Investigation of the effect of curcumin on oxidative stress, local inflammatory response, COX-2 expression, and microvessel density in Trichinella spiralis induced enteritis, myositis and myocarditis in mice

The genus Trichinella, family Trichinellidae, comprises several encapsulating and non-encapsulating species. The most important species infecting man is Trichinella spiralis (T. spiralis), which exists in its adult stage as the largest known intracellular infectious agent, and in its larval form in well-protected collagen capsules surrounding complex nursing cells. The involvement of several developmental stages of T. spiralis in disease pathogenesis and the targeting of different body systems, makes trichinellosis a rich and peculiar model for host-parasite interaction and antiparasite immunity (Gottstein et al., 2009).

Intestinal and muscular damage is induced by the direct invasive potential of adult worms and larvae, respectively, in addition to the immunopathology resulting from the host’s immune reponse. In comparison, T. spiralis-induced myocarditis results from an eosinophilic inflammatory reaction, since parasite stages do not reside in cardiac muscle tissue (Hidron et al., 2010). Benzimidazole derivatives, particularly albendazole and mebendazole, are the main antiparasitic agents employed for the treatment of trichinellosis. Albendazole acts by binding to cuticular tubulin, thus causing structural damage to the parasite (Pozzio et al., 2001). While the efficacy of albendazole is pronounced against adult worms in the small intestine, it is less potent against encysted larvae in skeletal muscles (Attia et al., 2015).

Fig 1

Herbal remedies have been studied as alternative or adjuvant treatment options against parasitic infections, including trichinellosis (Ullah et al., 2020). Curcuma longa, also known as tumeric, is one of the most widely used medicinal plants. It belongs to the Curcuma genus, family Zingiberaceae. It contains polyphenolic curcuminoid compounds such as diferuloylmethane and curcumin, which give the plant its therapeutic potential (Cheraghipour et al., 2018). Curcumin is known to exert antioxidant properties through the expression of antioxidant proteins and the scavenging of free radicles (Mallo et al., 2020). It is also known for its anti-inflammatory effects as it selectively inhibits the expression of cyclooxygenase-2 (COX-2) enzyme mRNA (Goel et al., 2001). Additional targets of curcumin include growth factors and their receptors, enzymes, cytokines and cell signaling pathways (Shahiduzzaman and Daugschies, 2011). Moreover, curcumin targets factors that promote angiogenesis such as vascular endothelial growth fatcor (VEGF), fibroblast growth factor (FGF) and matrix metalloproteinases (MMPs) (Wang and Chen, 2019). Angiogenesis is important for nurse cell formation during muscular trichinellosis, thus infection with T. spiralis up-regulates VEGF by inducing thymosin β4 (Kang et al., 2011). The degree of vascularization can be evaluated by assessing the microvessel density (MVD), which depends on the intercapillary space controlled by both pro-and anti-angiogenic factors (Hlatky et al., 2002). MVD can be determined by endothelial markers such as CD34 and CD105 (Goldiş et al., 2015).CD34 is a highly glycosylated cell surface sialomucin, that is expressed on the surface of many cell types including haematopoetic progenitor cell, small vessel endothelial cells, muscle satellite cells, mature mast cells, eosinophils and intestinal fibroblasts. CD34 plays an important role in cell adhesion, migration and trafficking (Grassl et al., 2010).

In the current study, we evaluated the benefit of curcumin and its combination with albendazole in the treatment of T. spiralis-induced enteritis, myositis and myocarditis. This was achieved by investigating the effect of curcumin on parasite load, oxidative stress, tissue inflammation, and local COX-2 and CD34 expression in the small intestine, skeletal muscle and heart of infected mice.

The experimental design and methods were implemented in strict accordance with approved national and institutional guidelines and were approved by the Research Ethics Committee for experimental studies at the Faculty of Pharmacy, Modern University for Technology and Information, Egypt, (Permit number: ES (881), 2020).

