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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 47  |  Issue : 2  |  Page : 52-61

Preliminary study on the role of toll-like receptor-4 antagonist in treatment of Trichinella spiralis infection


1 Medical Parasitology Department, Faculty of Medicine, Tanta University, Egypt
2 Pathology Department, Faculty of Medicine, Tanta University, Egypt

Date of Submission01-Apr-2018
Date of Acceptance01-Apr-2019
Date of Web Publication18-May-2020

Correspondence Address:
Dina A Elguindy
Demonstrator at Medical Parasitology Department, Faculty of Medicine, Tanta University
Egypt
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DOI: 10.4103/tmj.tmj_17_18

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  Abstract 


Background Trichinella spiralis is one of the most widespread zoonotic parasitic nematodes in the world. There is an increasing interest in developing new antihelminthic drugs for Trichinella. Toll-like receptor-4 (TLR4) is closely related to T. spiralis infection, and its deficiency is associated with rapid expulsion of T. spiralis worms.
Aim The aim of this study was to explore the effect of TLR4 antagonist (curcumin) on the course of experimental trichinellosis.
Materials and methods Mice were divided into two main groups. Group I was the control group (90 mice), which was subdivided into the following: subgroup Ia (10 mice), with noninfected nontreated mice (negative control); subgroup Ib (40 mice), with infected nontreated mice (positive control); and subgroup Ic (40 mice), with noninfected treated with curcumin. Group II was the infected and treated group (50 mice), where infected mice received curcumin starting 2 h after infection and continued for 10 successive days after infection. For each group, total adult and muscle larval count were estimated, and the small intestines and muscles were removed for further studies.
Results This results showed a significant decrease in the mean number of adults and larvae in the infected treated group compared with the infected control mice, with an extremely significant percentage of reduction of 53%. Regarding the histopathological changes, there was a marked increase in the inflammatory reaction surrounding the adult worms in the small intestines and the encysted larvae in muscles of the infected treated group compared with the infected nontreated group. Curcumin leads to degeneration of the capsule around the larvae in the skeletal muscles of the infected treated group. There was a significant increase in nuclear factor-κB expression by small intestinal tissues in T. spiralis infected treated group (group II) as compared with the infected nontreated group (group Ib).
Conclusion This study revealed that curcumin has an antiparasitic activity against both stages of T. spiralis. Thus, it could be a promising drug for treatment of T. spiralis infection.

Keywords: curcumin, nuclear factor-κB, Toll-like receptor-4, Trichinella spiralis


How to cite this article:
Elguindy DA, Ashour DS, Shamloula MM, Aboul Assad IA. Preliminary study on the role of toll-like receptor-4 antagonist in treatment of Trichinella spiralis infection. Tanta Med J 2019;47:52-61

How to cite this URL:
Elguindy DA, Ashour DS, Shamloula MM, Aboul Assad IA. Preliminary study on the role of toll-like receptor-4 antagonist in treatment of Trichinella spiralis infection. Tanta Med J [serial online] 2019 [cited 2020 Oct 20];47:52-61. Available from: http://www.tdj.eg.net/text.asp?2019/47/2/52/284494




  Introduction Top


Trichinellosis is a zoonotic disease caused by eating undercooked or raw meat harboring the infective Trichinella larvae [1]. It infects many mammalian, avian, and reptile host species, in which the adult worms and the larvae reside in the small intestinal and muscle tissues, respectively [2].

Trichinella infection in the human host is divided into two phases: an intestinal (or enteral) phase and a muscular (or parenteral) phase. Worms migrating in epithelium of the intestine can lead to traumatic damage to tissues of the host [3]. The invading Trichinella newborn larvae produce muscle cell damage that triggers the activation of satellite cells undergoing proliferation and re-differentiation, thus producing nurse cell and encapsulation of the larvae with surrounding capillary rete [4],[5]. The resulting inflammation in the intestine and the muscles is associated with activation of nuclear factor-κB (NF-кB) signaling pathways [6]. Although NF-κB promotes T-cell activation and differentiation, the function of NF-κB is paradoxical, as it is also involved in the generation of T regulatory (Treg) cells, thus has a protective role maintaining integrity of tissues [7],[8]. However, the persistence of infection indicates that Trichinella has manipulated the NF-κB signaling pathway to evade the host immune response [6].

