LAPORAN PENELITIAN
HIBAH PENELITIAN STRATEGIS NASIONAL
TAHUN 2009
JUDUL
Peran Ekstrak Juwet (Syzygium Cumini) terhadap Faktor Transkripsi
pada Model HUVECs Preeklampsia: Study in-vitro Penurunan Lipid
Peroksidasi melalui Ekspresi NF-κB dan kadar TNF-α
Nama Peneliti Utama Dan Anggota Ketua : Dr.dr. Siti Candra Windu B, SpOG(K)
Anggota : 1. Dr. dr. Nurdiana, M.Kes 2 . Wahyudha Ngatiril Lady, S.Si
Dibiayai Oleh Direktorat Jenderal Pendidikan Tinggi, Departemen Pendidikan Nasional, Sesuai Dengan Surat Perjanjian Pelaksanaan Hibah Penelitian Strategis Nasional, Nomor : 0174.0/023-04.2/XV/2009, tanggal 31 Desember 2008 dan berdasarkan SK
Rektor Nomor : 160/SK/2009, tanggal 7 Mei 2009.
UNIVERSITAS BRAWIJAYA
NOPEMBER 2009
Ringkasan
Latar belakang: Preeklampsia merupakan 1 diantara 3 penyebab kematian pada ibu hamil. Pada penderita Preeklampsia didapatkan kelainan disfungsi endotel. Adanya stress oksidatif adalah faktor penting sebagai patogenesis pada Preeklampsia. Stress oksidatif bisa dikendalikan dengan pemberian antioksidan. Buah mempunyai kandungan antioksidan bermacam – macam. Juwet (Syzygium Cumini) mengandung banyak antioksidan yang belum banyak dieksplorasi. Disamping itu Juwet banyak dipedesaan dan kejadian ibu hamil yang mengalami Preeklampsia bayak dari pedesaan.
Tujuan penelitian: Untuk membuktikan pada model HUVECs Preeklampsia terdapat peningkatan lipid peroksidasi (F2-isoprostan dan MDA), ekspresi NFkβ-p50 dan kadar TNF- α. Untuk mendapatkan dosis ekstrak Juwet (Syzygium Cumini) yang optimum dan tepat dalam menurunkan lipid peroksidasi pada model HUVECs Preeklampsia. Untuk membuktikan peranan ekstrak Juwet (Syzygium Cumini) dalam penurunan ekspresi NFkβ-p50 dan kadar TNF- α. Untuk memperjelas penurunan Lipid Peroksidasi pada Model HUVECs Preeklampsia melalui penurunan Ekspresi NFkβ-p50 dan kadar TNF-α. Metode penelitian adalah Penelitian dilakukan di laboratorium Biomedik Fakultas Kedokteran Universitas Brawijaya, merupakan penelitian eksperimental dengan menggunakan HUVEC`s yang dipapar dengan 2% plasma kehamilan Preeklampsia sebagai model HUVEC`s Preeklampsia in vitro.Pada kelompok 1 (kontrol); HUVECs. 2; Model HUVECs Preeklampsia. 3; Model + Ekstrak Juwet 50 ppm, 4; Model + Ekstrak Juwet 100 ppm. 5. Model + Ekstrak Juwet 200 ppm. 6; Model + Ekstrak Juwet 200 ppm. Inkubasi dilakukan selama 6-16 jam, kemudian dilakukan pemeriksaan MDA, F2
isoprostan, ekspresi NFkβ-p50 dan TNF-α. Pemeriksaan F2 isoprostan dan TNF-α
menggkunakan KIT dan dianalisa dengan ELISA, MDA dengan Spectrofotometri dan ekspresi NFkβ-p50 dengan immunohistokimia. Analisa statistik menggunakan SPSS 15, dilakukan uji beda One Way Anova dengan tingkat kepercayaan 95% untuk MDA, F2
isoprostan, dan TNF-α. dan ekspresi NFkβ-p50 dilakukan uji beda Two Way Anova kemudian untuk mengetahui pemaparan mana yang menunjukkan perbedaan dilakukan uji Tukey dengan tingkat kepercayaan 95%.
Hasil: Kadar MDA, F2 isoprostan, TNF-α dan ekspresi NFkβ-p50 lebih tinggi secara
signifikan pada model HUVECs Preeklampsia dibandingkan kontro (p < 0,05). Pengaruh ekstrak Juwet pada model HUVECs Preeklampsia terhadap kadar MDA, pada dosis 100, 200 dan 400 ppm menunjukkan terjadi penurunan signifikan dibandingkan pada model. Sedangkan F2 isoprostan, dosis ekstrak Juwet 50 dan 400 ppm yang optimal.
