Pratama, MirzaZaka (2015) Pengaruh Pemberian Kurkumin terhadap Persentase T Helper 17 dan T Regulator pada Kultur Sel T CD4+ Pasien Lupus Eritematosus Sistemik. Magister thesis, Universitas Brawijaya.
Abstract
Lupus eritematosus sistemik (LES) merupakan suatu penyakit autoimun sistemik yang dapat mengakibatkan kerusakan multiorgan. Patomekanisme LES sendiri hingga saat ini masih belum diketahui dengan jelas. Terdapat suatu paradigma baru dalam patomekanisme LES yaitu adanya ketidakseimbangan antara aktivitas T Helper 17 (Th17) dan T Regulator (Treg) yang mengakibatkan terjadinya kerusakan jaringan pada LES. Meskipun demikian, terapi LES konvensional masih belum bekerja secara spesifik dan memberikan hasil yang maksimal. Selain itu, efek samping yang tidak menyenangkan juga sering muncul akibat pemakaian jangka panjang. Oleh karena itu, dibutuhkan suatu terapi baru untuk mengatasi permasalahan LES tersebut. Salah satu zat yang memiliki sifat sebagai imunomodulator dan berpotensi sebagai obat bagi LES adalah kurkumin. Kurkumin telah banyak dibuktikan mampu memodulasi berbagai faktor transkripsi seperti signalling transducer and activator of transcriptions (STAT), proliferator-activated receptors γ (PPARγ), aryl hydrocarbon receptor (AhR), dan vitamin D receptor (VDR) Meskipun demikian, penelitian yang membuktikan pengaruh kurkumin terhadap Th17 dan Treg pada LES masih belum pernah dilakukan. Penelitian ini dilakukan dengan tujuan untuk membuktikan pengaruh pemberian kurkumin terhadap persentase Th17, persentase Treg, serta rasio Th17/Treg yang terbentuk dari kultur sel T CD4+ pasien LES dan kontrol sehat sebagai pembanding. Metode penelitian yang dilakukan pada penelitian ini meliputi isolasi sel T CD4+ dari pasien LES dan kontrol sehat dengan kit human CD4+ enrichment cocktail (RosetteSep, Stemcell Technologies) yang kemudian dilakukan konfirmasi menggunakan flowsitometri. Sel T CD4+ yang terisolasi kemudian dikultur sebanyak 5x105 sel/ml pada 96 microwell yang telah dilakukan coating dengan anti-CD3 5 μg/mL (Biolegend) tiap well sebelumnya. Kultur distimulasi menggunakan sitokin rekombinan untuk mengarahkan diferensiasinya menjadi Th17 menggunakan 5 μg/ml anti-CD-28 (Biolegend), 10 ng/mL IL-6 (Biolegend), 5 ng/mL TGF-β1 (Biolegend), 10 μg/mL anti-IFN-γ (Biolegend), dan 10 μg/mL anti-IL-4 (Biolegend). Kultur dibagi menjadi empat kelompok berdasarkan dosis kurkumin yang diberikan, yaitu P0 (tanpa pemberian kurkumin), P1 (0,1 μg/ml), P2 (1 μg/ml), dan P3 (10 μg/ml). Penentuan dosis kurkumin tersebut berdasarkan penelitian pendahuluan yang dilakukan sebelumnya. Kurkumin diberikan pada hari yang sama pada saat dilakukan stimulasi kultur, yaitu hari ke-0. Sel T CD4+ yang telah diberikan stimulasi