Pramipexole Exerts Beneficial Effects in a Rat Model of Acetic Acid-Induced Colitis via Modulating Inflammation

Abstract

Background:

Inflammatory bowel disease (IBD) is a serious public health problem worldwide. The existing therapy options for IBD are limited and can cause severe difficulties, and thus require more research on alternative therapeutic techniques. Pramipexole is a dopamine receptor agonist with anti-inflammatory effects that was recently discovered. Given the importance of dopaminergic pathways in ulcerative colitis inflammation, we tested pramipexole’s efficacy in a rat colitis model in this study.

Materials and Methods:

Colitis was induced by administering 3% acetic acid intrarectally. Rats were randomly assigned to one of six groups: normal, colitis control, dexamethasone (1 mg/kg; i.p.), and pramipexole (0.25, 0.5, and 1 mg/kg; i.p.). In intestinal samples, macroscopic and microscopic lesion ratings, pro-inflammatory cytokine levels (tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta), and myeloperoxidase (MPO) activity were evaluated.

Results:

Compared to the colitis control group, pramipexole (0.5 and 1 mg/kg) substantially reduced macroscopic and microscopic intestinal damage, pro-inflammatory cytokine levels, and MPO activity. Furthermore, the indices mentioned above were considerably lower in the dexamethasone treatment group compared to the colitis control group.

Conclusions:

Our findings indicate that pramipexole has favorable benefits in treating experimental colitis; however, further research is required to determine its clinical value as an IBD therapeutic agent.

INTRODUCTION

Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) characterized by mucosal and submucosal inflammatory alterations in the colon and extra-intestinal symptoms. UC produces a variety of gastrointestinal (GI) symptoms, such as stomach discomfort, cramps, bloody diarrhea, and fever.[1] The incidence of the disease is influenced by genetic makeup, environmental circumstances, and the gut microbiota, but none of these variables are sufficient to cause it.[2] The pathophysiology of IBD is yet unknown; however, it seems that an increase in pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and a decrease in anti-inflammatory cytokines play essential roles in mediating inflammation in the illness.[3,4] Currently, popular treatments for UC include aminosalicylates and corticosteroids. Alternative therapy includes immunosuppressive drugs and bioresponse modulators.[5] Despite recent advances in treating UC, no definitive solution is available. Furthermore, existing medications have several adverse effects, including immunosuppression, increased risk of infection, GI disturbances, and long-term metabolic complications.[6] Consequently, more research is required to develop effective medications with fewer side effects and greater efficacy.

Dopamine (DA) is a catecholamine neurotransmitter widely generated in the central nervous system and certain peripheral regions such as the GI, cardiovascular, and renal systems.[7] This neurotransmitter is generated in the GI tract by the enteric nervous system and non-neuronal cells such as intestinal epithelial cells, immune cells, and bacteria.[8] It has also been discovered that DA may affect neutrophil, monocyte, and lymphocyte activities by activating DA receptors.[9,10] The interaction of the DA system and immune cells makes DA receptors a possible target for immunological-mediated illnesses. Because deficiencies in the dopaminergic system and immune system are linked to some of the most severe neurological diseases, such as Parkinson’s disease, depression, and schizophrenia, the influence of DA on the immune system becomes even more fascinating.[11,12] Multiple lines of evidence point to the central and peripheral dopaminergic systems, particularly D2 receptors, involved in IBD pathophysiology.[13,14] L-3,4-dihydroxyphenylalanine (L-DOPA) and DA levels were lower in the colon mucosa of patients with IBD.[15] Magro et al.[15] discovered an inverse connection between inflammation and tissue DA levels in experimental colitis. They found that the decrease in DA levels was presumably due to the inhibitory action of Interferons on L-DOPA absorption in GI epithelial cells. Furthermore, Brudek et al.[16] observed that the risk of Parkinson’s disease is more significant in patients with IBD, which could be attributed to degradation of the dopaminergic system. It is worth noting that DA receptors are widely expressed in the colon, with D1 and D5 receptors found primarily in the epithelial layer and D2 and D4 receptors found primarily in the lamina propria layer.[8] The protective impact of DA agonists on intestinal mucosa has been established, although the mechanism of this protective action is yet unknown.[17] The fact that DA increases mucus production by colonic goblet cells through a D5 receptor-mediated route may explain this. Another potential mechanism is that dopaminergic agonists exert vasodilatory effects, enhancing GI blood flow. This improved perfusion may contribute to the protective effects against mucosal injury.[18] As a result, it seems that DA is a vital regulator of GI mucosal function.

