Impact Factor 2018 : 1.587 (@Clarivate Analytics)
  • Users Online: 266
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 1  |  Page : 42-46

Anti-microsporidial effect of thymoquinone on Encephalitozoon intestinalis infection in vitro


1 Halil Bayraktar Health Vocational College, Erciyes University, Kayseri, Turkey
2 School of Medicine, Department of Pharmacology; Genkok Genome and Stem Cell Centre, Department of Molecular Biology and Genetics, Erciyes University, Kayseri, Turkey
3 Life Science Research Centre, Faculty of Science, Ostrava University, Ostrava, Czech Republic

Date of Submission04-Sep-2019
Date of Decision08-Oct-2019
Date of Acceptance09-Dec-2019
Date of Web Publication24-Dec-2019

Correspondence Address:
Ulfet Cetinkaya
Halil Bayraktar Health Vocational College, Erciyes University, Kayseri
Turkey
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2221-1691.273093

Get Permissions

  Abstract 


Objective: To evaluate the anti-microsporidial effects of the active component of Nigella sativa seeds, thymoquinone, against Encephalitozoon intestinalis using an in vitro model.
Methods: Anti-microsporidial effect of thymoquinone against Encephalitozoon intestinalis was evaluated by using various concentrations of thymoquinone (0, 1, 5, 10, 15, 20, 30, 35, and 40 μΜ) and sterile dimethyl sulfoxide. Real time PCR was used to evaluate the inhibitory effects of thymoquinone on the life cycle of Encephalitozoon intestinalis.
Results: The cytotoxic effect of thymoquinone on HEK293 cell line was observed with 30, 35, and 40 μM concentrations of thymoquinone after 24, 48, and 72 hours of incubation. It was observed that 10, 15, 20, and 30 μM concentrations of thymoquinone decreased the spore density compared with the control; however, it was significant only at 30 μM.
Conclusions: Thymoquinone shows potent anti-microsporidial effects against Encephalitozoon intestinalis in the in vitro model; however, the toxic concentrations of thymoquinone are also toxic to the host cells.

Keywords: Microsporidia; Thymoquinone; Nigella sativa; Anti-parasitic; Encephalitozoon intestinalis


How to cite this article:
Cetinkaya U, Sezer G, Charyyeva A. Anti-microsporidial effect of thymoquinone on Encephalitozoon intestinalis infection in vitro. Asian Pac J Trop Biomed 2020;10:42-6

How to cite this URL:
Cetinkaya U, Sezer G, Charyyeva A. Anti-microsporidial effect of thymoquinone on Encephalitozoon intestinalis infection in vitro. Asian Pac J Trop Biomed [serial online] 2020 [cited 2020 Jan 25];10:42-6. Available from: http://www.apjtb.org/text.asp?2020/10/1/42/273093




  1. Introduction Top


Microsporidia are obligate intracellular, spore-forming parasites that infect both vertebrates and invertebrates. There are 14 microsporidia species that cause infection in humans. Encephalitozoon spp. and Enterocytozoon bieneusi are considered to be the clinically important pathogens and cause opportunistic infection in immunocompromised individuals[1],[2],[3].

Treatment of microsporidia infections is not easy since they are intracellular parasites and have natural resistance from a spore wall. Albendazole and fumagillin are the two most widely used drugs in the treatment of microsporidiosis since they are more effective than other alternative therapeutic agents and less toxic to living cells[4],[5]. Albendazole binds to tubulin and inhibits the polymerization of tubulin to microtubules. It remains the drug of choice in treatment although it does not have desirable activity against some microsporidians and in some cases, recurrences are reported[6]. Fumagillin is effective in the treatment of Enterocytozoon bieneusi infections in AIDS patients, so, its use is limited to people with HIV in some countries and has been associated with undesirable effects such as thrombocytopenia and neutropenia[7],[8].