T. spiralis can adjust the immune system to its advantage by activating or negatively regulating Toll-like receptor (TLRs) [9]. It was found that Toll-like receptor-4 (TLR4)-deficient mice expel worms more rapidly, proving that TLR4 plays an important role in T. spiralis infection [10]. It was suggested that ES antigens of T. spiralis interact with TLR4, inducing a tolerogenic status in dendritic cells (DCs) and as a result shift T-cell polarization toward the regulatory type. Activated Tregs can inhibit the specific response against the parasite as well as the autoantigens [11].

Curcumin which is extracted from the rhizome Curcuma longa has anti-inflammatory, anti-infection, antioxidant, and antitumor properties [12],[13]. Curcumin was found to be a TLR4 antagonist [14]. Therefore, this study aimed to explore the role of curcumin on the enteral and parenteral phases of experimental T. spiralis infection, postulating that its effect potentiates it to be used as a safe treatment for trichinellosis.


  Materials and methods Top


Parasite and animals

This study was conducted on 140 laboratory-bred male Swiss albino mice that were housed and fed according to the national guidelines. The strain of T. spiralis is maintained in the laboratory of Tanta Medical Parasitology Department by consecutive passages through rats and mice.

Drugs

Curcumin (Sigma Aldrich, Cairo, Egypt) was given orally at 100 mg/kg/day dissolved in distilled water for 10 successive days [15].

Experimental design

The experiment was approved by the Research Ethics Committee, Quality Assurance Unit, Tanta Faculty of Medicine, Egypt. Mice were infected with T. spiralis larvae orally in a dose of 200 larvae per mouse according to Dunn and Wright [16]. Animals were divided into two main groups: group I was the control group (90 mice), which was further subdivided into the following: subgroup Ia (10 mice) included noninfected nontreated mice, subgroup Ib (40 mice) included infected nontreated mice, and subgroup Ic (40 mice) included noninfected treated with curcumin for 10 days. Group II was the infected and treated group (50 mice), where infected mice received curcumin starting 2 h after infection and continued for 10 successive days after infection.

Five mice from each of the two infected groups (infected nontreated and infected treated) were killed twice weekly for 4 weeks starting from the fourth day after infection. The small intestine was removed, longitudinally opened, and cleaned with saline. Overall, 1 cm from the middle third of the small intestine was preserved in 10% formol-saline for histopathological and immunohistochemical studies. Five weeks after infection, muscle samples from the thigh muscles were obtained for histopathological and immunohistochemical studies. The rest of the skeletal muscle was used for total larval count. The negative control group (noninfected nontreated and noninfected treated) was killed once at the end of the experiment. All experiments were done in duplicate.

Adult worms count in small intestine

The remainder of the intestines of the infected treated and the infected nontreated groups were cut into 2 cm pieces and placed in a beaker full of normal physiological saline at 37°C for 3–4 h. After that, the intestine was shaken well in the solution and rinsed with saline. All the fluid was collected and centrifuged at 1500 rpm for 5 min. The worms were counted in the reconstituted sediment drop by drop at a magnification of ×40 [17].

Total larval burden in muscles

Five weeks after infection, five animals from each infected group were euthanized. The total muscle larvae were counted according to Wranicz et al. [18].

Histopathological study

Tissue samples (intestinal and skeletal muscle) from the studied groups were prepared by routine histological processing, paraffin embedding, sectioning at 5-µm thickness, and staining by hematoxylin and eosin, and then routine evaluation of the histopathological characteristics and comparative descriptive analysis of the studied experimental groups were done [19].