Pramipexole, a non-ergot DA receptor agonist, is an effective drug for the treatment of Parkinson’s disease and other neurodegenerative motor disorders.[19] All DA receptors interact with pramipexole; however, it has a 7-fold greater affinity for D3-receptors than for D2-receptors.[18,20] Following different chemically induced inflammation tests, pramipexole’s anti-inflammatory efficacy has recently been proven; however, the specific mechanisms behind its anti-inflammatory capabilities are still unknown.[21]

To our knowledge, no research has been done on the possible health benefits of pramipexole for UC. It seems that pramipexole’s anti-inflammatory qualities could justify investigating how it affects UC. The current research aimed to evaluate pramipexole’s ability to reduce colitis inflammation in an experimental IBD model.

MATERIALS AND METHODS

Animals

Thirty-six adult male Wistar rats (250 ± 20 g, 12-week-olds) were provided for this study. The animals were randomly divided into six groups of six rats each, housed per cage with free access to food and water, under standard conditions with a 12/12 h light-dark cycle and a constant temperature of 22°C.

Chemicals

We bought dexamethasone and pramipexole from Sobhan Darou Co. (Tehran, Iran). The following substances were purchased from Sigma-Aldrich Corp.: bovine serum albumin, benzethonium chloride, hexadecyltrimethylammonium bromide (HTAB), aprotinin A, ethylenediaminetetraacetic acid (EDTA), o-dianisidine dihydrochloride, and phenylmethylsulfonyl fluoride (St. Louis, MO, USA). We purchased glacial acetic acid, diethyl ether, and 35% formaldehyde solution from Merck (Darmstadt, Germany). TNF-α, interleukin-1 beta (IL-1β), and IL-6 concentrations in the rat’s colon were determined using ELISA kits (ZellBio, GmbH, Germany).

Grouping

Six groups of rats were created at random: group I (control group): the rats received a 0.9% solution of sterile saline intrarectally rather than acetic acid; group II (colitis control group): normal saline was administered i.p. 24 hours before colitis induction; group III (dexamethasone treatment): dexamethasone (1 mg/kg, i.p.) was given 24 hours before colitis induction; groups IV–VI (pramipexole treatment): pramipexole was administered at doses of 0.25, 0.5, and 1 mg/kg (i.p.) 24 hours before colitis induction in groups IV, V, and VI, respectively. Daily therapy continued for 4 days. Earlier research was used to define the drug dosage and sample size.[21,22]

Induction of colitis

A technique developed by Mahdavi et al.[23] was used to induce colitis with minor modification. Before induction of colitis, animals fasted for 36 hours with unlimited access to water. Rats were lightly anesthetized intraperitoneally using ketamine/xylazine (60/5 mg/kg). A polyethylene catheter was placed 8 cm distally to the anus and used to give 2 mL of acetic acid (3% v/v) intrarectally. Rats with unrestricted access to food and water returned to their cages after inducing colitis. Instead of giving the normal group an acetic acid enema, 2 mL of normal saline was used.

Macroscopic assessment

All animals were weighed daily, and the percentage of body weight lost was determined. Three days after inducing colitis, the animals were euthanized via CO₂ asphyxiation. The weight/length ratio (mg/cm) was assessed from the first 8 cm of the distal colon (from the anus), along with the ulcer area and percentage of necrosis after removal of the distal colon. Furthermore, the degree of macroscopically visible colon injury was graded using our methodology discussed previously.[24] After that, the tissues were cut into four equal pieces. The surviving fragment was preserved in a 10% formalin solution for histopathologic study, while the other three parts were immediately frozen in liquid nitrogen for biochemical investigation.

