Immunomodulating properties of Sabah Seaweeds Kappaphycus Striatus and Eucheuma Denticulatum

Pan, Shing Yi (2016) Immunomodulating properties of Sabah Seaweeds Kappaphycus Striatus and Eucheuma Denticulatum. Doctoral thesis, Universiti Malaysia Sabah.

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Abstract

Seaweeds which are high in vitamins, minerals, proteins, polysaccharides and dietary fibers have been consumed traditionally in many East Asia countries as functional food and as sources of medicinal drugs for health promotion and disease prevention. Despite of the vast benefits, documentation on the immunological properties of Sabah seaweeds is scarce. Immunomodulators are capable to augment the immune system in order to provide protection against infections and improve our health. Thus, this study investigated the water and ethanolic extracts of Kappaphycus striatus and Eucheuma denticulatum that are widely cultured in Sabah and of economic importance. Ex vivo experiments were conducted using Balb/c mice spleens for NK cells activities, splenocytes proliferation and serum was used for Th-1 cytokine IFN-y and Th-2 IL-4. Macrophage proliferation assay, nitric oxide production and wound healing assay were evaluated in vitro using RAW 264.7 and 3T3 cells. The K. striatu5 water extracts and ethanolic extracts of both seaweed samples could significantly increase the proliferation of RAW264.7 cells in a concentration-dependent manner (50 - 200 IJg/mL). Both water extracts at 12.5 - 200 IJg/mL were able to significantly increase considerable amounts of nitrite, while the production of nitrite was significantly inhibited when LPS-induced RAW 264.7 were incubated with the water extracts. This indicates that the water extracts of K. striatu5 and E denticulatum possess anti-inflammatory activity. £ denticulatum ethanolic extracts at 100 IJg/mL stimulated the migration of 3T3 fibroblasts in scratch assay indicated they exhibited wound healing properties. In addition, Balb/c mice administered with 50 mg/kg of £ denticulatum ethanolic extracts showed significant proliferation in all ex vivo tests suggesting that it is the best concentration to significantly immunomodulate the immune system. Gene expression profile were further investigated in mice treated with £ denticulatum ethanolic extracts at 50 mg/kg and 100 IJg/mL to further understand the molecular mechanism involved in the immunomodulation. Gene expression results showed that the ethanolic extracts were involved in T-cell Signaling pathway via the CDBBl and CD3G cell surface receptors. This lead to subsequent multiple downstream signaling pathways including calcium-calcineurin-NFAT, MAPK and NF-KB signaling pathways which could further activate downstream targets P38, NFAT, AP-l, NF-KB to induce cell proliferation, differentiation and production of cytokines. Increased expressions were shown in CDBBl and CD3G surface receptor genes, at a foldchange of 1.60 (,0=0.039) and 1.49 (,0=0.026) respectively whereas slight increased expressions which were not significant were shown in NFATl, at 1.21 (,0=1.53) and IFN-y, at 1.54 (,0=0.14) indicating that the extracts serve as a strong modulator in activating the surface receptors which could lead to the proliferation and activity of lymphocytes (T cells and NK cells), macrophage activation which mediate calcium-calcineurin-NFAT signaling pathway. In addition, the molecular mechanisms of cell-adhesion activities were also investigated. Wound healing activity involves the signal transduction and interaction of ECM to cell adheSion receptor integrins, cytoplasmic kinases, growth factors and cytokines. SPP1, an ECM protein with a fold-change of 2.11 (,0=2.11) which interacts and bind to ITGB3 cell surface integrins, with a fold-change of 1.44 (,0=0.004) followed by stimulating cell-to-cell and cell-to-ECM adheSion, were found to be significantly expressed in Seaweeds which are high in vitamins, minerals, proteins, polysaccharides and dietary fibers have been consumed traditionally in many East Asia countries as functional food and as sources of mediCinal drugs for health promotion and disease prevention. Despite of the vast benefits, documentation on the immunological properties of sabah seaweeds is scarce. Immunomodulators are capable to augment the immune system in order to provide protection against infections and improve our health. Thus, this study investigated