The Effect Of Propolis Extract From Trigona Spp. On Vascular Inflammatory Mediators And Its Molecular Mechanism In TNF-Alpha-Stimulated Endothelial Cells
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Date
2019-08
Authors
Asem, NorNaimah
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Abstract
Propolis is one of the bee products which contained multiple chemical compounds, namely polyphenols, waxes, steroids and terpenoids. Its contents may be influenced by the plant sources, geographical area as well as the bee species. A wide array of natural products have been used as anti-inflammatory and healing agents, with propolis being a remarkable option. Therefore, the objective of this study is to examine the phytochemical properties of the selected propolis extracts and its effects on the expression of adhesion molecules, as well as the molecular mechanisms involved, particularly the investigations on the nuclear factor-kappa B (NF-ĸB) signalling pathways in cultured endothelial cell. In the present study, the phytochemical screening of ethanolic extract of propolis (EEP) from local stingless bee species namely Tetrigona apicalis, Geniotrigona thoracica and Heterotrigona itama were investigated using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analysis. The total phenolic and flavonoid contents, and antioxidant activities which include 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assay were explored. Subsequently, the flow cytometry analysis was carried out to identify the potency of EEP in inhibiting the production of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on tumor necrosis factor-alpha (TNF-α) stimulated human umbilical vein endothelial cell (HUVEC). The molecular mechanism at protein and gene level of EEP from G. thoracica effects on NF-κB signalling pathway was examined using western immunoblot assay. In addition, the gene expression study of mRNA level of NF-κB1 and adhesion molecules was also performed using the 2 steps Reverse Transcription-quantitative Real Time PCR (RT-qPCR) analysis. The phytochemical screening by HPLC exhibited a chromatographic characteristic of each EEP from the 3 species but not similar to reference standards. Terpenoids were the major compounds among the volatile substances found in all EEP of GC-MS analysis. In addition, the 2-methoxy-4-vinylphenol (2M4VP) was the major compound found only in EEP from G. thoracica. A strong correlation was observed between the total phenolic and flavonoid content, and its antioxidant properties (range between R2 = 0.763 and R2 = 0.971); and the production of ICAM-1 (range between R2 = 0.730 and R2 = 0.976) and VCAM-1 (range between R2 = 0.835 and R2 = 0.983). The western blot analysis shows the percentage of EEP from G. thoracica inhibited the IKK, IκB and NF-κB p65 protein expression about 34.7%, 87.3% and 38.2%, respectively as compared to the TNF-α-stimulated control. Moreover, the RT-qPCR results demonstrated that the EEP from G. thoracica reduced the expression of VCAM-1- and NF-κB1-mRNA to the basal levels after the stimulation of TNF-α on HUVEC. In conclusion, EEP from G. thoracica is believed to down-regulate the protein and gene expression through NF-κB signalling pathway. Therefore, EEP is revealed to be a potential anti-inflammatory agent and have beneficial characteristics for the treatment of vascular diseases.
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Medicine