Sasaran aksi kurkumin dan pentagama vunon-0 pada steroidogenesis :: Kajian kadar cAMP, fosforilasi extracellular signal regulated kinase (ERK) dan ekspresi sitokrom P450SCC pada kultur sel luteal

PURWANINGSIH, Endang, PromotorProf. dr. Sri Kadarsih Soejono, MSc., PhD

2009 | Disertasi |

Steroidogenesis sel luteal melibatkan aktivitas hormon antara lain LH dan PGF2α. LH bersifat gonadotropik sedangkan PGF2α bersifat antigonadotropik. Dengan rangsangan hormon-hormon tersebut, sel luteal dapat mensekresi progesteron dengan melibatkan aktivitas adenilat siklase, pembentukan cAMP, fosforilasi MAP Kinase ERK dan ekspresi enzim steroidogenik sitokrom P450scc. Senyawa kurkumin (1,7 bis (4’-hidroksi-3’metoksifenil)-1,6 heptadiena-3’,5’-dion) dan analog kurkumin Pentagamavunon-0/PGV-0 (2,5-bis (4’-hidroksi-3’-metoksi-benzilidin) siklopentanon dapat menghambat steroidogenesis kultur sel luteal dengan menghambat produksi progesteron. Letak kerja kurkumin dan PGV-0 pada steroidogenesis pada sel luteal belum diketahui. Tujuan penelitian adalah mengkaji letak kerja kurkumin dan PGV-0 terhadap kadar cAMP, fosforilasi MAP Kinase ERK dan ekspresi enzim sitokrom P450scc oleh kultur sel luteal. Subyek penelitian adalah korpus luteum umur 4 hari (fase mid luteal) dari tikus galur Sprague Dawley umur 28 hari setelah diinduksi dengan pregnant mare’s serum gonadotropin (PMSG)10 IU. Korpus luteum selanjutnya didispersi secara enzimatik dengan kolagenase. Sel luteal hasil isolasi dikultur dalam cawan kultur 24 dan 96 sumuran yang mengandung.minimal essential medium (MEM) dan fetal bovine serum (FBS) 10 % hingga tercapai pertumbuhan konfluen. Kurkumin dan PGV-0 dosis 100 μM diberikan sesaat setelah stimulasi LH dan atau PGF2α dengan atau tanpa teofilin /forskolin. Pada kelompok kontrol diberikan senyawa pelarut kurkumin/PGV-0. Semua perlakuan diinkubasi selama 24 jam. Untuk menilai aktivitas steroidogenesisnya, kadar progesteron dalam media kultur diukur dengan metode Radioimmunoassay (RIA). Sampel sel luteal yang melekat pada coverslip dipulas dengan metode immunohistochemistry (IHC) untuk menilai fosforilasi ERK dan ekspresi sitokrom P450scc. Kadar cAMP sel luteal dinilai dengan metode Enzyme Linked Immunosorbent Assay (ELISA). Stimulasi LH (50 ng/ml) pada kultur sel luteal meningkatkan kadar cAMP, fosforilasi ERK, ekspresi sitokrom P450scc dan diikuti peningkatan bermakna produksi progesteron. Teofilin meningkatkan akumulasi cAMP diikuti peningkatan produksi progesteron hampir setinggi stimulasi LH. Forskolin meningkatkan fosforilasi ERK diikuti peningkatan bermakna produksi progesteron. Kurkumin (100 μM) mampu menghambat produksi progesteron basal dan menghambat stimulasi LH dalam meningkatkan produksi progesteron. Forskolin meningkatkan produksi progesteron meskipun diberikan kurkumin. Pada kelompok yang distimulasi LH forskolin tidak mampu meningkatkan produksi progesteron dengan adanya kurkumin. Berbeda dengan pemberian PGV-0 forskolin dapat meningkatkan produksi progesteron dengan adanya stimulasi LH. Kurkumin memperkuat hambatan produksi progesteron oleh PGF2α (0,56 μM). Kurkumin juga menunjukkan hambatan pada akumulasi cAMP oleh stimulasi LH maupun teofilin. Akumulasi cAMP oleh PGF2α tidak dihambat oleh kurkumin. PGV-0 tidak menyebabkan hambatan pada akumulasi cAMP kelompok pelarut maupun kelompok yang distimulasi LH maupun teofilin. PGV-0 menghambat fosforilasi ERK dan ekspresi sitokrom P450scc kelompok pelarut maupun kelompok yang distimulasi LH dan atau PGF2α. Berdasarkan hasil penelitian ini disimpulkan bahwa pada steroidogenesis kultur sel luteal, kurkumin menghambat sinyal transduksi di up stream adenilat siklase, up stream cAMP, up stream MAP Kinase ERK dan up stream sitokrom P450sc. PGV-0 menghambat sinyal transduksi di down stream adenilat siklase, down stream cAMP, up stream MAP Kinase ERK dan di up stream sitokrom P450scc.

