EFEK KOMBINASI CURCUMIN DAN 6-SHOGAOL TERHADAP KADAR HEMOGLOBIN F PADA SEL LINE ERITROLEUKEMI K562

JOKO SETYONO, Prof. Dr. Mustofa, M.Kes, Apt; dr. Ahmad Hamim Sadewa, Ph.D; Prof. Dr. Edy Meiyanto, MSi, Apt

2019 | Disertasi | DOKTOR ILMU KEDOKTERAN DAN KESEHATAN

Latar Belakang. Talasemia-Beta merupakan penyakit genetik herediter dimana tidak ada produksi atau hanya sedikit produksi rantai Beta-Globin dan terjadi kelebihan rantai alfa-Globin, sehingga terjadi kerusakan eritrosit sampai timbul anemia berat yang memerlukan transfusi darah sepanjang hidupnya dan berpotensi penurunan kualitas hidup maupun komplikasi multi organ akibat timbunan zat besi serta berisiko kematian cukup tinggi. Oleh karena itu, perlu pencegahan akibat terapi transfusi darah tersebut. Salah satu pendekatan terapi terkini yang potensial pada talasemia-Beta yaitu stimulasi peningkatan produksi rantai gamma-Globin, yang dapat menangkap rantai alfa-Globin bebas membentuk hemoglobin fetus (Hb F, alfa2gamma2). Penelitian menggunakan senyawa murni kurkumin dan 6- shogaol serta kombinasinya, bertujuan mengungkap efek senyawa tersebut terhadap peningkatan kadar Hb F melalui jalur sinyaling pada ekspresi mRNA gena BCL11A, gena STAT3, gena gamma-Globin, dan p38 MAPK terfosforilasi (p-p38 MAPK). Hasil penelitian diharapkan dapat memberikan gambaran potensi terapeutik terkini talasemia untuk meningkatkan kualitas hidup penderita talasemia-Beta dan mengungkap jalur mekanisme molekuler yang mendasari pemahaman regulasi ekspresi gena hemoglobin fetal. Metode. Penelitian menggunakan model sel line eritroleukemia K562 dengan desain Posttest-only control group design with repeated measurements. Penelitian dimulai dengan uji sitotoksisitas untuk memperoleh nilai IC50, dilanjutkan dengan uji kombinasi berdasar nilai IC50 untuk memperoleh dosis/konsentrasi kombinasi terbaik yang menghasilkan viabilitas sel tertinggi. Sel K562 secara acak dibagi ke dalam 5 kelompok dengan ulangan masing-masing sebanyak 3 kali yaitu kelompok kontrol tanpa perlakuan; kelompok kontrol positif; kelompok perlakuan kombinasi kurkumin dengan 6-shogaol; kelompok perlakuan kurkumin tunggal, dan kelompok 6-shogaol tunggal. Sampel uji diambil dalam serial waktu 72 jam dan 96 jam. Sampel uji untuk ekspresi mRNA gena STAT3, gena BCL11A dan gena gamma-Globin diukur dengan metode qRT-PCR. Sampel uji untuk kadar p-p38 MAPK dan kadar Hb F diukur dengan metode ELISA. Hasil. Ekspresi mRNA gena STAT3 lebih rendah bermakna pada serial waktu 72 jam (p<0,05) di kelompok kurkumin (p<0,01), kelompok 6-shogaol (p<0,05). Ekspresi mRNA gena gamma-Globin, pada serial waktu 72 jam (p>0,05) berturut-turut dari paling tinggi ke rendah di kelompok 6-shogaol, kurkumin, kombinasi dan kelompok kontrol positif. Kadar p-p38 MAPK, pada serial waktu 72 jam (p<0,05) berturut-turut dari yang paling lebih rendah di kelompok kurkumin (p<0,05) disusul 6-shogaol (p>0,05). Kadar Hb F, pada serial waktu 96 jam (p<0,05) berturut-turut dari paling tinggi ke rendah di kelompok kurkumin (p<0,05), 6-shogaol (p>0,05). Semua variabel dianalisis dibandingkan dengan kelompok kontrol tanpa perlakuan. Ekspresi mRNA gena BCL11A tidak ada data karena tidak didapatkan ekspresinya. Kesimpulan. Kombinasi kurkumin dengan 6-shogaol tidak berefek terhadap induksi Hb F, namun senyawa kurkumin tunggal dan 6-shogaol tunggal berefek terhadap induksi Hb F, sehingga berpotensi dapat dikembangkan sebagai terapi penyakit talasemia. Jalur sinyaling yang kuat dalam induksi Hemoglobin F oleh senyawa kurkumin dan 6-shogaol melalui jalur STAT3.

