Zulaikah, Siti (2014) Pioglitazone Menurunkan Produksi Nitric Oxide melalui Peningkatan Ekspresi mRNA PPARγ oleh Kondrosit yang Diinduksi IL-1β. Magister thesis, Universitas Brawijaya.
Abstract
Osteoarthritis (OA) merupakan salah satu penyakit sendi yang paling banyak diderita pada orang dewasa. Di Indonesia tercatat 8,1% dari total penduduk adalah penderita OA. Di Kabupaten Malang dan Kota Malang ditemukan prevalensi OA sebesar 10% dan 13,5%. Interleukin 1β merupakan sitokin proinflamasi yang berperan pada patogenesa OA. Pada kondrosit OA banyak menghasilkan IL-1β yang akan menginduksi peningkatakan sekresi matriks metalloproteinase (MMPs) dan nitric oxide (NO) . Produksi NO yang berlebih akan meningkatkan sintesis MMP, menghambat sintesis kolagen dan proteoglikan dimana hal ini akan berakibat degradasi matriks semakin meningkat. Penelitian sebelumnya menemukan bahwa pada kondrosit pasien OA yang diberi perlakuan IL-1β terjadi penurunan ekspresi peroxisome proliferator-activated receptor γ (PPARγ). PPARγ merupakan suatu reseptor nukleus yang berperan dalam respon inflamasi. Berbagai penelitian untuk menemukan kandidat terapi OA, salah satunya menggunakan ligan atau agonis PPARγ. Pemberian pioglitazone, salah satu ligan sintetis PPARγ, pada hewan model OA mampu menurunkan sintesis MMP-13 dan IL-1β. Berdasarkan hal tersebut perlu dilakukan penelitian mengenai pengaruh pemberian pioglitazone dalam menurunkan produksi NO pada kondrosit yang diinduksi IL-1β. Proposal penelitian ini telah diajukan ke Komite Etik Penelitian Kesehatan Fakultas Kedokteran Universitas Brawijaya dan telah mendapatkan Surat Kelayakan Etik. Desain penelitian ini adalah eksperimental laboratorik dengan membandingkan hasil yang didapat sesudah perlakuan dengan kontrol ( post - test only control group design ). Penelitian ini menggunakan sel line normal human articular chondrocyte (NHAC - kn ). Beberapa prosedur yang dilakukan adalah kultur kondrosit sel line pada kultur monolayer, redifferensiasi sel kondrosit menggunakan alginat beads untuk mengembalikan sifat kondrosit yang telah berubah menjadi kondrosit yang menyerupai fibroblas, pengukuran konsentrasi kolagen tipe 2 sebagai marker kondosit normal, pemberian IL-1β pada kondrosit normal untuk menginduksi menjadi kondrosit yang menyerupai kondrosit OA, pengukuran konsentrasi MMP-13 dan pengamatan hipertrofi kondrosit sebagai marker kondrosit OA. Pemberian pioglitazone tiga dosis yaitu 0,1 μM,1 μM dan 10 μM pada kondrosit yang diberi perlakuan IL-1β untuk memberikan efek terapi, pengukuran konsentrasi NO dengan metode colorimetry assay , Isolasi RNA dan sintesis cDNA untuk pengukuran ekspresi mRNA PPARγ. Ekspresi mRNA PPARγ diukur dengan menggunakan metode real time PCR (Roche). Analisa statistik menggunakan uji one way anova untuk mengetahui perbedaan pengaruh pioglitazon berbagai dosis terhadap ekspresi mRNA PPARγ dan kadar NO, dilanjutkan uji post hoc tukey untuk membandingkan antar perlakuan. Selanjutnya dilakukan uji korelasi Pearson untuk mengetahui hubungan antara dosis pioglitazone dengan peningkatan ekspresi mRNA PPARγ, hubungan antara dosis pioglitazone dengan penurunan kadar NO dan hubungan antara peningkatan ekspresi mRNA PPARγ dengan penurunan kadar NO. Uji regresi linear digunakan untuk menentukan model regresi yang dapat digunakan untuk memprediksi di masa mendatang peningkatan atau penurunan ekspresi mRNA PPARγ dan kadar NO dengan penambahan atau pengurangan dosis pioglitazone. Hasil penelitian selama kurun waktu Desember 2013 sampai dengan Juni 2014 adalah pengukuran konsentrasi kolagen tipe 2 pada kondrosit yang telah diredifferensiasi lebih tinggi (77,9 ng/ml) dan berbeda nyata dibandingkan konsentrasi kolagen tipe 2 pada kondrosit yang menyerupai fibroblas (15,6 ng/ml). Hal ini menunjukkan bahwa kondrosit yang menyerupai fibroblas telah kembali menjadi kondrosit normal. Pada pengukuran konsentrasi MMP-13 