Therapeutic benefits of Indole-3-Carbinol in adjuvant-induced arthritis and its protective effect against methotrexate induced-hepatic toxicity

Rheumatoid Arthritis (RA) is a progressive, chronic, autoimmune disease that is associated with inflammation affecting the lining of the joints, articular cartilage and bones. [1]. Infiltration of several mononuclear cells results in the release of an array of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β). The imbalance between pro and anti-inflammatory cytokines in RA eventually causes the synovial membrane to form a thickened area called pannus that has the ability to invade nearby cartilage and bone [2]. Moreover, the excessive release of cytokines can lead to systemic inflammation which can lead to deleterious consequences on several organs including the liver [3]. The overproduction of pro-inflammatory cytokines stimulates neutrophils and activates macrophages to secrete reactive oxygen species (ROS) in the synovial fluid, which acts as mediators of tissue injury [4].

Currently there is no panacea for RA, but contemporary treatments aim to reduce inflammation, relieve pain (NSAID, corticosteroid) or alter the course of the disease such as disease-modifying antirheumatic drugs (DMARDs) or biologic therapies [5, 6]. Methotrexate (MTX) which is an antimetabolite and a DMARD is generally recognized as the first-line treatment for RA. It is designed in a way that synovitis and systemic inflammation are addressed effectively. However, that efficacy is hindered by the adverse effects it generates on the liver, rendering compliance to the treatment negligible. The toxic effects of MTX are suggested to be due to the accumulation of methotrexate polyglutamates in the liver resulting in the depletion of hepatic folate stores and inhibiting purine and pyrimidine precursor synthesis. Prolonged use of MTX renders the body defenseless against the sustained production of toxic free radical by overwhelming and eventually depleting the antioxidant defense system and in the process aiding in the initiation and progression of hepatotoxicity [6, 7]. Consequently, complementary and alternative natural agents are nevertheless desired and strongly recommended.

Of these natural agents, Indole-3-carbinol (I3C) is of interest. It is a naturally occurring compound found in cruciferous vegetables of the Brassica genus (all species of cabbage plants, black radish, garden radish, and mustard) [8, 9]. Several researches have confirmed that I3C has the following therapeutic actions: cancer chemopreventive [9‐15], anti-inflammatory [9, 13‐16] and anti-oxidant [16, 17]. I3C is usually given in diet or administered as a supplement considering the active constituent is formed under acidic conditions in the stomach. [11‐13]. I3C acts as a potent anti-inflammatory agent by suppressing immune cells infiltration and pro-inflammatory cytokine production such as IL-1β, IL-6 and TNF-α in rodent models of inflammatory diseases [8]. It is one of several vegetable components that might protect against cancer. Considerable evidence shows that I3C inhibits experimentally induced carcinogenesis at different sites in the colon, lung, skin, liver, cervix and mammary gland in mouse and rat models [9, 13]. Furthermore, pre-treatment with I3C can attenuate experimentally- induced oxidative stress [8].

Although I3C possess many potential therapeutic activities, it was never tested on experimental arthritic models or used in conjugation with MTX to examine the therapeutic benefit of such combination. Adjuvant-induced Arthritis (AIA) is a well known experimental model of rheumatoid arthritis [18, 19]. The present study was designed to examine the therapeutic properties of I3C in AIA model on 1) the aberrant inflammation initiated in AIA rat model by testing its anti-inflammatory and anti-arthritic properties in compassion with MTX and 2) on the hepatotoxicity generated by the systemic consequences of arthritis and MTX treatment by assessing its anti-oxidant and hepatoprotective properties.

Induction of arthritis increased significantly the oxidative stress which was evident as an increase in MDA levels when compared to normal rats (P < 0.0001). However, weekly injections of MTX led to further significant increase in MDA levels compared to arthritic non-treated rat group on days 23 (P < 0.001) and 44 (P < 0.0001). Treatment with I3C decreased the MDA levels significantly on day 44 compared to arthritic non-treated model group rats and to the positive control group – MTX (P < 0.0001) with no significant difference when compared to normal rats (P > 0.05). The combination treatment reduced significantly MDA levels compared to the positive control group on day 23 and on day 44 by approximately 3-folds (P < 0.0001) (Fig. 7a).