Nigella sativa (N. sativa), grown in the Eastern Europe, Middle East, and Western and Central Asia, is used to treat numbers of conditions and diseases such as eczema, asthma, diabetes, hypertension, inflammation, bronchitis, cough, fever, headache, influenza, and dizziness[9],[10]. Thymoquinone is an active compound extracted from N. sativa essential oil[11], and has been shown to have anti-bacterial[12], anti-fungal[13], and anti-viral[14] characteristics in various studies. Its anti-parasitic activity has been also investigated against many parasites[15-18]. However, there is no information about the effectiveness of thymoquinone against Encephalitozoon intestinalis (E. intestinalis) or any other microsporidia.

The aim of this study was to investigate whether the active component of N. sativa seeds, thymoquinone, has any anti-microsporidial effects against E. intestinalis using an in vitro model.


  2. Materials and methods Top


2.1. Parasite strain and in vitro infection

The E. intestinalis reference strain (50506) was obtained from the American Type Culture Collection (ATCC). The parasites were cultured in HEK293 cells (human kidney epithelial cells, ATCC) as previously reported[19].

2.2. Chemicals

Thymoquinone was obtained from Sigma-Aldrich (St. Louis, MO) and was dissolved in sterile dimethyl sulfoxide (DMSO, Sigma- St. Louis, MO). After filtration, aliquoted solutions were stored at -20°C. The final concentration of DMSO did not exceed 0.1% in the DMSO treated or thymoquinone treated experimental groups. Albendazole (Sigma-St. Louis, MO) was used as a positive control for assaying the life cycle of the E. intestinalis.

2.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

HEK293 cells were cultured in DMEM-HG (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco), 1% L-glutamine (Gibco), 1% streptomycin (10 mg/mL)/penicillin (10 000 units/ mL) (Gibco) medium, and incubated under humidified conditions at 37°C and 5% CO2. To establish experimental groups, 3×103 cells were seeded into the wells of the 96-well plate with 100 μL fresh medium and incubated overnight. The cytotoxic effect of thymoquinone against HEK293 cells was evaluated by using various concentrations of thymoquinone (0, 5, 10, 15, 20, 30, 35, and 40 μM), and DMSO after incubation for 24, 48, and 72 h. At the end of each period, 10 μL MTT (5 mg/mL stock) solution was added to each well and incubated for 2 h at 37°C. Then, 100 μL of DMSO was added to solubilize the purple formazan crystals and the cells were incubated for 20 min at 37°C. The absorbance was measured at 560 nm using a microplate reader (Promega Multireader, Glomax, USA).

2.4. Effects of thymoquinone on the life cycle of E. intestinalis

To evaluate the inhibitory effects of thymoquinone on the life cycle of E. intestinalis, HEK293 cells were used as a host cell. HEK293 cells were cultivated in a 6-well tissue culture plate and incubated at 37°C in 5% CO2 overnight. After removal of non-adherent cells, E. intestinalis spores were added and incubated at 37°C in 5% CO2 overnight for infection. After incubation, non-adherent spores were removed with the medium. Fresh medium containing various concentrations of thymoquinone (0, 1, 5, 10, 15, 20 and 30 μM), albendazole as a positive control (8 and 16 ng/mL), or DMSO was added to each well and incubated at 37°C in 5% CO2 for 10 d with the media changed twice a week. Ten days after the infection, spores started to be detectable in the medium and the experiments were terminated. To quantify parasite density, all media and host cells were removed from the cell culture plate with trypsin (Sigma-St. Louis, MO) and collected in a tube. The tubes were then centrifuged at 4000×g for 10 min and the supernatant was removed. The pellet was used for DNA isolation. DNA was prepared using the GeneAll® Exgene Cell SV Mini Kit (GeneAll Biotechnology, Seoul, South Korea) according to the manufacturer’s recommendation. The parasite load was evaluated quantitatively by real-time PCR using the Roche LightCycler® 480 system (Germany).

