorded by the difference in the diameter of the paw in the metatarsal area, measured with a caliper (millimeters), 1, 2, 3, 4, 5 and 6 h after the induction of inflammation relative to the diameter before the introduction of the carrageenan solution.
Diclofenac sodium (Novartis Pharma Stein AG, Switzerland) was used in doses of 1 and 10 mg/kg; etoricoxib (Merck Sharp & Dohme B.V., Netherlands) was used in doses of 1 mg/kg and 10 mg/kg; Mexidol (NPK PHARMASOFT LLC, Russia) was used at a dose of 25 mg/kg; combinations of diclofenac sodium at a dose of 1 mg/kg and mexidol at a dose of 25 mg/kg, etoricoxib at a dose of 1 mg/kg and mexidol at a dose of 25 mg/kg were used; saline (control group) was administered orally 1 h before the induction of inflammation.
Statistical processing of the experimental results was carried out using the R version 4.0.3 programming language. The normal distribution of the data was checked by the Shapiro-Wilk test, followed by the assessment of the intergroup equality of variances by the Levene's test. In the statistical processing of experimental data, parametric and nonparametric methods of analysis were used: analysis of variance (ANOVA), Student's test, Dunnett's test, Kruskal-Wallis test and Mann-Whitney test. According to the data obtained in both experiments for each experimental group, a graph of the dependence of the change in the edema of the paws of animals on the time recorded, respectively, for 4 h when simulating carrageenan edema in rats and for 6 h when simulating carrageenan edema in mice was plotted. For each graph, the area under the curve (mm 2 ) was calculated using the trapezoidal method. The results in the graphs are presented as mean values; those in Table 1 are as the mean ± the error of the mean (standard deviation). Differences between groups were considered statistically significant at p ≤ 0.05.