dan kurkumin diinkubasi di dalam inkubator selama 72 jam sekaligus dilakukan pengamatan kultur di bawah mikroskop setiap harinya. Sel yang dipanen dilakukan penghitungan persentase Th17 dan Treg menggunakan flowsitometri. Persentase Th17 diukur dengan pelabelan antibodi FITC antihuman CD4 (Biolegend) dan PerCP/Cy5.5 antihuman IL-17A (Biolegend) yang sebelumnya distimulasi menggunakan phorbol myristate acetate (PMA), ionomycin, dan golgiplug ii selama minimal 4 jam. Persentase Treg diukur dengan pelabelan antibodi PerCP antihuman CD4 (Biolegend), antibodi PE antihuman CD25 (Biolegend), dan antibodi FITC antihuman FoxP3 (Biolegend). Rasio Th17/Treg diukur dari pembagian antara persentase Th17 dan Treg. Analisis hasil penelitian menunjukkan bahwa ketika masih belum diberikan kurkumin, terdapat peningkatan yang bermakna persentase Th17 yang terbentuk pada kultur sel T CD4+ pasien LES dibandingkan dengan kontrol sehat. Sebaliknya, persentase Treg didapatkan menurun secara bermakna pada pasien LES dibandingkan dengan kontrol sehat. Pemberian kurkumin dengan dosis 0,1, 1, dan 10 μg/ml dibuktikan menurunkan persentase Th17 yang terbentuk pada kultur sel T CD4+ pasien LES secara bermakna dengan dosis 1 μg/ml memberikan hasil yang paling rendah. Sebaliknya, persentase Th17 tidak didapatkan perbedaan yang bermakna pada kultur sel T CD4+ kontrol sehat yang diberikan kurkumin dosis 0,1 dan 1 μg/ml. Akan tetapi, pemberian dosis 10 μg/ml pada kultur sel T CD4+ kontrol sehat justru memberikan efek meningkatkan persentase Th17. Kurkumin juga dibuktikan meningkatkan persentase Treg pada kultur sel T CD4+ pasien LES secara bermakna pada setiap kelompok perlakuan, baik 0,1, 1, dan 10 μg/ml. Persentase Treg pada kultur sel T CD4+ kontrol sehat tidak didapatkan peningkatan pada dosis 0,1 dan 1 μg/ml akan tetapi peningkatan Treg secara bermakna ditemukan pada pemberian kurkumin dosis 10 μg/ml. Rasio Th17/Treg didapatkan menurun secara bermakna pada pasien LES sedangkan penurunan rasio Th17/Treg tidak didapatkan pada kultur sel T CD4+ kontrol sehat. Berdasarkan analisis data yang telah dilakukan, dapat disimpulkan bahwa pemberian kurkumin dapat menurunkan persentase Th17 sekaligus meningkatkan persentase Treg dan menurunkan rasio Th17/Treg pada kultur sel T CD4+ pasien LES. Selain itu, pemberian kurkumin dalam dosis yang rendah (0,1 dan 1 μg/ml) ternyata tidak dibuktikan mampu menurunkan persentase Th17 namun pemberian kurkumin dosis yang lebih tinggi (10 μg/ml) justru meningkatkan persentase Th17. Kurkumin juga mampu meningkatkan persentase Treg pada kontrol sehat namun dengan dosis yang berbeda dengan pasien LES, yaitu pada dosis 10 μg/ml. Kurkumin terbukti tidak menurunkan rasio Th17/Treg pada kontrol sehat.