2.5. Real-time PCR

The previously reported primer pair was used to detect microsporidia[20]. The total volume of the reactions were 20 μL containing 5 μL of extracted DNA, 1 μL of both forward and reverse primers (10 pmol), 10 μL 2×SYBR-Green Master (Roche Diagnostic, Germany), and 3 μL of PCR grade water. The thermal cycles for real-time PCR reaction consisted of the following steps: initial incubation at 95°C for 5 min, 45 cycles at 95°C for 10 s, 60°C for 10 s and 72°C for 10 s, followed by 1 cycle at 95°C for 5 s, 65°C for 1 min, and finally 1 cycle at 40°C for 30 s.

The dilution series prepared from the plasmid encoding E. intestinalis 16S SSU rRNA region were used as a positive control. The number of copies in the main stock dilution was calculated to be 2.8×108. Then, ten different dilutions were prepared by making a ten-fold dilution from the main stock.

2.6. Statistical analysis

All experiments were carried out in triplicate and data was presented as mean ± standard error of the mean (SEM). Statistical analyses were carried out with the SPSS statistical software package (version 21.0, Inc., Chicago, IL, USA) and graphics were prepared with the Prism GraphPad (San Diego, CA, USA). After checking for the homogeneity of variance using the Levene’s test, one-way ANOVA was used, followed by post hoc Tukey test to compare the absorbance and parasite load values of different thymoquinone concentrations with control group or albendazole treatment. P<0.05 was considered statistically significant.


  3. Results Top


3.1. Cytotoxic effect of thymoquinone on HEK293 cells

The cytotoxic effect of thymoquinone on HEK293 cell line was observed at 30, 35, and 40 μM concentrations of thymoquinone after 24, 48, and 72 hours of incubation period. Compared to the control group, DMSO, or thymoquinone at the concentration less than 30 μM; did not have significant effect on cell viability (P>0.05) [Figure 1].
Figure 1: Viability of HEK293 cells after 24, 48, and 72 hours of incubation with thymoquinone (TQ) at 0 (control), 5, 10, 15, 20, 30, 35, and 40 μM concentrations and with DMSO using MTT assay. DMSO was used to dissolve TQ. *P<0.05 compared to the control at 24, 48, and 72 h. Data is presented as mean ± SEM.

Click here to view


3.2. Effects of thymoquinone on life cycle of E. intestinalis

As shown in [Figure 2], serial dilutions were appropriate to determine the density of the parasites. After 10 d, the experiments were terminated and the parasite load was assessed by quantitative real time PCR.
Figure 2: Real-time PCR results. Dilution series prepared from the plasmid encoding of Encephalitozoon intestinalis 16S SSU rRNA region were used as a positive control. The number of copies in the main stock dilution was calculated to be 2.8×108. Then, ten different dilutions were prepared by making a ten-fold dilution from the main stock.

Click here to view


It was observed that 10, 15, 20, and 30 μM concentrations of thymoquinone decreased the spore density compared with the control [Figure 3]; however, it was significant only at 30 μM (P<0.05) [Figure 3]. The effect of 30 μM thymoquinone was similar to 8 ng/mL albendazole (P>0.05).
Figure 3: Real-time PCR evaluation of the inhibitory effects of thymoquinone (TQ) on the life cycle of Encephalitozoon intestinalis after ten days of incubation with TQ at 0 (control), 1, 5, 10, 15, 20, and 30 μM concentrations, albendazole (Alb) at 8 and 16 ng/mL of concentrations, and DMSO. DMSO was used to dissolve TQ and albendazole. *P < 0.05 compared to the control on the 10th day.