The management of T. spiralis is challenging due to the diversity of tissue involvement, immune response mechanisms and effectors cells during the different stages of infection. During the intestinal phase of trichinellosis, albendazole has shown to be effective in the near complete adult worm expulsion, whereas limited bioavail- ability reduces its efficacy during the muscular phase (Attia et al., 2015).

Many studies have investigated the potential use of both synthetic and natural compounds as alternative or adjuvant therapeutic agents to benzimidazole therapy in trichinellosis (Soliman et al., 2011). Herbal remedies have attracted special attention in the management of parasitic diseases (Basyoni and El-Sabaa, 2013; Attia et al., 2015). Curcumin extracted from Curcuma longa has proven to be effective against a number of parasites including Leishmania spp., Plasmodium spp., Trichomanas vaginalis, Giardia lamblia, Schistosoma mansoni and Fasciola spp. (Cheraghipour et al., 2018). In our study, we investigated the effect of curcumin on parasitic load, inflammatory response, and COX-2 and CD34 expression in small intestine, skeletal muscle, and heart of T. spiralis-infected mice. This was achieved by administering 2 doses of curcumin, 150 mg/kg and 300 mg/kg. In addition, albendazole was administered at a dose of 50 mg/kg/day either alone or in combination with curcumin 150 mg/kg. Drug were given for 3 days to investigate their effect during the intestinal phase and for 3 weeks to investigate their effect during the muscle phase.

During the intestinal phase, the worm burden was reduced by 32.99 % in mice receiving curcumin 150 mg/kg and by 65.35 % in mice receiving curcumin 300 mg/kg. After giving albendazole alone, the intestinal worm burden was significantly reduced by 90.78 %. The addition of curcumin 150 mg/kg to albendazole did not change the reduction rate achieved by albendazole monotherapy. As for the muscle phase, the larval count per gram muscle tissue of mice receiving curcumin 150 mg/kg was reduced by 23.46 %. Interestingly, the administration of curcumin 300 mg/ kg lead to the highest reduction rate of larval burden by 70.67 % reduction, showing a greater effect than albendazole during the muscle phase, which lead to a reduction of larval count by 56.49 % as compared to the positive control group (p<0.05). The addition of curcumin 150 mg/kg to albendazole reduced the parasite count more than when albendazole was given alone, though not to a statistical significance.

Different studies have reported different efficacy rates for albendazole, according to the dose, duration of administration, timing of animal sacrifice and stage of infection. Fadl et al. (2020) examined the effect of albendazole during the intestinal phase of infection. The drug was administered at a dose of 50 mg/kg on day 2 post-infection for 3 days and a reduction rate of 90.91 % was achieved. Siriyasatien et al. (2003) examined the effect of albendazole 20 mg/kg in reducing muscle larval count when given early as compared to when given late during the infection. They found that albendazole achieved a 100 % reduction rate when given for 15 days starting on day 7 post-infection and mice were sacrificed 7 days after treatment. The efficacy of albendazole was reduced to 71 % when albendazole was administered for 30 days and larval count was assessed 7 days after treatment. The authors suggested that the decrease in efficacy could be due to the development of tolerance secondary to the longer duration of therapy.

Various studies have reported the beneficial effect of curcumin as an antiparasitic agent. El-Ansary et al. (2007) demonstrated that curcumin was effective in lowering Schistosoma mansoni egg output, in addition to having a protective effect on liver. In another study, curcumin was found to reduce the size of the granuloma surrounding S. mansoni eggs (El-Banhawey et al., 2007). The schistosomicidal effect of curcumin was studied in vitro by Magalhães et al. (2011), where it was found to induce uncoupling of male and female schistosomes and to reduce egg output. The in vitro effect of curcumin was also studied by Ullah et al. (2017) on Fasciola gigantica, where it was found to decrease worm motility, induce structural damage, and decrease the activity of the antioxidant enzymes of the parasite. We have found only one study investigating the effect of curcumin on experimental trichinellosis. This study conducted by Elguindy et al. (2019) assessed the antinematode action of curcumin on T. spiralis in light of its effect on nuclear factor kappa B (NFκ-B) expression, which is an important regulator of immune response during infection. Curcumin was given at a dose of 100 mg/kg for 10 days starting 2 hours after infection. The authors observed that the intestinal adult count declined by 45.84 % in mice sacrificed 2 weeks after infection, and by 81.44 % in mice sacrificed 4 weeks after infection. The muscle larval count was reduced by 53 % in mice sacrificed 35 PI. This effect of curcumin on reducing parasitic load was accompanied by a marked increase in inflammatory reaction in small intestine and muscle and increased expression of NFκ-B.