sebagai biomarker kondrosit OA, konsentrasi MMP-13 pada kondrosit yang diberi perlakuan IL-1β lebih tinggi (7,947 ng/ml) dan berbeda nyata dibandingkan konsentrasi MMP-13 pada kondrosit normal (1,421 ng/ml) (p 0,05). Pemeriksaan mikroskopis kondrosit yang diberi perlakuan IL-1β, terlihat kondrosit mengalami hipertrofi, ditandai dengan kondrosit semakin besar ( swelling ). Ekspresi mRNA PPARγ pada kondrosit yang diiinduksi IL-1β mengalami penurunan (0,64) dan penurunannya berbeda nyata (p 0,05, uji post hoc LSD ) dibandingkan pada kondrosit normal (6,11). Perlakuan pioglitazone dengan tiga dosis yang berbeda mampu meningkatkan ekspresi mRNA PPARγ dan berbeda nyata dibandingkan dengan ekspresi mRNA PPARγ kondrosit yang diinduksi IL-1β saja, namun antar dosis perlakuan tidak berbeda nyata (p 0,05, uji post hoc LSD ). Ekspresi mRNA PPARγ pada kondrosit yang diinduksi IL-1β ditambah pioglitazone dosis 0,1=5,48, dosis 1μM=5,59 dan dosis 10μM=6,15. Uji korelasi Pearson menunjukkan bahwa terdapat hubungan yang kuat antara pemberian pioglitazone ketiga dosis dengan peningkatan ekspresi mRNA PPARγ, dan hasil uji regresi linear untuk memprediksi peningkatan
English Abstract
Osteoarthritis (OA) is a joint disease that is most common in adults. In Indonesia recorded 8.1% of total population are people with OA. In Malang Malang and found prevalence of OA by 10% and 13.5%. Interleukin-1β is a proinflammatory cytokine that plays a role in pathogenesis of OA. In OA chondrocytes produce IL-1β much higher who would increase secretion of matrix metalloproteinase (MMPs) and nitric oxide (NO). Excess NO production will increase MMP syn sis, inhibiting syn sis of collagen and proteoglycans that this will result in increased matrix degradation. Previous research found that in patients with OA chondrocytes treated with IL-1β expression of peroxisome proliferator decline-activated receptor γ (PPARγ). PPARγ is a nucleus receptor that plays a role in inflammatory response. Various studies to find candidate OA rapy, one of m using PPARγ ligands or agonists. Pioglitazone, a PPARγ syn tic ligand, in animal models of OA was able to decrease syn sis of MMP-13 and IL-1β. Based on this research needs to be done about effect of pioglitazone in reducing NO production in IL-1β induced chondrocytes. This research proposal has been submitted to Ethics Committee for Health Research UB School of Medicine and has received Letter of Eligibility of Ethics. design of this study was to compare laboratory experimental results obtained after treatment with control (post-test only control group design). This study used a cell line of human normal articular chondrocytes (NHAC-kn). Some of procedures performed are chondrocyte culture in monolayer culture, chondrocytes redifferensiasi cells using alginate beads to restore properties of chondrocytes that have been transformed into chondrocytes fibroblasts like cells, collagen concentration measurement as a marker of type 2 normal chondrocytes, administration of IL-1β in normal chondrocytes to induced normal chondrocytes into like OA chondrocytes, MMP-13 concentration measurements and observations as a chondrocyte hypertrophy marker OA chondrocytes. Three doses of pioglitazone at 0.1 mM, 1 mM and 10 mM in chondrocytes treated with IL-1β to provide rapeutic effects, measurements of NO concentration by method of colorimetry assay, RNA isolation and cDNA syn sis for measurement of mRNA expression of PPARγ. PPARγ mRNA expression was measured using real time PCR method (Roche). Statistical analysis using one way ANOVA test to determine differences in effect of various doses of pioglitazone on PPARγ mRNA expression and NO levels, followed Tukey post hoc test to compare between treatments. Pearson correlation test was n performed to determine relationship between dose of pioglitazone with increased mRNA expression of PPARγ, relationship between dose of