RESULTS AND DISCUSSION
Carrageenan edema in rats increased within 3 h after the injection of phlogogen, reaching a maximum equal to an increase in the diameter of the metatarsus of animals by 2.1 mm relative to background values, and decreased by 4 h after induction of inflammation (Fig. 1). The nonselective COX inhibitor diclofenac sodium, when administered orally, had a dose-dependent anti-inflammatory effect. This drug at a dose of 1 mg/kg caused statistically significant reduction of the degree of edema of the paws of rats 1, 3 and 4 h after administration of the carrageenan solution by 28.0, 37.1, and 21.4%, respectively; at a dose of 10 mg/kg, its effect was characterized by a significant decrease in exudation by 52.0, 33.0, 50.5, and 35.9% after 1, 2, 3 and 4 h, respectively, after administration of a carrageenan solution compared to the control group. The areas under the curve for the groups of diclofenac sodium at a dose of 1 and 10 mg/kg were MOSCOW  lower than that of the control group by 27.9 and 42.5%, respectively. Mexidol, when administered orally at a dose of 25 mg/kg, did not reduce the severity of exudative inflammation in rats. Administration of mexidol at a dose of 25 mg/kg in combination with diclofenac sodium at a dose of 1 mg/kg increased the antiexudative efficacy of diclofenac sodium. A significant decrease in the edema of the paw of rats relative to the indicators of the control group was observed throughout the experiment: it was 42.4%1 h after the administration of carrageenan, 34.5% after 2 h, 60.0% after 3 h, and 82.4% after 4 h. This led to a decrease in the area under the curve of changes in carrageenan edema in rats, which were injected with a combination of diclofenac sodium and mexidol, by 49.3% compared with the control group (Fig. 1, Table 1). At the same time, the combination of mexidol at a dose of 25 mg/kg and diclofenac sodium at a dose of 1 mg/kg more noticeably reduced the index of swelling of the paws of rats 4 h after the induction of inflammation compared with the use of diclofenac alone: an increase in the diameter of the metatarsus of animals that received the combination was statistically significant 4.5 times less than with the introduction of the drug at a dose of 1 mg/kg. The areas under the curve in the groups of diclofenac sodium at doses of 1 mg/kg and 10 mg/kg and its combination with mexidol did not differ significantly. Etoricoxib at doses of 1 mg/kg and 10 mg/kg, as well as diclofenac sodium at these doses, reduced the severity of exudative inflammation in rats with carrageenan paw edema; no statistically significant difference in the effectiveness of these NSAIDs was recorded. Unlike diclofenac sodium, no pronounced dose dependence of the antiexudative effect was observed with etoricoxib. Etoricoxib at a dose of 10 mg/kg more strongly reduced the edema of the paws of animals 1 and 4 h after administration of the carrageenan solution than at a dose of 1 mg/kg. With its use, compared with the control group, the decrease in edema was 51.2, 29.5, 33.8, and 36.6% after 1, 2, 3 and 4 h, respectively, after the induction of inflammation and the decrease in the area under the curve was 33.1%. Etoricoxib at a dose of 1 mg/kg, compared with the control group, caused statistically significant reduction of edema 1, 2, and 3 h after administration of carrageenan solution by 24.8, 38.0, and 40.0%, respectively, and the area under the curve by 36.1% (Fig. 2, Table 1). The combination of etoricoxib at a dose of 1 mg/kg with mexidol at a dose of 25 mg/kg more strongly suppressed edema 1 and 4 h after the administration of carrageenan: the difference compared with the control group after 1 h was 58.4% and that after 4 h was 58.8% (Fig. 2). The maximum edema of the paws in mice with carrageenan edema was recorded 2 h after the induction of inflammation and corresponded to an increase in the diameter of the metatarsus of the animals by 1.75 mm; from 3 to 6 h of observation, it gradually decreased to an indicator equal to 0.99 mm (Fig. 3). Mexidol at a dose of 25 mg/kg had no effect on the swelling of the paws of mice. The dose-dependence of the antiexudative effect of diclofenac in this model was observed more clearly than when the drug was administered to rats. Thus, with the use of a nonselective COX inhibitor at a dose of 1 mg/kg, a statistically significant decrease in the edema of the paws of animals was recorded only 1 h (by 30.0%) after the introduction of phlogogen; the area under the curve did not significantly decrease compared with the control group (Fig. 3, Table 1). Diclofenac at a dose of 10 mg/kg caused statistically significant suppression of the swelling of the paws of mice 1, 3, 4, 5, and 6 h after the induction of inflammation relative to the indicators of the control group by 37.1, 34.7, 28.1, 36.1, and 49.5%, respectively, and the area under the curve decreased by 34.7% (Fig. 3, Table 1). Diclofenac at a dose of 1 mg/kg in combination with mexidol at a dose of 25 mg/kg had the most pronounced antiexudative effect: a significant decrease in the diameter of the metatarsus of animals by 42.9-60.6% compared with the control group was recorded throughout the entire experiment, while the area under the curve was 45.2% smaller (Fig. 3, Table 1). At the same time, the effect of the combination of diclofenac and mexidol did not differ from the effect of diclofenac at a dose of 10 mg/kg and statistically significantly exceeded the effect of the drug at a dose of 1 mg/kg from 2 to 6 h of the experiment by 21.3-54.7% (and area under the curve decreased by 36.1%).
Etoricoxib in the model of carrageenan edema in mice, as in the model of carrageenan edema in rats, at doses of 1 and 10 mg/kg caused a comparable antiexudative effect, as evidenced by the equal values of the areas under the curve for these groups. At the same time, the anti-inflammatory effect of the drug at these doses was not as significant as when using diclofenac at a dose of 10 mg/kg. Compared with the control group, when using etoricoxib at a dose of 10 mg/kg, a statistically significant decrease in edema was observed only 2 and 6 h after the introduction of phlogogen by 20.6 and 44.4%, respectively, at a dose of 1 mg/kg: after 1 and 6 h, by 54.3 and 37.4%, respectively, and the area under the curve was lower by 27.6% (Fig. 4, Table 1).
Mexidol (25 mg/kg) enhanced the antiexudative effect of etoricoxib (1 mg/kg) in a model of carrageenan edema in mice: the area under the curve when using this combination of drugs was lower than that of the groups that received only the selective COX-2 inhibitor in both studied doses by 23.8% (Table 1). At the same time, the edema of the paws of mice injected with the combination of drugs was also statistically significantly less pronounced compared with the edema in the group of etoricoxib at a dose of 1 mg/kg 2 (by 40.6%) and 3 (by 27.9%) h after induction of inflammation and compared with edema in the group of   The results obtained indicate the ability of mexidol in a low dose (25 mg/kg) to enhance the anti-inflammatory (antiexudative) effect of the nonselective COX inhibitor diclofenac sodium (1 mg/kg) and the selective COX-2 inhibitor etoricoxib (1 mg/kg) when administered orally on models of carrageenan edema in rats and mice. These results are consistent with our earlier data that the combination of mexidol (100 mg/kg, intraperitoneally) with diclofenac sodium (5 mg/kg, intraperitoneally) has an antiinflammatory effect of the same level as when only this NSAID was administered at a dose of 10 mg/kg [18]. In addition, it is known that the combined use of succinic or lipoic acid, as well as vitamins C and E with a number of NSAIDs (acetylsalicylic acid, indomethacin, naproxen, diclofenac, niflumic acid, butadione, piroxicam) with a 5-day regimen (oral) leads to an increase in their antiexudative efficacy in a model of carrageenan edema in mice [19], and with a single intragastric administration of acetylsalicylic acid (100 mg/kg) in combination with antihypoxant/antioxidant hypoxene (50 mg/kg) there is a decrease in leukocytosis and ESR in the blood of rats with carrageenan edema of the paw [20].
It has been shown that mexidol potentiates the action of not only NSAIDs but also neuropsychotropic drugs [21]. The ability of mexidol to enhance the effect of NSAIDs and neuropsychotropic drugs is determined, first of all, by its antioxidant, membrane stabilizing, and microcirculation normalizing properties. Thus, the antiexudative effect of combinations of mexidol (25 mg/kg) with the nonselective COX inhibitor diclofenac sodium (1 mg/kg) and the selective COX-2 inhibitor etoricoxib (1 mg/kg) with their single oral administration is not inferior to the effect of NSAIDs at a dose of 10 mg/kg, and, therefore, the dose of NSAIDs in combination with mexidol can be reduced by ten times, which will help to reduce the unwanted side effects of these drugs.

FUNDING
The work was carried out within the framework of the State Assignment on the topic, no. 0521-2019-0007.

COMPLIANCE WITH ETHICAL STANDARDS
Conflict of interests. The authors declare that they have no conflict of interests.
Statement on the welfare of animals. The experiments were conducted in compliance with the ethical standards of working with animals and approved by the local ethical committee.