Microscopic assessment

Rat colon tissues were preserved in a 10% formalin solution for 24 hours. They were then put into a 70% ethanol solution. All materials were dehydrated, paraffin-embedded, and sectioned into 4-m-thick pieces. A pathologist blind to the experimental groups evaluated them histologically after staining them with hematoxylin and eosin. Furthermore, the total colitis index was calculated using the Cooper et al.[25] and Dieleman et al.[26] The technique was determined by adding three scores for the severity, the extent of the inflammation, and the degree of crypt destruction.

Measurement of myeloperoxidase activity in rat colon

The modified technique published by Bradley et al.[27] was used to quantify the myeloperoxidase (MPO) activity, a sign of neutrophil infiltration into the intestinal mucosa during acetic acid-induced colon lesions. The MPO activity rate was calculated and expressed as units per 100 mg of the moist weight of the tissue sample.

Determination of cytokine in the rat colon

Based on our previous study, levels of IL-1β, IL-6, and TNF-α in colon tissue were measured by ELISA assay.[24]

Statistical analysis

SPSS statistics software was used to perform the statistical analysis (version 17). The mean ± standard error of the mean (SEM) was used to represent all data. One-way analysis of variance (ANOVA), followed by the Tukey post-hoc test, was used to compare the groups. The Kruskal-Wallis and post-hoc Mann-Whitney U tests were used to assess nonparametric data. Statistical significance was defined as a P value of < 0.05.

RESULTS

Pramipexole reduced the percentage of body weight loss and macroscopic injuries in rat colon tissue with acetic acid-induced colitis

According to Table 1, the rats in the colitis control group lost weight significantly more than the rats in the normal group 3 days after colitis was induced (P < 0.001). Although the animals treated with dexamethasone and pramipexole (0.5 and 1 mg/kg) showed significant weight loss compared to the normal group, their body weight loss percentage was significantly lower than that of the acetic acid-treated group (P < 0.01, P < 0.05, and P < 0.01, respectively).

Table 1.

Effects of pramipexole on weight changes and macroscopic and histological rat colon injuries in acetic acid-induced colitis

Group		Body weight loss after 3 days (%)		Colonic weight/length ratio (mg/cm)		Ulcer severity (0–15)		Total colitis Index (0–10)		Ulcer area (cm2)		Necrosis (%)
Normal		−3.8±1.0***		95.8±4.9***		0±0***		0±0***		0±0***		0±0***
Colitis control		9.4±0.8		268.3±14.0		10.7±0.7		8.8±0.40		5.7±0.2		46.9±1.4
Dexamethasone		4.8±0.7**		194.2±5.5**		5.7±0.8***		4.7±0.7***		3.2±0.3**		27.4±3.7**
Pramipexole (0.25 mg/kg)		7.7±0.8		236.7±16.5		8.7±1.2		7.0±0.5		4.5±0.56		35.3±2.1
Pramipexole (0.5 mg/kg)		5.6±0.4*		205.0±14.8*		6.7±0.5**		6.2±0.60*		3.5±0.5**		28.0±3.7**
Pramipexole (1 mg/kg)		5.1±0.6**		195.0±12.8**		6.0±0.5**		5.5±0.8**		3.5±0.4**		26.8±5.5**

Values are means±SEM; n=6. Statistical analysis was performed using ANOVA followed by Tukey’s post-hoc test. ***P˂0.001, **P<0.01, and *P<0.05: Significant difference compared to the colitis control group

Table 1 shows the results of the macroscopic evaluations for all treatment groups, including the weight/length ratio of the colon, the severity index of the ulcer, the area of the ulcer, and the percentage of necrosis. Compared to the normal group, the distal colon of the colitis control group had significant bleeding, ulceration, inflammation, necrosis, and an increase in the thickness of the colon wall (P < 0.001). Compared to the colitis control group, animals given pramipexole (0.5 and 1 mg/kg) and dexamethasone had significantly lower levels of the parameters as mentioned above.

