Mekanisme aktivitas kurkumin dan pentagamavunon-0 terhadap steroidogenesis dan apoptosis pada kultur sel luteal

HADI, Restu Syamsul, Promotor Prof. dr. Sri Kadarsih Soejono, M.Sc., Ph.D

2009 | Disertasi |

Kurkumin (1,7 bis (4’-hidroksi-3’-metoksifenil)-1,6-heptadiena-3’,5’-dion adalah senyawa utama yang terkandung dalam kunyit (Curcuma longa,L) yang telah dapat disintesis dan teruji aktivitas biologisnya. Aktivitas dan mekanisme kurkumin pada sistem reproduksi khususnya terhadap steroidogenesis dan apoptosis sel uteal belum dapat dijelaskan. Pentagamavunon-0 (2,5-bis-(4’-hidroksi-3’-metoksi-benzilidin) siklopentanon merupakan senyawa analog kurkumin dengan perubahan struktur pada gugus ß diketon menjadi gugus monoketon.Pentagamavunon-0 (PGV-0) dilaporkan mempunyai aktivitas biologis lebih baik daripada senyawa asalnya. Tujuan penelitian ini adalah untuk mengkaji mekanisme kurkumin dan senyawa analognya (PGV-0) terhadap steroidogenesis dan apoptosis pada kultur sel luteal.Subyek kajian ini adalah korpus luteum umur 3 hari yang diperoleh dari tikus Sprague-Dawley umur 28 hari setelah diinduksi dengan pregnant mare’s serum gonadotropin (PMSG) 10 IU. Korpus luteum selanjutnya didispersi secara enzimatik menggunakan collagenase. Sel luteal hasil isolasi ditanam dalam cawan kultur 24 dan 96 sumuran yang mengandung minimal essential medium (MEM) dan fetal bovine serum (FBS) 10 % sampai tercapai pertumbuhan konfluen. Kurkumin dan PGV-0 dengan onsentrasi 100 μM dan 400 μM diberikan sesaat setelah stimulasi dengan luteinizing hormone (LH), prostaglandin-F2a (PGF2a) dan LH+PGF2a. Pada kelompok kontrol diberikan senyawa pelarut kurkumin. Semua perlakuan diinkubasi selama 24 jam. Untuk menilai aktivitas steroidogenesisnya kadar progesteron (P4)yang terkandung dalam medium kultur diukur dengan menggunakan metode radioimmunoassay (RIA). Sampel sel luteal yang melekat pada coverslip dipulas menggunakan metode mmunositokimia untuk menguji ekspresi protein Bcl-2 dan Bax. Apoptosis sel luteal dengan kerusakan DNA dianalisis dengan menggunakan metode terminal deoxy-nucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). Viabilitas sel diukur dengan metode trypan blue dye exclusion.Pemberian LH (50 ng/ml) pada kultur sel luteal meningkatkan produksi P4.Kurkumin (100 μM) maupun PGF2a (0,56 μM) menghambat peningkatan produksi P4 oleh stimulasi LH. Kurkumin (400 μM) mampu menurunkan produksi P4 basal maupun menghambat stimulasi LH dalam meningkatkan produksi P4. Pemberian PGV-0 (100 μM ) mampu meningkatkan produksi P4 basal. Pemberian PGV-0 (400 μM) maupun PGF2a (0,56 μM) menghambat stimulasi peningkatan produksi P4 oleh LH. Pemberian LH mampu meningkatkan ekspresi protein Bcl-2, menurunkan ekspresi protein Bax dan menurunkan rasio Bax/Bcl-2. Kurkumin (100 μM) maupun PGF2a (0,56 μM) menghambat peningkatan ekspresi protein Bcl-2 oleh stimulasi LH. Pemberian PGF2a dan PGV-0 (100 μM) menurunkan ekspresi protein Bcl-2 dan meningkatkan ekspresi protein Bax. PGV-0 (400 μM) menurunkan ekspresi protein Bcl-2, meningkatkan ekspresi protein Bax, meningkatkan rasio Bax/Bcl-2 dan menginduksi terjadinya apoptosis sel luteal. Berdasarkan hasil penelitian ini disimpulkan bahwa mekanisme kurkumin dan PGV-0 terhadap steroidogenesis pada sel luteal dengan menghambat stimulasi produksi P4 oleh LH pada fase luteal awal. Kemampuan kurkumin menghambat produksi P4 lebih kuat daripada PGV-0.Kurkumin dan PGV-0 memiliki aktivitas sebagai proapoptosis pada sel luteal dengan cara menurunkan hambatan apoptosis oleh LH tergantung dosis. Kurkumin dan PGV-0 memperlihatkan potensi untuk dikembangkan dalam pengaturan kesuburan.