Terhadap faktor inflamasi TNF-α. Dosis ekstrak Juwet 50 ppm dapat menurunkan kadar TNF-α meskipun tidak berbeda signifikan p= 0.560 (p > 0,05). Ekstrak Juwet pada ekspresi NFkβ-p50 dapat menurunkan mulai dosis mulai 50, 100, 200 dan 400 ppm. Penurunan secara signifikan mulai dosis 100, 200 dan 400 ppm (p < 0,05). Penurunan ekspresi NFkβ-p50 akan berpengaruh menurunkan kadar MDA dan F2 isoprostan (Lipid
Peroksidasi) dengan nilai r2 = 0,87 dan 0,16. Pengaruh penurunan kadar TNF-α terhadap penurunan lipid peroksidasi (MDA dan F2 isoprostane) berpengaruh kuat
dengan r2= 0,60 yang sama. Kesimpulan:
Terdapat peningkatan lipid peroksidasi (F2-isoprostan dan MDA), TNF- α dan ekspresi NFkβ-p50 pada model HUVECs Preeklampsia. Ekstrak Juwet dapat menurunkan kadar F2-isoprostan dan MDA), TNF- α dan ekspresi NFkβ-p50. Ekstrak Juwet dapat menurunkan Lipid Peroksidasi diduga melalui Ekspresi NFkβ-p50 dan penurunan TNF-α.
Melanjutkan penelitian untuk mengeksplorasi terhadap kandungan dari Juwet. Penelitian dilanjutkan ke in vivo dengan membuat model tikus Preeklampsia.
summary
Background: Preeclampsia is one out of three causes of pregnant mothers’ death. We may find endothelium dysfunction on the patient of preeclampsia. Oxidative stress is an important factor as a pathogenesis in preeclampsia. Oxidative stress can be controlled by giving antioxidant. Fruits has a variety of antioxidant. Juwet Extract (Syzygium Cumini) contains much antioxidant that had not been explored well. Beside that, Juwet or Syzygium can be found easily on the villages, and many pregnant mothers with preeclampsia come fron the villages.
The objective of the research: To prove that on HUVECs Preeclampsia model there is an increase of peroxidation lipid (F2 isoprostane and MDA), NFkβ-p50 expression and TNF-α level. It is also to get the optimum and proper dosage of Syzygium extract in decreasing peroxidation lipid on HUVECs Preeclampsia model. And also to prove the role of Syzygium extract in decreasing NFkβ-p50 expression and TNF-α level. Beside that, it is to clarify the decrease of peroxidation lipid on HUVECs Preeclampsia model through the decrease of NFkβ-p50 expression and TNF-α level.
The method of the research: The research was conducted at Biomedic Laboratory of Medicine Faculty of Brawijaya University, and it was an experimental research using HUVEC`s being exposed to 2% plasma of preeclampsia pregnancy as the model of HUVEC`s Preeclampsia in vitro. In group 1 (controlled group); HUVECs. 2; Model HUVECs Preeclampsia. 3; Model + Syzygium Extract 50 ppm, 4; Model + Syzygium Extract 100 ppm. 5. Model + Syzygium Extract 200 ppm. 6; Model + Syzygium Extract 200 ppm. Incubation was conducted for 6-16 hours, then there will be examinartions of MDA, F2 isoprostane, NFkβ-p50 and TNF-α expression. The eximantion of F2
isoprostane and TNF-α used KIT and was analyzed using ELISA, MDA using Spectrophotometri and NFkβ-p50 expression using immunohistochemistry. The statistical analysis is conducted using SPSS 15, by conducting differential test of One Way Anova with 95% realibility for MDA, F2 isoprostane, and TNF-α. For NFkβ-p50 expression,we
conducted the diffrential test of Two Way Anova. Then to identify which exposures indicate any differences, Tukey test with the reliability of 95% was conducted.
The results: The levels of MDA, F2 isoprostane, TNF-α, and NFkβ-p50 expression are
significantly higher on HUVECs Preeclampsia model compared to the controlled group (p < 0.05). The influence of Syzygium extract on HUVECs Preeclampsia model to the MDA level, on the dosage of 100, 200, and 400 ppm shows that there is a significant decrease compared to the model. Meanwhile for F2 isoprostane, the dosages of 50 and
400 ppm of Syzygium extract are optimal. On the inflammation factor of TNF-α. The Syzygium extract dosage of 50 ppm can decrease TNF-α level although it is not significantly different p= 0.560 (p > 0.05). The Syzygium extract on NFkβ-p50 expression can decrease from the dosages of 50, 100, 200, and 400 ppm. Significant decreases start from 100, 200, and 400 ppm (p < 0.05). The decrease of NFkβ-p50 expression will influence in decreasing the levels of MDA and F2 isoprostane (Peroxidation Lipid) by the
values of r2 = 0.87 and 0.16. The impact of TNF-α level decrease to the decrease of peroxidation lipid (F2 isoprostane and MDA) strongly influences by of r2 = 0.60.
Conclusion:
There is an increase of peroxidation lipid (F2-isoprostane and MDA), NFkβ-p50 expression and TNF- α level on HUVECs preeclampsia model. The extract of Juwet (Syzygium Cumini) can decrease levels of F2-isoprostane and MDA, TNF- α and NFkβ-p50 expression. It is assumed that the decrease of Peroxidation Lipid through the decrease of NFkβ-p50 Expression and TNF-α level
Suggestions:
We have a reccommendation to explore the content of Juwet. The research to be continued in vivo by making rat model of Preeclampsia.
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