Kajian Terhadap Ekspresi Receptor Advanced Glycation End Products (RAGE), EKSPRESI ENZIM SIKLOOKSIGENASE-2 (COX-2) dan Kadar Testosteron Kultur Sel Leydig Tikus Sprague Dawley yang Diinduksi Advanced Glycation End Products dan Diinkubasi dengan gamma mangostin

DICKY MOCH. RIZAL, DR., M.KES., SP.AND , Prof. dr. Sri Kadarsih, MSc, PhD; Prof. dr. Moch. Anwar HSA., MMedSc, SpOG(K)

2015 | Disertasi | S3 Ilmu Kedokteran

Salah satu permasalahan reproduksi pria penurunan produksi hormon testosteron merupakan penyebab dari infertilitas, gangguan seksual, gangguan metabolik, osteoporosis maupun penuaan dini.Hipogonadisme yang terkait dengan hiperglikemia berkaitan dengan adanya peran Advanced Glycation End Products (AGE) sebagai salah satu patofisiologi terjadinya diabetes mellitus.Paparan AGE dapat menyebabkan peningkatan ekspresi receptor AGE (RAGE) yang selanjutnya membentuk ikatan AGE-RAGE. Ikatan inilah yang kemudian mengaktifkan sinyal pembentukan NFkB yang salah satu efeknya adalah peningkatan ekspresi enzim siklooksigenase 2 (COX-2) yang memicu terjadinya proses inflamasi dan dapat berakhir dengan gangguan fungsi sel antara lain jika mengenai sel Leydig akan mengakibatkan penurunan produksi testosteron. Gamma mangostin, salah satu komponen utama ekstrak kulit manggis, diketahui punya efek anti inflamasi.Perlu dibuktikan paparan AGE dapat meningkatkan ekspresi RAGE (yang selanjutnya membentuk ikatan AGE-RAGE), ekspresi COX-2 dan mengakibatkan penurunan fungsi sel Leydig menghasilkan testosteron serta gamma mangostin dapat menghambat penurunan testosteron melalui penghambatan ekspresi RAGE serta COX-2. Subyek penelitian ini adalah kultur sel Leydig tikus Sprague Dawley jantan umur 3-4 bulan. Setelah hasil isolasi dan kultur sel Leydig konfluen, maka sel Leydig dipindahkan ke dalam sumuran dengan jumlah sel 105untuk masing-masing sumuran. Dibuat 13 kelompok yang terdiri dari kelompok kontrol (tanpa AGE-BSA) dan kelompok perlakuan (diinduksi AGE-BSA saja dan diinduksi AGE-BSA+diinkubasi gamma mangostin selama 24 jam). Ekspresi RAGE, COX-2 dan kadar testosteron diperiksa menggunakan metode ELISA dari cairan supernatan dan medium kultur. Data yang didapatkan adalah rerata +- SEM masing-masing kelompok. Analisis statistik menggunakan uji Mann Whitney untuk mengetahui perbedaan rerata+- SEM antar kelompok Rerata+-SEM ekspresi RAGE bebas kelompok tanpa AGE-BSA lebih tinggi dibandingkan kelompok dengan AGE-BSA 100,200,400ug/ml (14,4704 +-0,0523 vs13,9498+- 0,4184; 11,8703+-0,231; 13,4775+-0, 098 ng/105 sel/24 jam). (p<0,05) Rerata ekspresi RAGE bebas kelompok diinduksi AGE-BSA saja lebih tinggi dibandingkan kelompok yang diberi AGE + gamma mangostin. (p>0,05) Rerata+-SEM ekspresi COX-2 kelompok tanpa AGE-BSA lebih rendah dibandingkan dengan kelompok AGE- BSA100,200,400ug/mL(8,1410+-0,1586vs8,4907+-0,7831;10,9336;11,0909 ng/105 sel/24 jam (p>0,05) Rerata+-SEM ekspresi COX-2 kelompok AGE-BSA saja dosis 100,200,400ug/ml lebih rendah dibandingkan kelompok AGE-BSA+gamma mangostin dosis 5,10,20uM. (p>0,05) Kadar testosteron kelompok tanpa AGE lebih tinggi dibandingkan dengan kelompok AGE-BSA 100,200,400ug/ml (1,4387+-0,0270vs1,3761+-0,0252;1,3547+-0,018;1,3413+-0,0624 ng/105 sel/24 jam (p>0,05).Kadar testosteron kelompok yang diinduksi AGE-BSA +gamma mangostin 5,10,20uM lebih rendah dibandingkan kelompok AGE-BSA (p>0,05). Kesimpulan penelitian ini adalah ekspresi RAGE bebas kultur sel Leydig yang tidak diinduksi AGE-BSA lebih tinggidibandingkan yang diinduksi AGE-BSA. Ekspresi COX-2 dan kadar testosteron kultur sel Leydig yang tidak diinduksi AGE- BSA tidak berbeda dibanding kultur sel Leydig yang diinduksi AGE-BSA.Ekspresi RAGE bebas, COX-2 dan kadar testosteron kultur sel Leydig yang diinduksi AGE- BSA saja tidak berbeda dibanding kultur sel Leydig yang diinduksi AGE-BSA dan diinkubasi gamma mangostin