Oxidative damage contributes to parasite-induced immunopathology, since immune effector cells produce their cytotoxic effect in part by generating reactive oxygen species (ROS) (Abd Ellah, 2013). Increased activity of antioxidant enzymes has been demonstrated in mice infected with T. spiralis and was found to reach its maximum during the muscle phase of infection, since the larval stages induce the generation of free radicles by phagocytes (Derda et al., 2004). In the current study, the serum level of MDA was significantly higher during the muscle phase of infection. Both doses of curcumin and albendazole were effective in reducing serum MDA level during both the intestinal and muscle phases, where curcumin 150 mg/kg and combined albendazole/curcumin showed the best results. The benefit of antioxidants in the management of parasitic diseases has been reported against both protozoal and helminthic infections (Abd Ellah, 2013; Puente et al., 2018; Gouveia et al., 2019). Curcumin induces its antiparasitic effect in part by its property as an antioxidant (Mallo et al., 2020). Curcumin was found to be effective against different species of schistosomes by inducing structural damage to the parasite and reducing oviposition (Abou El Dahab et al., 2019; Gouveia et al., 2020). Antioxidants such as selenium and resveratrol were also found to be of value in the management of trichinellosis (Gabrashanska et al., 2019; Elgendy et al., 2020).

Adult T. spiralis worms lodge themselves in multi-intracellular niches most commonly at the crypto-villus junction. They move across the cells in a sinusoidal pattern damaging the cells they pass through, thus resulting in a local inflammatory reaction (Wright, 1979). In our study, histopathological examination of intestinal tissue sections from infected mice showed distorted intestinal villi, dense mononuclear inflammatory infiltration and goblet cell proliferation. In a study by Saracino et al. (2020) on the gut mucosal inflammatory response during intestinal trichinellosis in rats, epithelial destruction, goblet cell hyperplasia and inflammatory cellular infiltration characterized by eosinophilia and mastocytosis was reported. They demonstrated that the intestinal immune response against trichinellosis was initially a T-helper 1 response which subsequently polarized to a T-helper 2 response. They further stated that the helminthotoxic effect of this immune response was directed not only against the adult worms, but also against the newly born larvae, thus decreasing the larval burden reaching the host’s muscles. The anti-inflammatory action of curcumin has been studied in light of its activity against parasites. In a study by Mallo et al. (2020) on the in vitro effect of curcumin on Trichomonas vaginalis, it was found to decrease NO production and inhibit the expression of the pro-inflammatory mediators tumor necrosis factor α (TNFα) and interleukin-1beta (IL-1β) in macrophages.

