Tesis

Regulasi Ekspresi Antioksidan pada Hati Tikus Obesitas yang Diberi Ekstrak Etanol Biji Ketumbar (Coriandrum sativum L): Kajian In Silico dan In Vivo = Regulation of Antioxidant Expression on Liver of Obese Rats Treated with Ethanol Extract of Coriander Seeds (Coriandrum sativum L): In Silico and In Vivo Studies.

Latar belakang: Obesitas dapat meningkatkan spesies oksigen reaktif (ROS), sehingga memicu stres oksidatif. Biji ketumbar mengandung senyawa polifenol yang bertindak sebagai antioksidan alami untuk mengurangi stres oksidatif. Ekstrak etanol biji ketumbar terbukti menurunkan malondialdehid dan meningkatkan aktivitas katalase di hati tikus yang diinduksi diet tinggi lemak. Dengan demikian, biji ketumbar dianggap melindungi terhadap kerusakan oksidatif yang disebabkan oleh obesitas di hati. Namun, regulasi molekulernya belum terungkap. Nrf2 dan FOXO3 adalah faktor transkripsi yang meregulasi antioksidan seluler (misalnya superoksida dismutase/SOD, glutathione peroksidase/GPx, dan glutathione tereduksi/GSH). Nrf2 dan FOXO3 diatur secara negatif oleh protein Keap1 dan 14-3-3 untuk mempertahankan homeostasis selulernya. Penelitian ini bertujuan untuk menganalisis regulasi ekspresi antioksidan melalui studi in-silico dan in-vivo. Metode: Studi in silico menganalisis potensi penghambatan ekstrak etanol biji ketumbar terhadap Keap1 dan 14-3-3 menggunakan penambatan molekuler. Kemudian, pada tiga senyawa terbaik dilakukan prediksi drug-likeness, farmakokinetik, dan toksisitasnya. Sementara itu, studi in vivo menyelidiki bagaimana ekstrak etanol biji ketumbar memengaruhi kadar Nrf2, FOXO3, dan antioksidan hilirnya (T-SOD, MnSOD, GPx, dan GSH). Studi in vivo melibatkan lima kelompok tikus yang diinduksi oleh diet tinggi lemak dan ekstrak etanol biji ketumbar 100mg/kgBB selama 12 minggu. Hasil: Hasil in silico menunjukkan bahwa shionoside b memiliki potensi penghambatan terbaik terhadap protein Keap1 (ΔG=-8,90 kkal/mol; Ki=298,01 nM) dan protein 14-3-3 (ΔG=-6,85 kkal/mol; Ki=9,46 µM). Hasil in vivo menunjukkan bahwa kadar Nrf2 dan FOXO3 serta ekspresi mRNA MnSOD dan GPx berbeda secara signifikan antar kelompok (p < 0,05). Sementara itu aktivitas T-SOD, MnSOD, GPx, dan kadar GSH tidak berbeda secara signifikan antar kelompok (p > 0,05). Nrf2 berkorelasi secara signifikan dengan FOXO3. Nrf2 dan FOXO3 berkorelasi secara signifikan dengan aktivitas spesifik enzim T-SOD, MnSOD, GPx, dan kadar GSH. Nrf2 dan FOXO3 tidak berkorelasi dengan ekspresi mRNA MnSOD dan GPx. Kesimpulan: Biji ketumbar cenderung meningkatkan Nrf2 dan FOXO3 pada tikus obesitas namun tidak signifikan. Faktor transkripsi Nrf2 dan FOXO3 berkorelasi positif dengan aktivitas antioksidan enzimatik dan nonenzimatik hilirnya. Mekanisme yang mungkin terjadi adalah karena adanya interaksi komponen biji ketumbar dengan protein inhibitor (Keap1 dan 14-3-3) yang berkontribusi terhadap stabilitas dan mobilisasi Nrf2 dan FOXO3 di nukleus.
Kata Kunci: Coriandrum sativum L., FOXO3, Nrf2, obesitas, stres oksidatif


Background: Obesity can increase reactive oxygen species (ROS), thereby triggering oxidative stress. Coriander seeds contain polyphenolic compounds that act as natural antioxidants to reduce oxidative stress. Coriander seed ethanolic extract is proven to decrease malondialdehyde and increase the catalase activity in the liver of high-fatinduced rats. Thus, coriander seeds are thought to protect against obesity-induced oxidative damage in the liver. However, its molecular regulation has not been revealed. Nrf2 and FOXO3 are transcription factors for cellular antioxidant regulation (e.g. superoxide dismutase/SOD, glutathione peroxidase/GPx, and reduced glutathione/GSH). Nrf2 and FOXO3 are negatively regulated by Keap1 and 14-3-3 protein to maintain their cellular homeostasis. This study aimed to analyze the antioxidant expression regulation through in silico and in-vivo studies. Methods: The in-silico study assessed the inhibition potential of coriander seeds ethanolic extract against the Keap1 and 14-3-3 using molecular docking. Then, the three best compounds were analyzed for their drug-likeness, pharmacokinetics, and toxicity prediction. Meanwhile, the in vivo study investigated how coriander seed ethanolic extract impacts the level of Nrf2, FOXO3, and their downstream (T-SOD, MnSOD, GPx, and GSH). The in vivo study involved five groups of rats induced by a high-fat diet and 100mg/kgBW coriander seeds ethanolic extract for 12 weeks. Results: The in-silico revealed that shionoside b has the best inhibition potential against Keap1 protein (ΔG=-8.90 kcal/mol; Ki=298.01 nM) and 14-3-3 protein (ΔG=-6.85 kcal/mol; Ki=9.46 µM). The in vivo study showed that Nrf2, FOXO3, MnSOD mRNA expression, and GPx mRNA expression are significantly different between the groups (p < 0.05). Meanwhile, T-SOD, MnSOD, GPx, and GSH activity are not significantly different between the groups (p > 0.05). Nrf2 has a significant correlation with FOXO3. Nrf2 and FOXO3 have a significant correlation with T-SOD, MnSOD, GPx, and GSH. Nrf2 and FOXO3 have no correlation with the relative mRNA expression of MnSOD and GPx. Conclusion: The coriander seeds tend to increase Nrf2 and FOXO3 in obese rats, but this was not significant. Nrf2 and FOXO3 transcription factors are positively correlated with their downstream enzymatic and nonenzymatic antioxidant activity. The possible mechanism is due to the interaction between the coriander seed phytoconstituents and the protein inhibitor (Keap1 and 14-3-3), which contributes to the stability and nuclear mobilization of Nrf2 and FOXO3.
Keywords: Coriandrum sativum L., FOXO3, Nrf2, obesity, oxidative stress