Pramipexole reduced histopathological damage in rat colon tissue with acetic acid-induced colitis

As shown in Figure 1, the histological analysis of the colon segments revealed an intact architecture and unaltered epithelium in the colonic mucosa of the normal group. The colitis control group showed widespread necrosis, inflammatory granulomas, submucosal neutrophil infiltration, and/or severe and intense transmural inflammation. Compared to the colitis control group, rats treated with dexamethasone and pramipexole (0.5 and 1 mg/kg) showed a substantial improvement in histological alterations, infiltration of inflammatory cells in the lamina propria, and overall colitis index. In addition, epithelial regeneration of inflammatory colon tissues was observed in these two groups.

Figure 1.

Microscopic presentation of the colon in acetic acid-induced colitis in rats (hematoxylin and eosin staining; original magnification 10×). (a) Normal group: Mucus layer and crypts are normal; (b) Colitis control group: Epithelial destruction, architectural deformity of the crypts, and inflammatory cell infiltrates; (c) Dexamethasone (1 mg/kg): Mild to moderate mucosal and submucosal inflammation and mucosal inflammatory cell infiltrates; (d): Pramipexole (0.25 mg/kg): Destruction of mucosal architecture and neutrophil infiltration, (e and f) Pramipexole (0.5 and 1 mg/kg): Mild to moderate mucosal and submucosal inflammation and mucosal inflammatory cell infiltrates

Pramipexole decreased MPO activity in rat colon tissue with acetic acid-induced colitis

As shown in Figure 2, the activity of MPO in the colitis control group was significantly higher than in the normal group. Compared to the colitis control group, MPO activity was considerably decreased in the groups treated with dexamethasone and pramipexole (0.5 and 1 mg/kg) (P < 0.001, P < 0.05, and P < 0.01, respectively).

Figure 2.

Effects of the pramipexole (0.25, 0.5, and 1 mg/kg, i.p.) or dexamethasone (1 mg/kg, i.p.) on biochemical parameters of the rat colon in acetic acid-induced colitis. MPO = myeloperoxidase; TNF-α = tumor necrosis factor alpha; IL-6 = interleukin-6; and IL-1β = interleukin-1 beta. Data are presented as mean ± SEM, n = 6. Statistical analysis was performed using ANOVA followed by the Tukey post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the colitis control group

Pramipexole decreased pro-inflammatory cytokines in rat colon tissue with acetic acid-induced colitis

TNF-α level in the colon segment of the colitis control group increased considerably compared to the normal group, as seen in Figure 2 (P < 0.001). Compared to the colitis control group, the treatment with pramipexole (0.5 and 1 mg/kg) and dexamethasone significantly reduced the levels of this cytokine (P < 0.01, P < 0.01, and P < 0.001, respectively).

Figure 2 shows a significant difference between the colonic IL-6 level of the colitis control group and the normal group (P < 0.001). Compared to the colitis control group, animals given pramipexole (0.5 and 1 mg/kg) and dexamethasone had significantly lower levels of colonic IL-6 levels (P < 0.01, P < 0.001, and P < 0.001, respectively). Furthermore, the colonic level of IL-1β increased dramatically in the colitis control group compared to the normal group (P = 0.001). Compared to the colitis control group, the stated parameter was considerably reduced in rats treated with dexamethasone and pramipexole (0.5 and 1 mg/kg) (P < 0.01, P < 0.05, and P < 0.01, respectively)

DISCUSSION

In the current investigation, the anti-inflammatory effects of pramipexole were investigated in various dosages (0.25, 0.5, and 1 mg/kg) in rats with acetic acid-induced colitis. According to macroscopic, histological, and biochemical tests, pramipexole therapy significantly reduced colonic mucosal damage and inflammatory biomarkers and accelerated the healing process in an experimental model of colitis.

The pathophysiology of UC has recently been studied using several experimental animal models. In terms of the pathophysiological pattern and pro-inflammatory mediators involved, the acetic acid-induced UC model and clinical UC have many common characteristics of the disease.[28] Intrarectal administration of acetic acid results in non-transmural inflammation that is notable for having the characteristics of human colitis.[29] This inflammation is characterized by vasodilation, extensive mucosal and submucosal layers necrosis, edema, submucosal ulcers, increased neutrophils, and elevation of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α.