Immunohistochemistry for nuclear factor-кB expression

Paraffin tissue sections were immunostained for NF-кB transcription factor by the biotin streptavidin-peroxidase method using the NF-кB p65 (F-6) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA), which recognizes amino-terminal sequences of the p65 subunit. Nuclei were counterstained with hematoxylin. Substitution of the primary antibody with PBS served as the negative control. NF-кB staining was assessed by light microscopy and localized within the cell cytoplasm and/or nucleus. Positive reaction appears in the form of brownish staining with nuclear localization of NF-кB, which was categorized as positive or negative [20].

Statistical analysis

Quantitative data were presented as mean±SD. The probability of significant differences among groups was determined by one-way anal test. Differences were considered significant, when P value was less than 0.05. The statistical analyses were processed using statistical package of the social sciences (SPSS Inc., Chicago, Illinois, USA) software for Windows, version 10.0.


  Results Top


Adult worms count in small intestine

The mean adult worm count in the small intestine on day 4 after infection of the infected nontreated group (group Ib) was 67±8, with no significant difference with that of the infected and treated group (group II) (60±9). Two weeks after infection, the mean adult worm of the infected treated group greatly declined, reaching 30.33±1.53, with percentage of reduction of 45.84%, as compared with the infected nontreated group (56±4). By the end of the fourth week, the percentage of reduction reached 81.44% with an extremely statistically significant difference (P<0.001) between the infected treated and infected nontreated groups ([Table 1]).
Table 1 Adult worm count (mean±SD) of Trichinella spiralis in the small intestine of control and infected treated groups

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Total larval counts in muscles

On day 35 after infection, there was an extremely statistically significant reduction of total larval counts in the infected treated group (26.169±4.48) as compared with infected nontreated group (56.128±13.07) (P<0.001), with percentage of reduction of 53%.

Histopathological study

Small intestinal changes

The small intestinal sections of both noninfected nontreated and noninfected treated groups showed normal small intestinal structure, and no histopathological changes were observed. Small intestinal sections of the infected control group (group Ib) revealed the presence of adult worm sections within the mucosa with inflammatory cellular infiltration of the mucosa and the submucosa ([Figure 1]a). The infiltrate was mostly present in the core of the villi and in the submucosa and was mainly composed of lymphocytes, eosinophils, plasma cells, neutrophils, and fibroblasts together with villous blunting and edema and lymphoid follicle hyperplasia ([Figure 1]b). In addition, there was goblet cell hyperplasia ([Figure 1]c). Crypt abscess was also seen together with high mitosis in crypts ([Figure 1]d). Small intestinal sections of the infected treated group (group II) revealed similar histopathological changes; however, there was a marked increase in the inflammatory reaction surrounding the adult worms compared with the infected control group ([Figure 2]a–d).
Figure 1 Small intestinal section of Trichinella spiralis-infected control mouse (group Ib) (hematoxylin and eosin) showing the following: (a) adult worm (arrow) within the mucosal crypts surrounded by inflammatory cells (×100); (b) villous edema with inflammatory cellular infiltrate in the villi cores (×100), goblet cells hyperplasia (arrows) with inflammatory cellular infiltrate in the villi cores, and submucosa (×200); and (d) crypt abscess (arrow) (×200).

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Figure 2 Small intestinal section of Trichinella spiralis-infected treated mouse (group II) (hematoxylin and eosin) showing the following: (a) marked increase in the inflammatory reaction surrounding the adult worms (×100), (b) villous edema (×200), (c) excessive inflammatory cellular infiltrate (×200), and (d) high mitotic count in the crypts (arrows) (×200).