the water and ethanolic extracts of Kappaphycus striatus and Eucheuma denticulatum that are widely cultured in sabah and of economic importance. Ex vivo experiments were conducted using Balb/c mice spleens for NK cells activities, splenocytes proliferation and serum was used for Th-l cytokine IFN-y and Th-2 IL-4. Macrophage proliferation assay, nitric oxide production and wound healing assay were evaluated in vitro using RAW 264.7 and 3T3 cells. The K. striatus water extracts and ethanolic extracts of both seaweed samples could significantly increase the proliferation of RAW264.7 cells in a concentration-dependent manner (50 - 200 jJg/mL). Both water extracts at 12.5 - 200 jJg/mL were able to significantly increase considerable amounts of nitrite, while the production of nitrite was significantly inhibited when LPS-induced RAW 264.7 were incubated with the water extracts. This indicates that the water extracts of K. striatus and E denticulatum possess anti-inflammatory activity. E. denticulatum ethanolic extracts at 100 jJg/mL stimulated the migration of 3T3 fibroblasts in scratch assay indicated they exhibited wound healing properties. In addition, Balb/c mice administered with 50 mg/kg of E denticulatum ethanolic extracts showed significant proliferation in all ex vivo tests suggesting that it is the best concentration to significantly immunomodulate the immune system. Gene expression profile were further investigated in mice treated with E denticulatum ethanolic extracts at 50 mg/kg and 100 I.Ig/mL to further understand the molecular mechanism involved in the immunomodulation. Gene expression results showed that the ethanolic extracts were involved in T-cell Signaling pathway via the C0881 and C03G cell surface receptors. This lead to subsequent multiple downstream signaling pathways including calcium-calcineurin-NFAT, MAPK and NF-KB signaling pathways which could further activate downstream targets P38, NFAT, AP-l, NF-K8 to induce cell proliferation, differentiation and production of cytokines. Increased expressions were shown in C0881 and C03G surface receptor genes, at a foldchange of 1.60 (.0=0.039) and 1.49 (.0=0.026) respectively whereas slight increased expressions which were not significant were shown in NFAT1, at 1.21 (,0=1.53) and IFN-y, at 1.54 (,0=0.14) indicating that the extracts serve as a strong modulator in activating the surface receptors which could lead to the proliferation and activity of lymphocytes (T cells and NK cells), macrophage activation which mediate calcium-calcineurin-NFAT Signaling pathway. In addition, the molecular mechanisms of cell-adhesion activities were also investigated. Wound healing activity involves the signal transduction and interaction of ECM to cell adheSion receptor integrins, cytoplasmic kinases, growth factors and cytokines. SPP1, an ECM protein with a fold-change of 2.11 (.0=2.11) which interacts and bind to ITG83 cell surface integrins, with a fold-change of 1.44 (.0=0.004) followed by stimulating cell-to-cell and cell-to-ECM adheSion, were found to be significantly expressed in this study. Activation of SPPl protein and ITGBJ cell surface receptor could initiate the integrin-mediated signaling pathway followed by activation of downstream pathways such as cytoskeletal organization, PI3K-Akt signalling pathway and MAPK signalling pathway. Besides, SPPl and ITGBJ genes play an important role in cell proliferation, migration, differentiation, growth regulation which could accelerate fibroblast migration thus enhancing wound healing activity. Gene expression studies showed that Sabah seaweeds especially E denticulatum ethanolic extracts has immunomodulating activities by acting on cell surface receptors which could then mediate T-cell receptor Signaling and integrin-mediated signaling pathway. This study stipulated that all water and ethanolic seaweed extracts especially 50 mg/kg E denticulatum ethanolic extracts served as a strong immunomodulator in enhancing host defence mechanisms and immune system.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: Seaweeds, Sabah, E denticulatum ethanolic
Subjects: S Agriculture > SH Aquaculture. Fisheries. Angling
Divisions: FACULTY > Faculty of Science and Natural Resources
Depositing User: Noraini
Date Deposited: 03 Jan 2018 05:03
Last Modified: 03 Jan 2018 05:03
URI: http://eprints.ums.edu.my/id/eprint/17911

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