Click here to view



  4. Discussion Top


Albendazole is the most effective drug in the treatment of microsporidia to date. Recent studies have reported that this drug limits the parasite burden, however, fails in clearance of microsporidia especially in immunosuppressed individuals and can result in the reoccurrence of the disease after the treatment[21],[22]. Fumagillin is reported to be effective in the treatment of Enterocytozoon bieneusi but has some side effects[7],[8]. Some medications such as azithromycin, metronidazole, paromomycin, nitazoxanide, and cyclosporine have been used in treatment, but no effective results have been obtained[22],[23].

Plant extracts and compounds have been used as a valuable natural resource of traditional remedies for centuries. In previous studies, it was reported that thymoquinone shows an anti-parasitic effect against some parasites. In one study[18], it was reported that thymoquinone showed anti-leishmanial activity (Leishmania infantum, IC50: 1.47 mg/mL and Leishmania tropica, IC50: 1.16 mg/ mL) on promastigotes after 72 hours of incubation period. In the same study, it was observed that the parasites had less macrophage invasion ability and intracellular survival rate of intramacrophage amastigotes (Leishmania infantum, IC50: 2.6 mg/mL and Leishmania tropica, IC50: 2.1 mg/mL) in the presence of thymoquinone. Thymoquinone showed an inhibitory effect on Babesia and Theileria proliferation in both in vitro and in vivo. These results make thymoquinone a promising candidate for use in therapy. Other authors reported IC50 values of thymoquinone were (0.28 ± 0.016), (7.35 ± 0.17), (35.41 ± 3.60), (67.33 ± 0.94), and (74.05 ± 4.55) μM for Babesia divergens, Babesia bigemina, Babesia bovis, Babesia caballi, and Theileria equi, respectively[24]. In addition, Mahmoudvand et al.[17] revealed that thymoquinone can be a new scolicidal agent for use in hydatid cyst surgery. They reported that all of the protoscoleces died after exposure at the concentration of 1 mg/mL at 10 min. In this study, the anti-microsporidial effect of thymoquinone was evaluated for the first time. It was found that 30 μM thymoquinone inhibited the life cycle of E. intestinalis. In addition, the amount of spores was significantly decreased ten days later. In this study, unlike against leishmania[18], the lower concentrations of thymoquinone (30 μM for intracellular forms) were found to be more toxic for intracellular forms than extracellular spores. This can be explained by the spore structure of the parasite. While microsporidian parasites have highly resistant spores, they do not have a wall structure in its intracellular forms. For this reason, higher concentrations of thymoquinone and long term exposures are necessary to change the viability of spores. The cytotoxic effect of thymoquinone on host cell has been reported by many researchers[25],[26],[27] . The findings of the present study demonstrated that thymoquinone is cytotoxic on HEK293 cells at concentrations of 30 μM. Since microsporidia are obligatory intracellular parasites requiring host cells for their reproduction, the studies on the host cell and the life cycle of the parasite showed that thymoquinone has toxic effects at 30 μM concentrations. In this case, it is hard to understand whether the decreased number of spore was caused directly by the toxic effect of thymoquinone on the spore or host cells.

Despite studies demonstrating an anti-parasitic effect of thymoquinone, there are some studies reporting that thymoquinone has no significant effect on parasite survival. Khan et al. reported that N. sativa has a weak effect on balantidiasis in donkeys[15]. Similarly, Nasir et al. proved that N. sativa shows poor effect on treatment of Cryptosporidium parvum infection in calves[28]. This can be explained in several ways; (1) In some studies, N. sativa extracts were used instead of thymoquinone. Some studies reported that thymoquinone is more effective than N. sativa extracts, (2) Studies were conducted only in vivo. (3) It may have different effects on different parasites.

In summary, thymoquinone showed potent anti-microsporidial effects against E. intestinalis in the in vitro model, however, the toxic concentrations of thymoquinone are also toxic to the host cells. Thymoquinone in combination with other medications or formulations as nanoparticles can be evaluated for further studies. It should be noted that these are the main limitations in the present study. Despite these limitations, this study gives a general idea of the use of thymoquinone.