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Skeletal muscle changes

Normal muscle architecture was observed in the muscle sections of both noninfected nontreated and noninfected treated groups. However, muscle sections of the infected group (group Ib) revealed the presence of massive number of encysted T. spiralis larvae surrounded by thick capsule, which was surrounded by inflammatory cells such as lymphocytes, plasma cells, macrophages, histiocytes, neutrophils, and eosinophil’s ([Figure 3]a and b). Muscle sections of infected treated group (group II) showed much fewer numbers of encysted larvae and an increase in the inflammatory reaction surrounding the larvae, besides the capsule around the larvae showed splitting, thinning, and vacuolation ([Figure 3]c). Degenerative changes inside the muscles were seen in the form of loss of striation, hyaline appearance, and swelling ([Figure 3]d).
Figure 3 Muscle section of Trichinella spiralis-infected control mouse (a and b) and infected treated mouse (c and d) (hematoxylin and eosin) showing the following: (a) large number of encysted larva inside muscle fibers surrounded by thick capsule and inflammatory cellular infiltrate (arrows) (×100); (b) higher magnification of encysted larva inside the muscles (×200); (c) heavy inflammatory cellular infiltrate surrounding encysted larva and its capsule showed areas of vacuolation (curved arrow), thinning out, and splitting (arrow) (×400); and (d) degenerated muscles in the form of swelling, loss of striation, and hyaline appearance (×100). Insit: higher magnification of (d) (×200).

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Immunohistochemistry for nuclear factor-кB expression

Expression of nuclear factor-кB in small intestinal tissues

There was an increase in NF-кB expression in small intestinal tissues in the infected treated group (group II). The expression showed strong nuclear and/or cytoplasmic positivity in the enterocytes and inflammatory cells ([Figure 4]a and b). The infected nontreated group showed moderate nuclear and/or cytoplasmic positivity in the enterocytes and inflammatory cells ([Figure 4]c), with a highly statistically significant difference (P<0.001). However, the noninfected treated group showed weak expression ([Figure 4]d), and the noninfected nontreated was negative.
Figure 4 Immunohistochemical expression of nuclear factor-κB in small intestine of Trichinella spiralis-infected treated mice (a and b), infected nontreated mice (c), and noninfected treated (d) (immunoperoxidase ×400) showing the following: (a) strong positivity in inflammatory cells and enterocytes; (b) strong positivity in inflammatory cells; (c) moderate positivity in inflammatory cells and enterocytes; and (d) weak nuclear staining in the submucosal inflammatory cells only.

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Expression of nuclear factor-кB in the skeletal muscle tissues

There was an increase in NF-кB expression by the skeletal muscles in the infected treated group (group II). It showed strong positive expression (nuclear and/or cytoplasmic) in the sarcoplasm of the skeletal muscles as well as inflammatory cells ([Figure 5]a and b). However, infected nontreated and the noninfected treated groups showed mild nuclear positivity in the skeletal muscles sarcoplasm ([Figure 5]c). Regarding the noninfected nontreated group, the expression was negative.
Figure 5 Immunohistochemical expression of nuclear factor-κB in muscle of Trichinella spiralis-infected treated mice (a and b) and infected nontreated mice (c) (immunoperoxidase ×400) showing the following: (a) strong cytoplasmic positivity in the sarcoplasm of the striated muscles (b) strong positivity in the inflammatory cells and positivity in the skeletal muscles.

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  Discussion Top


Several studies have elucidated the mechanisms of Trichinella adult worm expulsion from the intestine based mainly on the immune response against T. spiralis and the inflammatory response that plays a crucial role in the parasite expulsion because it makes the habitat hostile, thus facilitating worm expulsion with subsequent regulation of the muscle phase [21],[22].

T. spiralis releases abundant glycoproteins throughout its life that activate immune cells or an immune response related to TLRs [23],[24]. The phosphorylcholine of the parasite binds to TLR4 [25]. TLR4 is very closely related to T. spiralis infection, as the gene expression of TLR4 was elevated in the intestinal and muscular phases of mice affecting cytokine production [9]. This study assessed the effect of TLR4 antagonist (curcumin) on the course of experimental trichinellosis.

Curcumin has been the subject of many research studies owing to its various pharmacological activities and biosafety [26]. Curcumin is a TLR4 antagonist, as it inhibits TLR4 homodimerization, and it is a potent inhibitor of many signaling molecules in the TLR4 pathway [27].

