The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation

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dc.contributor.advisorKaschula, Catherineen_ZA
dc.contributor.advisorSchäfer, Georgiaen_ZA
dc.contributor.advisorKatz, Arieh A.en_ZA
dc.contributor.authorHitchcock, Jessica Kaarien_ZA
dc.date.accessioned2015-12-09T14:44:54Z
dc.date.available2015-12-09T14:44:54Z
dc.date.issued2015en_ZA
dc.description.abstractCancer is a leading cause of death in the modern world. Chronic inflammation facilitates tumourigenesis and cancer progression by providing an environment conducive to cancer. Dysregulation of the immune response, and particularly inflammation, is an important part of this process. Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent, more recent epidemiological and experimental evidence shows that garlic has both cancer-preventative and immune system-enhancing effects. While garlic contains many bioactive compounds, garlic organosulfur compounds (OSCs) have been most widely studied for their anti-cancer properties. In this study, we hypothesize that garlic OSCs modulate the inflammatory immune response by downregulating pro-inflammatory while stimulating anti-inflammatory responses, preventing the formation of a cancer-friendly chronic inflammatory environment. To test this hypothesis we established and optimised an in vitro inflammatory model using lipopolysaccharide-stimulated RAW264.7 murine macrophages. Expression analysis of selected inflammatory genes was performed by qPCR on RNA harvested 4 h and 8 h post treatment, while protein expression was analysed by ELISA using cell culture supernatant samples harvested 8 h and 24 h post treatment. These experiments were complemented by gene and protein arrays. Results showed that allicin had a more pronounced upregulatory effect on LPS-induced gene expression 4 h post-LPS treatment. In contrast, Z-ajoene generally had mild downregulatory effects on the expression of LPS-induced genes. Conversely, Z-ajoene had pronounced downregulatory effects on LPS-induced inflammatory proteins after 24 h, while allicin showed mild up- or downregulatory effects. Overall, we found that allicin induced an initial pro-inflammatory gene response, while Z-ajoene induced a longer-lasting anti-inflammatory response at a protein level. Finally, as many of the inflammatory genes investigated are regulated by the transcription factor STAT3, we investigated the effects of allicin and Z-ajoene on STAT3 phosphorylation and hence activation. Western blot analyses showed that allicin increased LPS-induced STAT3 phosphorylation (2-8 h), while Z-ajoene was found to decrease the phosphorylation of STAT3 after 4 h. These effects on STAT3 phosphorylation are in agreement with the early pro-inflammatory effect of allicin and the later anti-inflammatory effect of Z-ajoene on LPS-induced gene and protein expression. Further, using Western blotting we showed that E/Z-ajoene directly interacts with and reversibly alkylates STAT3 via a thiol-disulfide reaction with a cysteine thiolate on STAT3.en_ZA
dc.identifier.apacitationHitchcock, J. K. (2015). <i>The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry. Retrieved from http://hdl.handle.net/11427/15735en_ZA
dc.identifier.chicagocitationHitchcock, Jessica Kaari. <i>"The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry, 2015. http://hdl.handle.net/11427/15735en_ZA
dc.identifier.citationHitchcock, J. 2015. The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Hitchcock, Jessica Kaari AB - Cancer is a leading cause of death in the modern world. Chronic inflammation facilitates tumourigenesis and cancer progression by providing an environment conducive to cancer. Dysregulation of the immune response, and particularly inflammation, is an important part of this process. Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent, more recent epidemiological and experimental evidence shows that garlic has both cancer-preventative and immune system-enhancing effects. While garlic contains many bioactive compounds, garlic organosulfur compounds (OSCs) have been most widely studied for their anti-cancer properties. In this study, we hypothesize that garlic OSCs modulate the inflammatory immune response by downregulating pro-inflammatory while stimulating anti-inflammatory responses, preventing the formation of a cancer-friendly chronic inflammatory environment. To test this hypothesis we established and optimised an in vitro inflammatory model using lipopolysaccharide-stimulated RAW264.7 murine macrophages. Expression analysis of selected inflammatory genes was performed by qPCR on RNA harvested 4 h and 8 h post treatment, while protein expression was analysed by ELISA using cell culture supernatant samples harvested 8 h and 24 h post treatment. These experiments were complemented by gene and protein arrays. Results showed that allicin had a more pronounced upregulatory effect on LPS-induced gene expression 4 h post-LPS treatment. In contrast, Z-ajoene generally had mild downregulatory effects on the expression of LPS-induced genes. Conversely, Z-ajoene had pronounced downregulatory effects on LPS-induced inflammatory proteins after 24 h, while allicin showed mild up- or downregulatory effects. Overall, we found that allicin induced an initial pro-inflammatory gene response, while Z-ajoene induced a longer-lasting anti-inflammatory response at a protein level. Finally, as many of the inflammatory genes investigated are regulated by the transcription factor STAT3, we investigated the effects of allicin and Z-ajoene on STAT3 phosphorylation and hence activation. Western blot analyses showed that allicin increased LPS-induced STAT3 phosphorylation (2-8 h), while Z-ajoene was found to decrease the phosphorylation of STAT3 after 4 h. These effects on STAT3 phosphorylation are in agreement with the early pro-inflammatory effect of allicin and the later anti-inflammatory effect of Z-ajoene on LPS-induced gene and protein expression. Further, using Western blotting we showed that E/Z-ajoene directly interacts with and reversibly alkylates STAT3 via a thiol-disulfide reaction with a cysteine thiolate on STAT3. DA - 2015 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2015 T1 - The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation TI - The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation UR - http://hdl.handle.net/11427/15735 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/15735
dc.identifier.vancouvercitationHitchcock JK. The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry, 2015 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/15735en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Medical Biochemistryen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedical Biochemistryen_ZA
dc.titleThe immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammationen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMSc (Med)en_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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