Decreasing of testosterone production is one of the male reproductive problems lead to infertility, sexual disorders, metabolic disorders, osteoporosis and premature aging. Hypogonadism associated with hyperglycemia refer to the role of Advanced Glycation End Products (AGE) as one of the pathophysiology of diabetes mellitus. AGE exposure can lead to increased expression receptor AGE(RAGE), which subsequently form the AGE-RAGE complex activates NFkB signaling stimulate the expression of the enzyme cyclooxygenase 2 (COX-2) that trigger the inflammatory process and may end up with impaired cell function, if it happen in the Leydigcells will lead to a decreasing testosterone production. Gamma mangostin, one of the main components of mangosteen peel extract, known to have anti- inflammatory effects. AGE induced Leydig cells need to be proven increased of RAGE expression(hereinafter bonding AGE-RAGE), COX-2 expression and resulted in a decrease in testosterone production and gamma mangostin can inhibit deterioration of testosterone by inhibiting the expression of RAGE and COX-2. The subjects are isolated Leydig cells Sprague Dawley rats aged 3-4 months followed by culturing. Once confluent, the Leydig cells were transferred into the wells with a cell number 105 per well. There were 13 groups consisting of a control group (without AGE-BSA) and the treatment group (induced AGE-BSA alone 100, 200, 400ug/ml and given AGE-BSA plus gamma mangostin 5,10,20uM) and incubated for 24 hours. RAGE expression, COX-2 and testosterone levels were measured using ELISA from the supernatant and culture medium. The data obtained are mean +- SEM of each group. Statistical analysis using the Mann Whitney test to determine differences between groups mean +- SEM. The expression of free RAGE of no AGE-BSA group were higher than the group with AGE 100,200,400ug /mL(14,4704 +-0,0523 vs 13,9498+- 0,4184; 11,8703+-0,231; 13,4775+-0, 098 ng/105 sel/24 hours). (p<0,05) The expression free RAGE of AGE-BSA groups were higher than the group induced AGE-BSA plus gamma mangostin5,10,20uM (p>0,05) . Expression of COX-2 no AGE-BSA group were lower than the AGE-BSA group (8,1410+-0,1586vs8,4907+-0,7831;10,9336;11,0909 ng/105 sel/24hours) (p>0,05) Expression of COX-2 from AGE-BSA 100ug/ml were lower than AGE-BSA100ug/ mlgroup+gamma mangostin 5,10,20uM. (p>0,05) Expression of COX-2 from AGE- BSA group were lower than AGE-BSA + gamma mangostin 5,10,20uM dose (p>0,05). Testosterone levels culture group without the AGE-BSA were higher than the AGE-BSA group (1,4387+-0,0270vs1,3761+-0,0252;1,3547+-0,018;1,3413+-0,0624 ng/105 sel/24 hours)(p>0.05). Testosterone levels in the AGE-BSA groups higher than that induced AGE-BSA + gamma mangostin 5,10,20uM, (p> 0.05). The conclusion of this study were the expression of free RAGE on Leydig cell culture AGE-BSA free higher compared with those induced AGE-BSA. No significant differences neither the expression of COX-2 or testosterone levels in Leydig cell cultures without AGE-BSA and induced AGE-BSA. No significant differences neither the expression of free RAGE, COX-2 or testosterone levels in Leydig cell cultures AGE-BSA induced only and incubation with gamma mangostin

Kata Kunci : kultur sel Leydig, Advanced Glycation End-products, gamma mangostin, testosterone, reproduksi pria , Leydig cell culture, Advanced Glycation End-products, gamma mangostin, testosterone, male reproductive