MEKANISME KEMOPREVENTIF EKSTRAK HEKSAN BIJI JINTEN HITAM (Nigella sativa Lor) PADA TIKUS Sprague Dawley DIINDUKSI 7,12 DIMETHYLBENZ(A)ANTRACENE: KAJIAN ANTIOKSIDAN DAN IMUNOMODULATOR

AKROM, Prof. dr. Marsetyawan HNES, MSc., PhD

2013 | Disertasi | S3 Kedokteran Umum

Senyawa 7,12-dimethylbenz(a)antracene (DMBA) terbukti bersifat genotoksik-karsinogenik, menekan hematopoiesis dan imunosupresan. Biji jinten hitam (BJH) secara empiris telah dimanfaatkan sebagai imunostimulan. Bagaimana mekanisme kemopreventif ekstrak heksan biji jinten hitam (EHBJH) pada tikus Sprague Dawley (SD) diinduksi DMBA belum jelas. Tujuan penelitian ini adalah untuk mengetahui pengaruh pemberian 7 minggu EHBJH terhadap aktivitas dan mekanisme kemopreventif pada tikus SD. Penelitian ini dilakukan pada 200 ekor tikus SD berumur 4-5 minggu, terbagi dalam 8 kelompok, masing-masing kelompok terdiri dari 25 ekor. Kelompok I (kontrol normal) diberi akuades dan makanan standar, kelompok II , III dan IV sebagai kelompok perlakuan dengan pemberian EHBJH setara timokuinon (EHBJHST) dosis 6,8, 68 dan 136 mg/kgBB, kelompok V sebagai kontrol timokuinon diberi timokuinon dosis 50 mg/kgBB, kelompok VI sebagai kontrol tamoksifen diberi tamoksifen dosis 0,6 mg/kgBB, kelompok VII sebagai kelompok DMBA diberi diet standar dan induksi DMBA dan kelompok VIII sebagai kelompok kontrol pelarut diberi diet standard an minyak jagung. Lima ekor tikus masing-masing kelompok dikorbankan pada minggu ke-2 dan ke-3 untuk uji respon imun dan aktivitas antioksidan, sisa tikus pada masing-masing kelompok diikuti sampai minggu ke-27. EHBJHST diberikan mulai dua minggu sebelum induksi DMBA dilanjutkan 5 minggu selama induksi. Pada minggu ke-3 semua kelompok diberikan DMBA dosis 20 mg/kgBB, 2x/minggu selama 5 minggu, kecuali kelompok I dan VIII. Pada minggu ke-27 hewan uji dikorbankan, dilakukan pengamatan aktivitas kemopreventif (hambatan pembentukan nodul, ekspresi gen p53, H-ras dan CYP1A1/1B1), aktivitas antioksidan (aktivitas enzim GST dan ekspresi GST) dan aktivitas imunomodulator (jumlah CD4, CD8 dan CD4CD25; jumlah dan aktivitas limfosit limpa dan jumlah dan aktivitas makrofag). Data dengan skala rasio dihitung perbedaan rata-rata antar kelompok dengan anova satu lengan, kemudian dilanjutkan uji perbedaan rata-rata antar kelompok dengan uji post hoc Least Significant Difference (LSD). Efek kemopreventif EHBJHST pada tikus SD diinduksi DMBA: menurunkan 45-55% insidensi nodul dan 70-90% adenokarsinoma; menurunkan 84%-95% ekspresi CYP1A1 dan 55%-64% ekepresi CYP1B1, menurunkan 88%- 93% ekspresi H-Ras dan meningkatkan 20%-30% ekspresi p53. Efek antioksidan EHBJHST: menurunkan 37%-53% kadar NO serum, meningkatkan 63%-105% aktivitas GST dan 105%-140% ekspresi GST. Efek imunomodulator EHBJHST: meningkatkan 3-4x jumlah CD4, 4-6x jumlah CD8 dan menurunkan 50% rasio CD4CD25/CD4; meningkatkan 3-5x jumlah limfosit limpa dan 16%-40% kadar IFNγ; meningkatkan 2,5-4x jumlah makrofag, 2-3x aktivitas fagositosis, 2-2x aktivitas sekresi ROI dan 3-4x aktivitas sekresi IL-12. EHBJHST meningkatkan 1-3x ekspresi p53 sumsum tulang dan 2-6x ekspresi FOXp3 limpa tetapi menurunkan 50% ekspresi IL-10. Efek kemopreventif, antioksidan dan imunomodulator dosis EHBJHST dosis 6,8 mg/kgBB sebanding dengan dosis EHBJHST dosis 68 mg/kgBB tetapi lebih aman. Aktivitas kemopreventif EHBJHST dosis 6,8 mg/kgBB sebanding dengan aktivitas kemopreventif timokuinon dan tamoksifen. Simpulan bahwa pemberian EHBJHST dosis 6,8 mg/kgBB memiliki efek kemopreventif akibat induksi DMBA pada tikus SD.