English Abstract
Systemic lupus ery matosus (SLE) is a chronic systemic autoimmune disease with multiple organ manifestations. re is a new paradigm on pathogenesis of SLE involving a balance between T helper 17 (Th17) and T regulator (Treg) activity. Conventional rapies of SLE including glucocorticoid and immunosuppresant are still ineffective againts SLE. Moreover, unpleasant side effects often arise as a result of long term use of treatments. refore, re is a need of a new rapy to overcome problems on SLE treatments. One of substances that have immunomodulatory properties and has a potency to be a drug for SLE is curcumin. Curcumin can modulate many transcription factors, such as signalling transducer and activator of transcriptions (STAT), proliferator-activated receptors γ (PPARγ), aryl hydrocarbon receptor (AhR), and vitamin D receptor (VDR). However, study to prove curcumin effects on Th17 and Treg in SLE is never conducted. aim of this research is to prove effects of curcumin on Th17 percentages, Treg percentages, and also Th17/Treg ratios from CD4+ T cells culture from SLE patients and healthy control as comparison. Research method was including isolation of CD4+ T cells from SLE patients and healthy controls using human CD4+ enrichment cocktail (RosetteSep, Stemcell Technologies). Isolated CD4+ T cells were cultured in 5x105 cells/ml on 96 microwells which had been coated by anti-CD3 5 μg/mL (Biolegend) for each well. Cultures were stimulated using recombinant cytokines to alter differentiation into Th17 using 5 μg/ml anti-CD-28 (Biolegend), 10 ng/mL IL-6 (Biolegend), 5 ng/mL TGF-β1 (Biolegend), 10 μg/mL anti-IFN-γ (Biolegend), and 10 μg/mL anti-IL-4 (Biolegend). Cultures were divided into four groups depended on dosages of curcumin given, namely P0 (without curcumin), P1 (0,1 μg/ml), P2 (1 μg/ml), and P3 (10 μg/ml). Doses determination was based on preliminary study which had been done before. Curcumin was given on same day with stimulation of cultures or on day 0. CD4+ T cells which had been stimulated by cytokines and curcumin were incubated for 72 hours and observed microscopically along incubation. Harvested cells were counted for Th17 and Treg percentages using flowcytometry. Th17 percentages were labelled by antihuman CD4 (Biolegend) and PerCP/Cy5.5 antihuman IL-17A (Biolegend) antibodies stimulated by phorbol myristate acetate (PMA), ionomycin, and golgiplug for 4 hours before. Whereas, Treg percentages were labelled by PerCP antihuman CD4 (Biolegend), PE antihuman CD25 (Biolegend), and FITC antihuman FoxP3 (Biolegend) antibodies. Th17/Treg ratios were counted from extraction of Th17 and Treg percentages. Results analysis were found that before curcumin treatments, re were signficant increases of Th17 percentages from CD4+ T cells cultures of SLE patients compared to healthy control. Whereas, Treg percentages from SLE patients were reduced significantly compared to healthy control. Administration of curcumin at iv concentration 0.1, 1, and 10 μg/ml could decrease Th17 percentages significantly with 1 μg/ml resulted lowest result. Administration of curcumin at concentration 0.1 and 1 μg/ml could not reduce of Th17 percentages significantly from CD4+ T cell cultures of healthy control. However, re was a significant enhancement of Th17 percentages on CD4+ T cell cultures of healthy control after administration of 10 μg/ml of curcumin. Curcumin also increased Treg percentages significantly on CD4+ T cells of SLE patients in every treatment groups, including 0,1, 1, and 10 μg/ml. Treatment of curcumin on CD4+ T cells cultures of healthy controls showed an increasing of Treg percentages significantly after administration of 10 μg/ml of curcumin however re were no significant differences of Treg percentages after treatment of 0.1 and 1 μg/ml of curcumin on CD4+ T cells of healthy controls. Th17/Treg ratios on CD4+ T cells of SLE patients were also reduced significantly. However, curcumin could not reduce Th17/Treg ratios on CD4+ T cells cultures of healthy controls. From se data, it can be concluded that curcumin administration can decrease Th17, increase Treg percentages, and also reduce Th17/Treg ratios on CD4+ T cells culture of SLE patients. Moreover, treatment of low doses of curcumin (0,1 and 1 μg/ml) were not able to decrease Th17 percentages on CD4+ T cells cultures of healthy control. However, treatment of higher doses of curcumin (10 μg/ml) on healthy controls resulted in increasing of Th17 percentages. Curcumin also increase Treg percentages significantly on CD4+ T cells cultures of healthy controls at different doses compared to SLE patients (10 μg/ml). Lastly, curcumin were not able to reduce Th17/Treg ratios on CD4+ T cells culture of healthy controls.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/615.324 39/PRA/p/041502085 |
| Subjects: | 600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.3 Organics drugs |
| Divisions: | S2/S3 > Magister Ilmu Biomedis, Fakultas Kedokteran |
| Depositing User: | Samsul Arifin |
| Date Deposited: | 15 Apr 2015 10:29 |
| Last Modified: | 15 Apr 2015 10:29 |
| URI: | http://repository.ub.ac.id/id/eprint/158139 |
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