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Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line

BACKGROUND: Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy. METHODS: In this study, we invest...

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Autores principales: Nemati, Sara, Mohammad Rahimi, Hanieh, Meyfour, Anna, Pazoki, Hossein, Asadzadeh Aghdaei, Hamid, Shahrokh, Shabnam, Mirjalali, Hamed
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10029279/
https://www.ncbi.nlm.nih.gov/pubmed/36941573
http://dx.doi.org/10.1186/s12866-023-02819-8
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author Nemati, Sara
Mohammad Rahimi, Hanieh
Meyfour, Anna
Pazoki, Hossein
Asadzadeh Aghdaei, Hamid
Shahrokh, Shabnam
Mirjalali, Hamed
author_facet Nemati, Sara
Mohammad Rahimi, Hanieh
Meyfour, Anna
Pazoki, Hossein
Asadzadeh Aghdaei, Hamid
Shahrokh, Shabnam
Mirjalali, Hamed
author_sort Nemati, Sara
collection PubMed
description BACKGROUND: Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy. METHODS: In this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed. RESULTS: The results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6. CONCLUSION: Taken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-023-02819-8.
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spelling pubmed-100292792023-03-22 Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line Nemati, Sara Mohammad Rahimi, Hanieh Meyfour, Anna Pazoki, Hossein Asadzadeh Aghdaei, Hamid Shahrokh, Shabnam Mirjalali, Hamed BMC Microbiol Research BACKGROUND: Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy. METHODS: In this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed. RESULTS: The results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6. CONCLUSION: Taken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-023-02819-8. BioMed Central 2023-03-21 /pmc/articles/PMC10029279/ /pubmed/36941573 http://dx.doi.org/10.1186/s12866-023-02819-8 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Nemati, Sara
Mohammad Rahimi, Hanieh
Meyfour, Anna
Pazoki, Hossein
Asadzadeh Aghdaei, Hamid
Shahrokh, Shabnam
Mirjalali, Hamed
Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line
title Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line
title_full Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line
title_fullStr Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line
title_full_unstemmed Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line
title_short Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line
title_sort evaluation of the mtorc activity in the presence of toxoplasma gondii and azathioprine in human monocyte cell line
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10029279/
https://www.ncbi.nlm.nih.gov/pubmed/36941573
http://dx.doi.org/10.1186/s12866-023-02819-8
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