pioglitazone with decreased levels of NO and relationship between increase in mRNA expression of PPARγ with decreased levels of NO. Linear regression was used to determine regression model can be used to predict future increase or decrease in PPARγ mRNA expression and NO levels with addition of pioglitazone or dose reduction. results of study during period December 2013 to June 2014 was measurement of concentration of collagen type 2 in chondrocytes that have redifferentiated higher (77.9 ng/ml) and significantly different than concentration of collagen type 2 in chondrocytes fibroblasts like cells (15.6 ng/ml). This suggests that chondrocytes fibroblasts like cells has returned to normal chondrocytes. In measurement of concentration of MMP-13 as a biomarker of OA chondrocytes, concentration of MMP-13 in chondrocytes treated with IL-1β was higher (7.947 ng/ml) and significantly different than concentration of MMP-13 in normal chondrocytes (1,421 ng/ml) (p 0.05). Microscopic examination chondrocytes treated with IL-1β, looks chondrocytes hypertrophy, characterized by greater chondrocytes (swelling). PPARγ mRNA expression in IL-1β induced chondrocytes decreased (0.64) and decrease was significantly different (p 0.05, LSD post hoc test) than in normal chondrocytes (6.11). Pioglitazone treatment with three different doses capable of increasing mRNA expression of PPARγ and significantly different compared with chondrocytes PPARγ mRNA expression induced IL-1β alone, but inter-dose treatment was not significantly different (p 0.05, LSD post hoc test). PPARγ mRNA expression in IL-1β induced chondrocytes plus pioglitazone dose of 0.1 = 5.48, = 5.59 and 1μM dose of 10μM dose = 6.15. Pearson correlation test showed that re was a strong relationship between three doses of pioglitazone with increased expression of PPARγ mRNA, and results of a linear regression to predict increase in PPARγ mRNA expression when coupled with a certain dose of pioglitazone is: mRNA expression of PPARγ = 5797.42 + 8119,57X , where X was dose. concentration of NO in IL-1β induced chondrocytes increased (56.9 mol/L) and significantly different from concentration of NO in normal chondrocytes (8 mol/L) (p 0.05). Pioglitazone treatment with three different doses able to reduce concentration of NO and significantly different in IL-1β induced chondrocytes (p 0.05, LSD post hoc test). concentration of NO in IL-1β induced chondrocytes plus pioglitazone dose of 0.1 mM = 29,4μmol/L, a dose of 1 mM = 17.1 mol/L and a dose of 10 mM = 13.1 mol/L. Pearson correlation test showed that re was a strong relationship between three doses of pioglitazone on reducing levels of NO, and results of a linear regression to predict decrease in NO levels when coupled with a certain dose of pioglitazone was: NO concentration = 64.621 - 14,122X, where X is dose of pioglitazone . Pearson correlation test results demonstrate that re was a strong relationship between increased expression of PPARγ mRNA with decreased NO levels (R = -0.866) indicating that 86.6% decrease in NO levels was affected by an increase in mRNA expression of PPARγ, while rest was due to o r factors. It can be concluded that administration of various doses of pioglitazone could increase PPARγ mRNA expression and decrease NO production in IL-1β induced chondrocytes, and increased expression of PPARγ mRNA affect decrease in NO production by IL-1β induced chondrocytes.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/616.722 3/ZUL/p/041407346 |
| Subjects: | 600 Technology (Applied sciences) > 616 Diseases > 616.7 Diseases of musculoskeletal system |
| Divisions: | S2/S3 > Magister Manajemen Rumah Sakit, Fakultas Kedokteran |
| Depositing User: | Endro Setyobudi |
| Date Deposited: | 10 Nov 2014 16:36 |
| Last Modified: | 10 Nov 2014 16:36 |
| URI: | http://repository.ub.ac.id/id/eprint/158391 |
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