In the present study, the administration of curcumin, especially at its higher dose, led to the improvement of inflammatory pathology in intestinal, muscle and cardiac tissue of infected mice. Novaes et al. (2016) investigated the effect of curcumin in combination with benznidazole on cardiac pathology in Trypanosoma cruzi (T.cruzi)-infected mice. The drug combination resulted in decreased parasitic load, diminished immunoreactivity to T.cruzi, decreased pro-inflammatory cytokines, improved local inflammation in the heart and diminished oxidative damage. Similar results were obtained by Hernández et al. (2018), where curcumin was found to exert an anti-inflammatory and vasculo-protective effect through the inhibition of endothelin -1 release, which is implicated in vasculitis during Chagas cardiomyopathy. The anti-inflammatory action of curcumin results from its regulatory effect on the eicosanoid signaling pathway, resulting in transcriptional and posttranslational regulation of cyclooxygenase and lipoxygenase enzymes (Rao, 2007). In a study by Barbara et al. (2001) on the effect of COX-2 on intestinal hypermotitliy secondary to trichinellosis, the expression of COX-2 mRNA, local expression of COX-2 protein and the level of PGE2 in the jejunal smooth muscles were assessed. They were all increased during acute infection and the up-regulation of COX-2 mRNA and protein expression were found to persist beyond the acute stage. COX-2 is also important for the early stages of muscle regeneration. In the current study we observed that mice receiving curcumin showed a significant improvement in skeletal muscle and myocardial fiber structure. Bondesen et al. (2004) demonstrated that COX-2 inhibitors interfered with muscle regeneration, while COX-1 inhibitors did not. This was supported by the finding that COX-2 -/- mice showed a decreased regenerative capacity. The action of COX-2 is explained by its effect on inducing satellite cell activation and myoblast proliferation. El-Aswad et al. (2020) measured the degree of local expression of COX-2 and IL-23 in the muscles of T. spiralis-infected mice in a time dependent manner. They observed a progressive increase in the expression of the two markers, reaching the highest degree on day 35 PI. This was associated with an initial mixed TH1/TH2 cytokine response, which became a predominantly TH2 cytokine response during the advanced stages of infection.

In addition to its role in inflammation, COX-2 is also in involved in tumour angiogenesis. Cervello et al. (2005) reported that COX-2 expression was positively correlated with CD34 expression in hepatocellular carcinoma. Angiogenesis is important for nurse cell formation during muscular trichinellosis, since it provides adequate nutrient supply and waste disposal. T. spiralis up-regulates VEGF by inducing thymosin β4, which is a multi-functional protein having anti-apoptotic, anti-inflammatory and anti-angiogenic effects (Kang et al., 2011; Ock et al., 2013). In our study, CD34 was markedly expressed in the small intestine, skeletal muscle and heart of infected non-treated mice. Ibrahim et al. (2019) demonstrated the increased expression of CD34 in myoendothelial cells around encysted larvae in skeletal muscle of T. spiralis-infected mice, which was efficiently reduced by the administration of artemether. In the current study, the administration of curcumin lead to a significant decrease in CD34 expression in all tissue samples. Fu et al. (2015) demonstrated that curcumin suppresses VEGF expression both in vitro and in vivo, through its effect on the VEGF-VEGFR2 (VEGF receptor 2) pathway. The study of angiogenesis during trichinellosis is focused on its role during nurse cell formation. However, angiogenesis is an important feature during intestinal inflammation. MVD has been reported to increase during ulcerative colitis and Crohn’s disease. Angiogenesis plays a crucial role in leucocyte recruitment, oxygen and nutrient supply and healing (Alkim et al., 2015).

Trichinosis induces both parasite-induced and immune-mediated tissue pathology, most noted in the small intestine, skeletal muscle, and heart. Curcumin was found to be of effect in reducing parasitic load during both intestinal and muscle phases of infection, where it has shown to be even more effective than albendazole during the muscle phase. We therefore recommend further dose and time-dependent studies to consolidate data on the effect of curucumin during the muscle phase of trichinellosis. Curcumin showed an additional beneficial effect in reversing muscle fibre degeneration and improving skeletal and cardiac muscle fiber structure. It was also effective in reducing the inflammatory response in the examined tissues. Curcumin exerted its effects by decreasing oxidative stress and inhibiting of local COX-2 expression. In addition, curcumin has an anti-angiogenic effect that adds to the reduction of intestinal inflammation and the interference with muscle nurse cell formation. Curcumin can also reduce cardiac inflammation and pathology. Conclusively, it can be of value as an adjuvant therapy to conventional antiparasitic agents and can produce promising results when used alone at higher doses.