Curcumin (1,7 bis (4’-hydroxy-3’-methoxyphenyl)-1,6-heptadiene-3’,5’-dione, a major constituent in turmeric (Curcuma longa,L), was synthesized and examined for its biological activity. The activity and mechanism in reproductive system especially in luteal steroidogenesis and apoptosis has never been elucidated. Pentagamavunon-0 (2,5-bis-(4’-hydroxy-3’-methoxy-benzylidene)cyclopentanone is a synthetic analog of curcumin with structure modification on ß diketone group of curcumin is carried out to yield monoketon group.Pentagamavunon-0 (PGV-0) is reported previously to have better biological activity than curcumin. The aims of this research were to study the mechanism of action of curcumin and PGV-0 on steroidogenesis and apoptosis in cultured rat luteal cells.Subject in this study was 3 days old corpus luteum of rat Sprague-Dawley at 28 days of age after induced by 10 IU pregnant mare’s serum gonadotropin (PMSG). Corpora lutea were dispersed enzimatically by collagenase. Isolated luteal cells were seeded in 24 and 96 well culture plates containing minimal essential medium (MEM) and 10 % fetal bovine serum (FBS) until cell had grown to confluence. Curcumin and PGV-0 in the concentrations 100 μM and 400 μM were immediately treated after they were stimulated by luteinizing hormone (LH),prostaglandin-F2a (PGF2a) and LH+PGF2a. Vehicle was administered as control group. All these treatments were incubated for 24 hours. The activity of steroidogenesis were calculated by measured progesterone (P4) levels contained in culture medium using the adioimmunoassay (RIA) method. To examine of Bcl-2 and Bax protein expression, sample of plated luteal cells on coverslip were stained by immunocytochemical method. Apoptotic luteal cells were analyzed for DNA break by terminal deoxy-nucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) method. Cell viability was measured by trypan blue dye exclusion method.The administration of LH (50 ng/ml) to luteal cell culture increased P4 levels.Treatment of curcumin (100 μM ) or PGF2a (0.56 μM) inhibited LH-increased P4 production. Curcumin (400 μM ) decreased basal P4 production and inhibited LHinduced P4 production. Administration of PGV-0 (100 μM) increased basal P4 production. Treatment of PGV-0 (400 μM) or PGF2a (0.56 μM) inhibited LHstimulated P4 production. LH administration increased Bcl-2 protein expression,decreased Bax protein expression and decreased Bax/Bcl-2 protein ratio. Curcumin (100 μM) or PGF2a (0.56 μM ) inhibited LH-stimulated Bcl-2 protein expression.Administration of PGF2a and PGV-0 (100 μM) decreased Bcl-2 and increased Bax protein expression. PGV-0 (400 μM) decreased Bcl-2 protein expression, increased Bax protein expression, increased Bax/Bcl-2 protein ratio and induced apoptotic luteal cells. Based on the result of the research, it was concluded that the mechanism of curcumin and PGV-0 on luteal cell steroidogenesis may caused by inhibited LH-stimulated progesterone synthesis in the early luteal phase. Curcumin had more potent to inhibited progesterone productions than PGV-0. Curcumin and PGV-0 has proapoptotic activity on luteal cell with decreased apoptotic inhibition by LH in dose dependent mechanism. The property of curcumin and PGV-0 suggests that these molecules could have a possible potential to develop on fertility regulation.

Kata Kunci : Pertumbuhan konfluen, Steroidogenesis, Sel luteal,Apoptosis sel luteal, Kurkumin, Pengaturan kesuburan