Conflict of interest statement

We declare that there is no conflict of interest.

Authors’ contributions

UC, AC and GS designed the study and made the critical revision of the article. UC and GS performed the in vitro experiment. Collecting test data, drafting the article and getting a final approval of the version to be published were done by UC and GS, as well as data analysis and interpretation was done by GS. In addition, UC was responsible for supervision, and project administration.



 
  References Top

1.
Anane S, Attouchi H. Microsporidiosis: Epidemiology, clinical data and therapy. Gastroen Clin Biol 2010; 34(8-9): 450-464.  Back to cited text no. 1
    
2.
Ambroise-Thomas P. Emerging parasite zoonoses: The role of hostparasite relationship. Int J Parasitol 2000; 30(12-13): 1361-1367.  Back to cited text no. 2
    
3.
Ferreira FM, Bezerra L, Santos MB, Bernardes RM, Avelino I, Silva ML. Intestinal microsporidiosis: A current infection in HIV-seropositive patients in Portugal. Microbes Infect 2001; 3(12): 1015-1019.  Back to cited text no. 3
    
4.
Costa SF, Weiss LM. Drug treatment of microsporidiosis. Drug Resist Updat 2000; 3(6): 384-399.  Back to cited text no. 4
    
5.
Didier PJ, Phillips JN, Kuebler DJ, Nasr M, Brindley PJ, Stovall ME, et al. Antimicrosporidial activities of fumagillin, TNP-470, ovalicin, and ovalicin derivatives in vitro and in vivo. Antimicrob Agents Chemother 2006; 50(6): 2146-2155.  Back to cited text no. 5
    
6.
Molina JM, Oksenhendler E, Beauvais B, Sarfati C, Jaccard A, Derouin F, et al. Disseminated microsporidiosis due to septata-intestinalis in patients with aids - Clinical-features and response to albendazole therapy. J Infect Dis 1995; 171(1): 245-249.  Back to cited text no. 6
    
7.
Molina JM, Tourneur M, Sarfati C, Chevret S, de Gouvello A, Gobert JG, et al. Fumagillin treatment of intestinal microsporidiosis. N Engl J Med 2002; 347(17): 1963-1969.  Back to cited text no. 7
    
8.
Champion L, Durrbach A, Lang P, Delahousse M, Chauvet C, Sarfati C, et al. Fumagillin for treatment of intestinal microsporidiosis in renal transplant recipients. Am J Transplant 2010; 10(8): 1925-1930.  Back to cited text no. 8
    
9.
Randhawa MA, Al Ghamdi MS. A review of the pharmaco-therapeutic effects of Nigella sativa. Pakistan J Med Res 2002; 41(2): 77-83.  Back to cited text no. 9
    
10.
El-Far AH, Al Jaouni SK, Li W, Mousa SA. Protective Roles of thymoquinone nanoformulations: Potential nanonutraceuticals in human diseases. Nutrients 2018; 10(10): 1369.  Back to cited text no. 10
    
11.
El-Dakhakhny M. Studies on the chemical constitution of Egyptian N. sativa L. seeds. Planta Med 1963; 11: 465-470 .  Back to cited text no. 11
    
12.
Morsi NM. Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-resistant bacteria. Acta Microbiol Pol 2000; 49(1): 6374.  Back to cited text no. 12
    
13.
Khan MA, Ashfaq MK, Zuberi HS, Mahmood MS, Gilani AH. The in vivo antifungal activity of the aqueous extract from Nigella sativa seeds. Phytother Res 2003; 17(2): 183-186.  Back to cited text no. 13
    
14.
Salem ML, Hossain MS. Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection. Int J Immunopharmacol 2000; 22(9): 729-740.  Back to cited text no. 14
    
15.
Khan A, Khan MS, Avais M, Ijaz M, Ali MM, Abbas T. Prevalence, hematology, and treatment of balantidiasis among donkeys in and around Lahore, Pakistan. Vet Parasitol 2013; 196(1-2): 203-205.  Back to cited text no. 15
    