Steroidogenesis of luteal cell involves the activity of hormones such as LH and PGF2α. LH is gonadotropic while PGF2α is antigonadotropic. By the stimulation of those hormones, luteal cell secretes progesterone by involving the activity of adenylate cyclase, the forming of cAMP, the phosphorylation of MAP Kinase ERK and the expression of steroidogenesis of cytochrome P450scc enzyme. Curcumin compound (1,7 bis (4’-hydroxy-3’methoxyphenyl)-1,6 heptadiene-3’, 5’-dione) and curcumin analog Pentagamavunon-0/PGV-0(2,5-bis (4’-hydroxy-3’- methoxy-benzylidene) cyclopentano-ne can inhibit steroidogenesis of luteal cell culture by inhibiting the production of progesterone. The action site of the curcumin and PGV-0 in steroidogenesis at luteal cells is still unknown. The objective of this study is to analyze the action site of curcumin and PGV-0 on the level of cAMP, the phosphorylation of MAP Kinase ERK and the expression of cytochrome P450scc enzyme by luteal cell culture. The subject was corpus luteum of rat Sprague Dawley aged 4 days (mid luteal phase) 28 days of age after induced with pregnant mare’s serum gonadotropin (PMSG)10 IU. The corpus luteum was then dispersed enzymatically with collagenase. The isolated result of luteal cell is cultured in culture plates 24 and 96 wells that contained minimal essential minimum (MEM) and fetal bovine serum (FBS) 10% until the confluent growth was reached. Curcumin and PGV-0 with a dosage of 100 μM was given shortly after the stimulation of LH and or PGF2α with or without theophyline/forskolin was given. The control group was given curcumin vehicle compound/PGV-0. All treatments were incubated for 24 hours. To assess the activity of steroidogenesis, the level of progesterone in culture media was measured with a method of Radioimmunoassay (RIA). Luteal cell sample attached to coverslip was smeared using a method of Immunohistochemistry (IHC) to assess phosphorylation of ERK and the expression of cytochrome P450scc. The level of cAMP of luteal cell was assessed using a method of Enzyme Linked Immunosorbent Assay (ELISA). LH stimulation (50 ng/ml) at luteal cell culture increased the level of cAMP, the phosphorylation of ERK, and the expression of cytochrome 450scc and followed by the increase of progesterone production significantly. Theophyline increased the accumulation of cAMP followed by the increase of progesterone production which was as high as the LH stimulation. Forskolin increased the phosphorylation of ERK followed by the increase of progesterone production significantly. Curcumin (100 μM) was able to inhibit the production of basal progesterone and inhibit LH stimulation in increasing the production of progesterone. Forskolin increased the production of progesterone although it was given curcumin. In the group stimulated by LH, forskolin was not able to increase the production of progesterone by the presence of LH stimulation. Curcumin strengthened the inhibition of progesterone production by PGF2α (0.56 μM). curcumin also showed the inhibition in the accumulation of cAMP by either LH or theophyline stimulation. The accumulation by PGF2α was not inhibited by curcumin. PGV-0 did not cause the inhibition in the accumulation of cAMP of either the vehicle group or the group stimulated by LH or theophyline. PGV-0 inhibited the phosphorylation of ERK and the expression of cytochrome P450scc in either the vehicle group or the group stimulated by LH or PGF2α. The conclusion of this study is that in steroidogenesis of luteal cell culture, curcumin inhibited the transduction signal in up stream adenylate cyclase, up stream cAMP, up stream MAP Kinase ERK and up stream sitokrom P450scc. PGV-0 inhibited the transduction signal in down stream adenilate cyclase, down stream cAMP, up stream MAP Kinase ERK and up stream cytochrome P450scc.

Kata Kunci : Steroidgenesis,Kurkumin dan Pentagamavunon-0 (PGV-0),Kadar cAMP,Fosforilasi Extracellular Signal Regulated Kinase (ERK),Ekspresi Sitokrom P450SCC,Kultur sel luteal