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Title:	Penentuan mekanisme anti-penuaan y-tokotrienol pada model 'stress-induced premature senescence (SIPS)' fibroblas kulit manusia
Authors:	Norhazira Abdul Rahim. (P42706)
Supervisor:	Prof. Madya Dr. Suzana Makpol
Keywords:	Penuaan Stress-induced premature senescence (SIPS) Y-Tokotrienol (GTT) Dissertations, Academic -- Malaysia
Issue Date:	14-Aug-2012
Description:	γ-Tokotrienol (GTT) merupakan salah satu sebatian diet yang berpotensi untuk melambatkan proses penuaan. Keupayaannya mengawalatur isyarat transduksi, pengekspresan gen, pengisyaratan sel dan apoptosis telah dilaporkan. Namun, mekanisme tindakan GTT dalam mencegah penuaan sel belum dijelaskan dengan tepat. Tujuan kajian ini adalah untuk menentukan mekanisme anti-penuaan oleh GTT dengan memberi tumpuan khusus kepada tapak jalan apoptosis pada model 'stress-induced premature senescence' (SIPS) sel diploid fibroblas manusia (HDFs). Pada awalnya, model SIPS dibangunkan untuk mewakili model penuaan in vitro, iaitu dengan mengaruh 20 μM hidrogen peroksida (H2O2) secara berpanjangan selama 2 minggu. Perlakuan GTT dilakukan dengan mengeram sel SIPS dengan 1 μM GTT. Seterusnya, kesan perlakuan GTT ke atas perubahan berkait-penuaan replikatif ditentukan melalui pemerhatian ke atas perubahan morfologi, aktiviti β-galaktosidase-berkaitan senesen (SA-β-gal), tahap kerosakan DNA, profil kitaran sel, panjang telomere, aktiviti telomerase dan pengekspresan gen-gen berkait-kerosakan DNA (TP53, PAK2, GADD45GIP1 dan RB1). Perubahan berkaitan apoptosis ditentukan dengan menilai pengaktifan enzim kaspase (kaspase-3, kaspase-8 dan kaspase-9), Annexin V-FITC, pembebasan sitokrom c dan pengekspresan gen-gen pro-apoptotik (BAX dan BID) dan anti-apoptotik (BCL2A1 dan BCL2L1). Analisis densitometri dijalankan untuk mengukur nisbah ekspresi protein Bax/Bcl-2. Aruhan berpanjangan H2O2 ke atas sel muda menyebabkan ciri-ciri perubahan molekul yang menyerupai sel senesen replikatif seperti pembesaran saiz sel, peningkatan aktiviti SA-β-gal, peningkatan kerosakan DNA, peningkatan taburan sel pada fasa G0/G1 serta pengurangan sel pada fasa S, pemendekan telomere, penurunan aktiviti telomerase dan peningkatan bilangan sel apoptotik. Perlakuan GTT terhadap SIPS menyebabkan penurunan aktiviti SA-β-gal, peningkatan aktiviti telomerase, peningkatan taburan sel pada fasa S serta pengurangan sel pada fasa G0/G1 dalam kitar sel dan penurunan aras GADD45GIP1 (p<0.05). Perlakuan GTT juga menyebabkan penurunan signifikan sel apoptotik peringkat awal yang ditunjukkan oleh pengurangan sel positif Annexin V-FITC, diikuti oleh penindasan pengaktifan kaspase-3 dan kaspase-9, penyusutan pembebasan sitokrom c dan penurunan aras ekspresi BAX (p<0.05). Selain itu, perlakuan GTT menyebabkan penurunan nisbah protein Bax/Bcl-2 pada sel SIPS (p<0.05). Secara keseluruhan, GTT melindungi sel senesen-aruhan H2O2 dengan mengawalatur perubahan molekul-berkait senesen yang melibatkan aktiviti telomerase, kitaran sel dan pengekspresan gen-gen berkait-kerosakan DNA, seterusnya menahan tapak jalan apoptosis-berperantaraan mitokondria melalui penyekatan kaskad apoptosis seiring dengan perencatan pengekspresan gen pro-apoptotik BAX. Penemuan ini mencadangkan mekanisme tindakan anti-penuaan oleh GTT adalah melalui perencatan tapak jalan apoptosis intrinsik mitokondria.,γ-Tocotrienol (GTT) is one of the potential dietary compounds that can delay aging. Its ability in modulating signal transduction, gene expression, cellular signaling and apoptosis were reported. However, its mechanism of action in preventing cellular senescence is not well defined. The aim of this study was to determine the anti-aging mechanism of GTT by focusing on apoptosis pathway in 'stress-induced premature senescence' (SIPS) model of human diploid fibroblasts (HDFs). Initially, SIPS model that represents in vitro model of cellular aging, was developed by prolong exposure to 20 μM hydrogen peroxide (H2O2) for 2 weeks. GTT treatment was conducted by incubating SIPS cells with 1 μM GTT. Then, the effects of GTT on replicative senescence-associated changes were observed by determining morphological changes, senescence-associated β- galactosidase activity (SA-β-gal), level of DNA damage, cell cycle profile, telomere length, telomerase activity and expression of DNA-damage associated genes (TP53, PAK2, GADD45GIP1 dan RB1). Apoptotic-related changes were determined by evaluating caspases enzyme activation (caspase-3,caspase-8 and caspase-9), Annexin VFITC, cytochrome c release and expression of pro-apoptotic genes (BAX and BID) and anti-apoptotic genes (BCL2A1 dan BCL2L1). Densitometric analysis was carried out to quantify the ratio of Bax/Bcl-2 protein expression. Prolong exposure of H2O2 to young fibroblasts caused molecular changes similar to replicative senescent cells such as enlargement of cells size, increased SA-β-gal activity, increased DNA damage, increased population of G0/G1 phase cells with reduced cells in S phase, shortened telomere, decreased telomerase activity and increased number of apoptotic cells. GTT treatment against SIPS resulted in reduction in SA-β-gal activity, increased telomerase activity, increased population of S phase cells with reduced G0/G1 cells and down-regulation of GADD45GIP1 expression level (p<0.05). GTT treatment also caused a significant reduction of early apoptotic cells as indicated by decreased Annexin V-FITC positive cells, followed by suppression of caspase-3 and caspase-9 activation, reduction of cytochrome c release and down-regulation of BAX expression level (p<0.05). In addition, GTT treatment caused a reduction of Bax/Bcl-2 protein ratio in SIPS (p<0.05). In summary, GTT protects against H2O2-induced cellular senescence by modulating molecular changes associated with senescence involving telomerase activity, cell cycle and DNA-damage related genes, which subsequently restrains the mitochondria-mediated apoptosis pathway through inhibition of apoptosis cascade with concomitant suppression of pro-apoptotic gene BAX. These findings suggested that the mechanism of action of GTT as an anti aging compound is via inhibition of intrinsic mitochondria apoptosis pathway.,Ph.D
Pages:	273
Call Number:	WR102 .N839p 2012 9
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Medicine / Fakulti Perubatan

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