This results showed a significant decrease in the mean number of adults in the infected treated group compared with the infected control mice, with percentages of reduction of 61.76 and 81.44% at the third and fourth weeks, respectively. Our results agree with Scalfone et al. [10] and Eckburg et al. [28] who stated that TLR4 deficiency produced rapid expulsion of T. spiralis worms, proving that these receptors are engaged in T. spiralis infection. Eckburg et al. [28] also mentioned that TLR4 deficiency impaired gut homeostasis that contributes to a “leaky gut,” thus promoting parasite expulsion.

In the same context, several studies have reported other antiparasitic effects of curcumin. Magalhaes et al. [29] mentioned that treatment with curcumin modulates humoral and cellular immune responses of Schistosoma mansoni-infected mice and leads to a significant reduction of parasite burden and liver pathology. Moreover, De Paula Aguiar et al. [30] mentioned that curcumin caused apoptosis and DNA fragmentation in adult worms of S. mansoni and induced the production of reactive oxygen species (ROS).

Lakkany and Seif El-Din [31] mentioned that in helminthes, numerous antioxidant systems regulate the concentrations of ROS inside the cell via different enzymes that play an essential part in the decomposition of ROS and protect the parasite from damage. De Paula Aguiar et al. [30] reported that the activities of these antioxidant enzyme were decreased in S. mansoni adult female and male worms incubated with curcumin; thus, the protein carbonyl content was highly elevated in adult worms. In other words, curcumin produces oxidative stress followed by apoptosis in adult S. mansoni worms, leading to parasite death. The same mechanism of action was reported in filariasis [32]. Curcumin was found to act as both a macrofilaricide and microfilaricide, as it causes significant reduction in the viability of both adults and microfilariae. Although not investigated in the current study, curcumin may accomplish its antiparasitic effect against T. spiralis in the same way.

In this study, the mean total larval count of the infected treated group was greatly reduced as compared with that of the infected nontreated group, with a significant percentage of reduction of 53%. We propose that the decreased adult count subsequently resulted in reducing the larval count. In addition, curcumin produces an impairment of embryogenesis as reported by De Paula Aguiar et al. [30]. They discovered that, under the effect of curcumin, some alterations took place, mostly in the vitellarium of S. mansoni female worms, which is the proliferative tissue that aids in the development of the embryo. Similarly, Mohapatra et al. [33] stated that curcumin induces apoptosis in embryonic stages of the nematode Setaria digitate. Curcumin causes chromatin condensation and DNA fragmentation in developing embryos and microfilariae in gravid female ROS leading to apoptosis. This promising strategy played by curcumin may produce effective antiparasitic measures [32].

Regarding the histopathological examination, intestinal sections of T. spiralis-infected nontreated group (group Ib) revealed inflammatory cells infiltrating the mucosa and the submucosa with villous edema. In addition, there were goblet cell hyperplasia, blunting of the villi, and lymphoid follicle hyperplasia. Crypt abscess was also seen together with high mitosis in crypts. These findings coincide with Airis et al. [34] who reported the same observations. Khan [35] stated that goblet cell hyperplasia takes place leading to increased secretion of mucus that helps in the defense mechanism by trapping the parasite in the mucus, thus decreasing the motility of the worm and in turn leads to parasite expulsion.

TLRs do not only promote the production of inflammatory molecules but they are also regulatory (anti-inflammatory) contributors [interleukin 10 (IL-10) and transforming growth factor-β (TGF-β)] [36],[37]. In other words, in T. spiralis infection, the modulated TLR expression in the small intestine is related to Treg cell-mediated immune responses and increased IL-10 and TGF-β gene expression [9]. Lipopolysaccharide-bound TLR4 engages different adapter mechanisms to induce anti-inflammatory cytokines such as IL-10 [38]. It was found that TGF-β works with IL-10 to control local inflammation, and their deficiency leads to severe inflammation in the muscles [39]. Thus, it explains our finding that there was a marked increase in the intestinal inflammatory reaction in response to decreased TLR4 in the infected treated group compared with the infected nontreated group.