16.
Mahmoud MR, El-Abhar HS, Saleh S. The effect of Nigella sativa oil against the liver damage induced by Schistosoma mansoni infection in mice. J Ethnopharmacol 2002; 79(1): 1-11.  Back to cited text no. 16
    
17.
Mahmoudvand H, Dezaki ES, Kheirandish F, Ezatpour B, Jahanbakhsh S, Harandi MF. Scolicidal effects of black cumin seed (Nigella sativa) essential oil on hydatid cysts. Korean J Parasitol 2014; 52(6): 653-659.  Back to cited text no. 17
    
18.
Mahmoudvand H, Tavakoli R, Sharififar F, Minaie K, Ezatpour B, Jahanbakhsh S, et al. Leishmanicidal and cytotoxic activities of Nigella sativa and its active principle, thymoquinone. Pharm Biol 2015; 53(7): 1052-1057.  Back to cited text no. 18
    
19.
Cetinkaya U, Charyyeva A, Gurbuz E. Evaluation of the reproductive potential of Encephalitozoon intestinalis in four different cell line. Mikrobiyol Bul 2018; 52(4): 390-400.  Back to cited text no. 19
    
20.
Cetinkaya U, Yazar S, Kuk S, Sivcan E, Kaynar L, Arslan D, et al. The high prevalence of Encephalitozoon intestinalis in patients receiving chemotherapy and children with growth retardation and the validity of real-time PCR in its diagnosis. Turk J Med Sci 2016; 46(4): 1050-1058.  Back to cited text no. 20
    
21.
Kotkova M, Sak B, Kvetonova D, Kvac M. Latent microsporidiosis caused by Encephalitozoon cuniculi in immunocompetent hosts: A murine model demonstrating the ineffectiveness of the immune system and treatment with albendazole. PLoS One 2013; 8(4): e60941.  Back to cited text no. 21
    
22.
Lallo MA, da Costa LF, de Castro JM. Effect of three drugs against Encephalitozoon cuniculi infection in immunosuppressed mice. Antimicrob Agents Chemother 2013; 57: 3067-3071.  Back to cited text no. 22
    
23.
Bicart-See A, Massip P, Linas MD, Datry A. Successful treatment with nitazoxanide of Enterocytozoon bieneusi microsporidiosis in a patient with AIDS. Antimicrob Agents Chemother 2000; 44(1): 167-168.  Back to cited text no. 23
    
24.
El-Sayed SAE, Rizk MA, Yokoyama N, Igarashi I. Evaluation of the in vitro and in vivo inhibitory effect of thymoquinone on piroplasm parasites. Parasit Vectors 2019; 12(1): 37.  Back to cited text no. 24
    
25.
ElKhoely A, Hafez HF, Ashmawy AM, Badary O, Abdelaziz A, Mostafa A, et al. Chemopreventive and therapeutic potentials of thymoquinone in HepG2 cells: Mechanistic perspectives. J Nat Med 2015; 69(3): 313-323.  Back to cited text no. 25
    
26.
Ali BH, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 2003; 17(4): 299-305.  Back to cited text no. 26
    
27.
Abukhader MM. The effect of route of administration in thymoquinone toxicity in male and female rats. Indian J Pharm Sci 2012; 74(3): 195200.  Back to cited text no. 27
    
28.
Nasir A, Avais M, Khan MS, Khan JA, Hameed S, Reichel MP. Treating Cryptosporidium parvum infection in calves. J Parasitol 2013; 99(4): 715717.  Back to cited text no. 28
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  2. Materials and...
  In this article
Abstract
1. Introduction
3. Results
4. Discussion
References
Article Figures

 Article Access Statistics
    Viewed143    
    Printed5    
    Emailed0    
    PDF Downloaded64    
    Comments [Add]    

Recommend this journal