Arthritic non-treated group rats exhibited compromised anti-oxidant defense system in the form of decreased CAT, SOD activities and GSH levels. Arthritic rats treated with MTX exhibited decreased anti-oxidant defense system (by more than 2 folds) with no significant statistical difference when compared to arthritic non-treated model group rats expect for catalase which was significantly lowered (P < 0.05). Rats treated with I3C and the combination treatment exhibited CAT, SOD activities and GSH levels similar to the normal rat groups on day 44 (P > 0.05) (Fig. 7b, c and d).

This study addressed the therapeutic benefits of I3C on the aberrant inflammation initiated by AIA model in comparison with MTX and its possible hepatoprotective and anti-oxidant effects against arthritis and the prolonged use of MTX. The main findings of this study were that daily administration of I3C for 21 days attenuates inflammation in a manner similar to MTX while alleviating the oxidative stress and hepatotoxicity generated by the prolonged use of MTX and by the systemic consequences of arthritis. Interestingly it even offers the possibility of prolonged protective benefits. Effects of the I3C on inflammation and oxidative stress have been previously addressed [14, 16, 31‐34], whereas to our knowledge, this is the first report of investigating the therapeutic benefits of I3C when combined with MTX or on AIA rat model.

Consistent with findings from previous studies [7, 35, 36], our results clearly demonstrated that the injection of (1 mg/ml) CFA was associated with prominent arthritis in the injected paw. This was confirmed macroscopically and histologically at the tibiotarsal joint in the ankle using several types of specific stains. The H&E stain revealed the presence of classical destructive features of arthritis: proliferative synovitis accompanied with massive amounts of cellular infiltrates, aggregate formation and severe growth of destructive layers of granulation tissue. The latter was able to erode the cartilage which was proven by toluidine blue O and Masson’s Trichrome stain. Many cell types such as macrophages, lymphocytes, synovial cells and neutrophils aggregate into the articular cavity, releasing mediators and inflammatory cytokines [37, 38]. These released materials establish a complex niche that aggravate joint damage through pannus formation and cartilage erosion. The inflammation induced in CFA injected rats was evident as expected by the significant upregulation of serum cytokine levels (TNF- α and IL-6). This was accompanied by an increase in the circulation of inflammatory cells, which was confirmed by an increase in spleen weight and erythrocyte sedimentation rate. It has also been shown that cytokines released by inflamed synovial tissue can reach systemic circulation and act on other organs as proven in this study and by others [7, 21, 39, 40]. The systemic manifestations of arthritis were confirmed by a dramatic drop in the rats’ body weight and by impairing the liver. RA in humans is usually accompanied with oxidative stress and lipid peroxidation which is seen in animal models as well [7, 24]. Free radical induced hepatic damage due to impaired dynamic balance between prooxidant and antioxidant can initiate lipid peroxidation which was evident in this study by increased MDA levels. Induction of arthritis even aberrantly impairs the anti-oxidant defense system by almost depleting as evident here the vital line of defenses (GSH, SOD and CAT) against toxic free radicals [6]. This was even confirmed by increased ALT and AST levels in serum. The raised activity of these enzymes in serum is structurally related to the damage targeting the liver which leads to increased membrane permeability to ions resulting in their release into the circulation [21, 24]. Even the intensity of liver injury was revealed at histological level with increased inflammatory infiltration. These results were in accordance with the results of other studies that have investigated the action of CFA-induced arthritis in rats [7, 20, 24, 41‐43].