It has been well documented that 7,12-dimethylbenz(a)antracene (DMBA) is a genotoxic carcinogenic able to suppress hematopoiesis and serves as immunosuppressant. Black cumin seed (BJH) has been empirically used as immunostimulant. How chemopreventive mechanisms of hexane extraction of black cumin seeds (EHBJH) on DMBA-induced rats has been unclear yet. The aim of the study was to investigate the effects of EHBJH on activity and mechanism chemopreventive in DMBA-induced rats. The study was conducted on 200 SD rats of 4-5 weeks, divided into 8 groups; each group consisted of 25 rats. Group I (normal control) were given with distilled water and standard diet; groups II, III and IV as treatment group received EHBJHST with doses of 6,8, 68 and 136 mg/kgBW; group V was a thymoquinon control group received thymoquinone 50 mg/kgBW, and group VI was tamoxifen control group received tamoxifen of 0,6 mg/kgBW; group VII served as DMBA group, given with standard diet and induced with 10x20mg/kgBW for 5 weeks and group VIII was a solvent control received standard diet and corn oil as a solvent. At weeks 2 and 3, five rats of each group were used for immune response and antioxidant activity assay; the remaining rats in each group were followed until week 27. EHBJH had been applied since two weeks before DMBA induction and followed 5 weeks during induction. At week 3, all groups were given with DMBA (20 mg/kgBW, 2x/week) for 5 weeks, excluded the groups I and VIII. At week 27 of experimental animal were sacrificed, chemopreventive activity, antioxidant activity (GST enzyme activity and GST relative expression) and immunomodulatory activities (CD4, CD8 and CD4CD25; total number and activity of spleenocyte and macrophages). Data with ratio scale was calculated in terms of difference average among groups with one-way ANOVA ; then, it was followed by significance test between groups with post hoc Least Significant Difference (LSD) test. EHBJH chemopreventive effects on DMBA-induced SD rats involved to reduce 45-55% of nodule appearance and 70-90% of adenocarcinoma; to lower 84% -95% of CYP1A1 and 55%-64% of CYP1B1 expression, 88% -93% of HRas expression and to increase 20% -30% p53 expression. EHBJH antioxidant effect was to reduce 37% -53% of NO serum level, to increase 63% -105% of GST activity and 105% -140% GST expression. The effect of EHBJH immunomodulatory was to increase 3-4 times of CD4 count, 4-6 times of CD8 count and to lower 50% of CD4CD25/CD4 ratio; to increase 3-5 times of total spleen lymphocytes and 16% -40% of IFNγ level; to increase 2.5-4 times of total macrophage , 2-3 times of phagocytic activity, 2-2 times of ROI secretion activity and 3-4 times of IL-12 secretory activity. EHBJH increased 3 times of p53 bone marrow expression and 2-6 times of spleen Foxp3 expression, but to lower 50% of IL-10 expression. The effects of chemoprevention, antioxidant and immunomodulatory of EHBJHST with dose of 6,8 mg/kgBW was equivalent to EHBJHST withdose of 68 mg/kgBW, however the previous one was safer. Chemopreventive activity of EHBJHST with dose of 6,8 mg/kgBW was comparable the activity of timquinone and tamoxifen. It was concluded that EHBJHST with dose of 6,8 mg/kgBW has a chemopreventive effects on DMBA- induced SD rats.

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