Muscle sections of the infected nontreated group (group Ib) revealed the presence of a massive number of encysted T. spiralis larvae surrounded by thick capsule and inflammatory cells, whereas the infected treated group (group II) showed much fewer numbers of encysted larvae and an increase in the inflammatory reaction surrounding the larvae with occasional abscess formation. Degenerative changes inside the muscles were seen in the form of loss of striation, hyaline appearance, and swelling, besides splitting, thinning, and vacuolation of the capsule around the larvae.

These results are similar to the findings of Bruschi and Chiumiento [22] which state that T. spiralis causes mechanical damage to the skeletal muscle cells as well as inflammatory cells accumulation, through the production of high levels of oxygen reactive species and other free radicals. Nurse cell formation is important for the larva to survive inside the skeletal muscle. Angiogenesis around the collagen capsule is required for maintaining the larvae within the host for long periods [40]. The main factor promoting angiogenesis in trichinellosis is the vascular endothelial growth factor [41]. Curcumin inhibits a variety of angiogenesis growth factors including vascular endothelial growth factor [42]. Curcumin was able to inhibit the angiogenic response to fibroblast growth factor-2 stimulation in mouse endothelial cells [43]. This interference with the angiogenesis process can deprive the larvae from their nutrition with accumulation of their wastes causing their death.

In this study, there was a marked increase in the inflammatory reaction in the muscles surrounding the larvae in response to decreased TLR4 in the infected treated group compared with the infected nontreated group. This marked increase in inflammation can explain the decrease in the number of both adult worms and larvae through the release of ROS and free radicals such as nitric oxide. Inflammatory cells release high levels of ROS and other free radicals [44].

There was a significant increase in NF-κB expression by small intestinal and muscle tissues in T. spiralis-infected treated group (group II) as compared with the infected nontreated group (group Ib). There was both nuclear and cytoplasmic positivity in the enterocytes, sarcoplasm of the skeletal muscles, as well as intestinal and muscle inflammatory cellular infiltrate. Although curcumin is reported to decrease NF-κB expression [42], our results showed that its expression was increased in the treated group mostly because of its massive expression from the increased inflammatory cells. These results agree with Ashour et al. [45] who stated that the degree of NF-κB activation correlates with the severity of inflammation. In this study, curcumin inhibited TLR4 and consequently increased inflammation, thus increasing NF-κB expression. NF-κB is located in the cytoplasm in most cell types, until induced by a stimulus to be activated and translocated in the nucleus [46].

In this study, curcumin was found to have an antiparasitic activity against T. spiralis and at the same time increase the inflammatory reactions both in the small intestine and muscles leading to significant reduction in the adult worm count and the larval burden, respectively.

Regarding the previous protocols used for treatment of T. spiralis, it has been proven that albendazole is active against the enteral and parenteral phase of the parasite in experimental trichinellosis but failed to act during the invasive and encystment phase, as it has a relatively low antiparasitic activity against encysted larvae [47]. In addition, the efficacy of mebendazole against muscle larvae depends on the time between infection and treatment and could be dose dependent [48].

Therefore, medicinal plants could be used as alternative target for managing T. spiralis infection such as Nigella sativa [49], Artemisia vulgaris [50], and myrrh and thyme [51]. The efficacy of curcumin in this study was higher than those of previous studies. This may be owing to its immunological effect as TLR4 inhibitor or because of the protocol used for treatment, that is, high doses, early administration, and during both the intestinal and muscular phases of infection for many successive days.


  Conclusion Top


This study suggests that curcumin may act as a TLR4 antagonist and can be used in the treatment of trichinellosis. Curcumin was able to decrease the adult worm count and the larval count. However, an important limitation hindering the clinical advancement of curcumin as a promising molecule for treatment of T. spiralis is its limited oral bioavailability though it has a high tissue distribution [52]. Thus, a successful enhancement of curcumin bioavailability is likely to bring this promising natural product to the forefront of therapeutic agents for the treatment of trichinellosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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