Background. Beta-thalassemia is a hereditary genetic disease in which there is no production or only a small amount of Beta-globin chain production and excess alfa-Globin chains occur, resulting in erythrocyte damage until severe anaemia occurs that requires blood transfusion throughout his life and has the potential to decrease the quality of life and multi-organ complications due to accumulation of substances iron and the risk of death is quite high. Therefore, prevention is needed due to blood transfusion therapy. One potential current therapeutic approach to thalassemia-Beta is the stimulation of increased production of the gamma- Globin chain, which can capture the free alfa-Globin chain to form fetal haemoglobin (Hb F, alfa2gamma2). The research using pure curcumin and 6- shogaol compounds and their combination, aims to reveal the effect of these compounds on increasing Hb F levels through signalling pathways in the expression of BCL11A gene mRNA, STAT3 gene, gamma-Globin gene, and phosphorylated MAPK p38 (p-p38 MAPK). The results are expected to provide an overview of the current therapeutic potential of thalassemia to improve the quality of life of patients with Beta-thalassemia and reveal the molecular pathways underlying the understanding of fetal haemoglobin expression regulation. Method. The study used a K562 erythroleukemia line cell model with a Posttest-only control group design with repeated measurements. The investigation began with a cytotoxicity test to obtain IC50 values, followed by a combination test based on IC50 values to obtain the best dose/concentration combination that produced the highest cell viability. K562 cells were randomly divided into five groups with three replications each, namely the control group without treatment; positive control group; a combination treatment group of curcumin with 6-shogaol; a single curcumin treatment group, and a single 6-shogaol group. Test samples were taken in 72-h and 96-h time series. Test samples for the expression of STAT3 mRNA, BCL11A mRNA and gamma-Globin mRNA genes were measured by qRT-PCR method. Test samples for p-p38 MAPK levels and Hb F levels were measured by the ELISA method. Results. The expression of STAT3 mRNA gene was significantly lower in the 72 hour time series (p <0.05) in the curcumin group (p <0.01), in the 6-shogaol group (p <0.05). gamma-Globin mRNA expression, in the 72 hour time series (p> 0.05) in a row from highest to lowest in the 6-shogaol, curcumin, combination and positive control groups. The level of p-p38 MAPK, in the 72 hour time series (p <0.05) in a row from the lowest in the curcumin group (p <0.05) followed by 6-shogaol (p> 0.05). Hb F levels, in the 96-hour time series (p <0.05), respectively from highest to lowest in the curcumin group (p <0.05), 6-shogaol (p> 0.05). All variables were analyzed compared to the control group without treatment. The BCL11A mRNA expression had no data because no expression was obtained. Conclusion. The combination of curcumin with 6-shogaol does not affect Hb F induction. However, the single curcumin and 6-shogaol compounds have an effect on Hb F induction, so that it can potentially be developed as a therapy for thalassemia. Secure signalling pathway in the induction of Hemoglobin F by curcumin and 6-shogaol compounds via the STAT3 pathway.

Kata Kunci : Efek Kombinasi, Kurkumin, 6-Shogaol, Hemoglobin F, sel K562