A standard monotherapy or supplementary drug for Parkinson’s disease is pramipexole, a non-ergot DA D2-receptor agonist.[19] Additionally, it is used as the primary therapy for moderate to severe restless leg syndrome.[30] Pramipexole has been shown to exhibit anti-inflammatory properties in three well-studied animal models of inflammation, including carrageenan and formalin-induced paw inflammation in rats and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice.[21] Although Sadeghi et al.[21] reported that pramipexole has anti-inflammatory properties against two phases of carrageenan-induced edema, they could not determine the precise mechanism of its effects. They assumed that pramipexole could modulate the synthesis or secretion of pro-inflammatory mediators during inflammation through unknown mechanisms. In mouse models of 2, 4-dinitrofuorbenzene (DNFB)-induced IBD, bromocriptine, a predominantly D2-receptor agonist, was shown to have a beneficial effect in reducing IBD-associated symptoms such as anorexia and diarrhea, gross and microscopic histological changes and mortality.[31] However, the DA antagonist domperidone made the condition worse. In rat models of UC and IL-10 deletion mice induced by iodoacetamide that spontaneously develop chronic IBD, Tolstanova et al.[13] revealed the beneficial effects of the D2-receptor agonists cabergoline and quinpirole in terms of clinical symptoms and histological characteristics. Downregulation of vascular permeability, avoidance of excessive vascular leakage, and decrease of inflammation were all associated with the action of D2-receptor agonists seen. These data point to the positive effects of D2-receptor agonists on UC in animal models. Furthermore, in both human and laboratory models of UC, neutrophil infiltration into the inflamed intestine increases MPO activity.[32] According to our findings, pramipexole dramatically reduced the level of pro-inflammatory cytokines and the activity of MPO. This medication seems to reduce neutrophil infiltration into the inflamed colon and stop the production and/or release of pro-inflammatory cytokines.

In this study, pramipexole was administered at three different doses, namely 0.25, 0.5, and 1 mg/kg, with a clear dose-dependent effect observed on the reduction of inflammation in the experimental colitis model. The results indicated that as the dose of pramipexole increased, there was a more significant reduction in the severity of colonic inflammation, suggesting that higher doses of pramipexole may be more effective in managing inflammatory processes. However, despite the dose-dependent effect, pramipexole did not show efficacy comparable to dexamethasone, often used as a positive control in similar experimental settings.[33] This difference in potency between pramipexole and dexamethasone is expected, as dexamethasone is a potent corticosteroid with well-documented anti-inflammatory effects. Nonetheless, the dose-dependent effects observed with pramipexole highlight its potential as an adjunct or alternative treatment option. Further investigation into the optimal dosing regimen is necessary to maximize its therapeutic benefits while minimizing potential side effects. Additionally, further research should focus on the underlying mechanisms responsible for the dose-dependent effects of pramipexole, particularly its interaction with the dopaminergic and other receptor pathways involved in inflammation. It should be noted that pramipexole, like some other D2-receptor agonists, may cross BBB,[34] and consequently, both the central and peripheral dopaminergic systems should be addressed to create an advantageous impact. It is important to note that DA agonists may cause side effects such as diarrhea, anorexia, and dyspepsia,[13] which should be considered for patients with a history of IBD. Therefore, exploring the long-term safety and efficacy of pramipexole at higher doses in chronic models of inflammatory diseases would provide valuable insights into its potential clinical application.

CONCLUSION

Based on the dose-dependent effects observed in this study, pramipexole shows promising potential as an adjunctive or alternative treatment for inflammatory conditions such as UC. Although its efficacy was not comparable to dexamethasone, pramipexole’s anti-inflammatory properties warrant further investigation into its clinical application in managing IBD. Additional studies are needed to optimize dosing strategies and explore pramipexole’s long-term safety and efficacy in chronic inflammatory conditions. This suggests that pramipexole may offer a new avenue for the treatment of IBD, especially in patients who may benefit from alternative therapeutic options with potentially fewer side effects than traditional corticosteroids.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Guilan University of Medical Sciences (Ethics No: IR.GUMS.REC.1398.321).

Availability of data and materials

The data are available from the corresponding author upon reasonable request.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

The research was financially supported by the Vice Chancellery of Research of Guilan University of Medical Sciences, Rasht, Iran (Contract Number: 559-279).