The ameliorating effect of MTX therapy was evident as expected. Its well-known anti-arthritic and anti-inflammatory effects were proven on AIA model in this study and by others, too [6, 7, 21, 43]. The polyglutamate form of MTX (MTXGlu) is known to inhibit the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide transformylase causing an increase in adenosine and cAMP levels that are responsible for the anti-arthritic and anti-inflammatory actions of MTX [21]. In this study, both MTX and I3C alleviated the severity of the disease by down regulating the pro-inflammatory cytokines (TNF-α, IL-6) and acting directly on the recruitment of inflammatory cells which was evident by a decreased ESR and spleen index. These cytokines play a pivotal role in recruiting immune cells, stimulating the release of matrix metalloproteinase and other proteinases to degrade cartilage, and upregulating the expression of pro-inflammatory mediators. Therefore, regulation of these cytokines in arthritic subjects is one of the approaches to treat arthritis [1, 44, 45]. This beneficial effect even extended to alleviating paw swelling and inflammation and inhibiting to a certain point the weight loss that was triggered by arthritis. The therapeutic effects of MTX and I3C were even evident microscopically at the histological level where they decreased the pathological changes (synovial hyperplasia, pannus formation, cellular infiltration and cartilage erosion) in the tibiotarsal joint in the ankle with better effects observed when I3C is used. The potent anti-inflammatory and anti-arthritic effects of I3C observed here corroborate with other studies. I3C has been shown to significantly decrease several inflammatory mediators including TNF-α and IL-6 in lipopolysaccharide-activated macrophages and in clonidine-induced neurotoxicity [8, 34].

Although proven quite effective, an adverse effect is known to limit the efficiency of MTX. Hepatotoxicity is known to occur in RA patients taking the therapeutic dose of MTX for a prolonged period of time as also evident in arthritic animal models [7, 21]. In this study, rats treated with MTX, exhibited increased lipid peroxidation (MDA) and decreased anti-oxidant defenses (CAT) compared to arthritic non-treated rats. This was even evident in the liver at a histological level with marked increase in the pathological scores. The toxic effects of MTX largely result from the depletion of hepatic folate stores (MTX is a folate antagonist) due to accumulation of methotrexate polyglutamates in the liver [46]. MTX is also known to increase the generation of toxic by-products such as reactive oxygen species (ROS) and inhibit the cofactors of several anti-oxidant enzymes ultimately decreasing the anti-oxidant defense mechanism known to protect the liver from damage [7, 21, 46]. This proves the need for alternative strategies to handle this deleterious adverse effect.

Although the concurrent treatment with MTX showed no synergistic activity regarding the inflammatory and arthritic markers assessed, I3C may have induced hepatoprotective and anti-oxidant effect against the well-known MTX toxicity and arthritis toxicity. I3C upregulated the anti-oxidant defenses (GSH, SOD and CAT) and in the process inhibited lipid peroxidation. This was even evident by decreased ALT and AST values. It should be noted that MTX treatment had no significant effect on ALT and AST levels in contrast to I3C treatment. I3C even attenuated the pathological changes affecting the liver at histological level that were induced by arthritis and MTX treatment (decreased cellular infiltration). These results are consistent with other studies that showed that I3C exhibits potent hepatoprotective effect against known hepatocarcinogenic agents such as diethylnitrosamine, 2-acetylaminofluorene and trabectedin) [47‐49]. I3C even exerts potent anti-oxidant effects against hyperglycemia-mediated oxidative stress and clonidine-induced neurotoxicity [8, 50].

Interestingly, in this study, I3C markedly attenuated the progression of the arthritic disease in manner similar to MTX but without impairing the liver or inducing oxidative stress as opposed to MTX. The potent therapeutic action of I3C largely revolves around its ability to significantly downregulate the release of inflammatory mediators and markers (TNF-α, IL-6 and ESR) from damaged tissues. I3C abrogated the oxidative stress and toxicity known to be induced by arthritis and MTX treatment. Considering the experimental model of arthritis used in this study shares several clinical and pathologic similarities to human RA [7, 35] then I3C could represent a novel therapeutic agent in the treatment of arthritis.