EFEK PROTEKTIF EKSTRAK ETANOL PEGAGAN (Centella asiatica (L.) Urban) TERHADAP PROLIFERASI SEL FORMASI HIPPOCAMPUS TIKUS DEWASA PADA KONDISI STRES KRONIS

VIDYA IRAWAN, Dr. drh. Tri Wahyu Pangestiningsih, MP.,

2012 | Tesis | S2 Sain Veteriner

Latar belakang Stres kronis dapat menyebabkan penurunan proliferasi sel serta kemampuan learning dan memori. Salah satu tanaman yang berpotensi mencegah penurunan sel akibat stres kronis adalah pegagan (Centella asiatica (L.) Urban). Tujuan dari penelitian ini adalah untuk mengetahui efek protektif ekstrak etanol pegagan terhadaptingkat proliferasi sel formasi hippocampus tikus dewasa pada kondisi stres kronis. Metode Penelitian Sebanyak 30 ekor tikus Sprague Dawley jantan umur 17-18 minggu dibagi menjadi kelompok kontrol non stres, kontrol stres, ekstrak ethanol pegagan dosis 150, 300, dan 600mg/kg dan antidepresan fluoksetin HCl 10mg/kg per oral yang diikuti restraint stress 6 jam/hari selama 21 hari. Hari ke-22 tikus diinjeksi BrdU 200 mg/kg (B5002, Sigmaaldrich) intra peritoneal, kemudian diuji Morris water maze setiap hari selama 6 hari setiap hari. Hari ke-28 tikus dieuthanasi dan dikoleksi sampel otak. Otak kiri diblok dan diiris koronal secara serial (ketebalan 12µm, interval pemotongan 40 irisan), diwarnai dengan anti BrdU mouse monoclonal IgG (1:100, NCL-BrdU, Novocastra). Sel immunoreaktif BrdU tiap irisan dihitung, diukur diameter inti, dan dievaluasi dengan rumus Abercrombie correction. Hasil dilaporkan sebagai rerata±SD dan dianalisis dengan ANOVA, Welch ANOVA, dan independent t-test. Hasil Penelitian Sel berproliferasi berupa cluster (mitotik) dan non cluster (post mitotik) baik neuron maupun non neuron dengan pola distribusi menyebar. Distribusi terbanyak terdapat pada cornu ammonis terutama di stratum radiatum-locunosum-moleculare. Pada bagian tengah formasi hippocampus (plate 64-77 atlas Paxinos dan Watson) terdapat peningkatan yang jumlah proliferasi sel secara signifikan pada kelompok fluoksetin HCl 10 mg/kg dibandingkan kontrol non stres. Pada lapisan polimorfik girus dentatus terdapat penurunan yang signifikan (p<0.05) pada kelompok pegagan 150 mg/kg dibandingkan kontrol non stres dan pada kelompok pegagan 300 mg/kg dan 600 mg/kg serta fluoksetin HCl 10 mg/kg tidak menunjukkan perbedaan secara signifikan. Hal ini menunjukkan pemberian pegagan dosis 300 dan 600 mg/kg serta fluoksetin HCl 10 mg/kg mampu mencegah penurunan proliferasi sel di lapisan polimorfik. Proliferasi sel yang terjadi diharapkan dapat menggantikan sel yang rusak atau apoptosis pasca stres kronis sehingga terbentuk neuron yang terintegrasi dan berfungsi dalam proses pembentukan memori. Simpulan Pemberian ekstrak etanol pegagan dosis 300 dan 600 mg/kg bb per hari yang diikuti restraint stress 6 jam/hari selama 21 hari mampu mencegah penurunan tingkat proliferasi sel di lapisan polimorfik girus dentatus dari formasi hippocampus.

Background Decrease cell proliferation rate in hippocampal formation have been reported to contribute in the suppression of learning and memory during chronic stress. Centella asiatica (L.) Urban has been known traditionally as having neuroprotective properties with therapeutic implications for patients with memory loss. The aim of this research for studying the protective effect of Centella asiatica etanolic extract againts cell proliferation in chronic stress condition. Methods Thirty 17-18 w male Sprague Dawley rats were divided into six groups i.e. non stress, stress, Centella asiatica ethamolic extract dose (CA) 150, 300 or 600 mg/kg bw + stress, fluoxetin 10 mg/kg bw groups + stress. For 21 day the rats received restraint stress for 6 h per day after administration of extract or fluoxetine. On day 22, 200 mg/kg BW BrdU (B5002, Sigmaaldrich) was injected i.p and behavior tests were started and last for 6 days. On day 28th, rats were sacrificed and the hippocampal formations were dissected out and prepared for paraffin embedding. Serial 12 mm coronary sections were performed and one every 40 slices were put on object glass and stained with BrdU mouse monoclonal IgG (1:100, NCL-BrdU, Novocastra) and visualized with diaminobenzidine. Immunoreactive cells were counted and their nuclei were measured. The cell number were calculated using Abercrombie correction formula. The result were reported as mean +/- SD and analyzed using ANOVA and independent t-test. Result The proliferation cells were observed as cluster (mitotic) or non cluster (post-mitotic) cells and have scattered distribution. The highest number of proliferation cells was observed in cornu ammonis especially in stratum lacunosum molecular. In middle of hippocampal formation (plate 64-77 Paxinos and Watson’s atlas), the number of cell proliferation at fluoxetin HCl group increased compared to stress control (p<0.05). In polymorphic cell layer of dentate gyrus, the number of cell proliferation CA 150 mg/kg decreased compared to non stres control (p<0.05) but in CA 300 mg/kg and 600 mg/kg bw and fluoxsetin HCl 10 mgkg bw were not showed significantly different compare to non stresss control groups (p>0.05). These results showed that the application of CA 300 mg/kg, CA 600 mg/kg and fluoxetin can inhibit the decreased of cell proliferation in polymorphic layer of dentate gyrus. The cell proliferation that differentiated into neuron or glia is suggested to replace the damaged or apoptotic cells after chronic stress and maintain the integrity of neuron so that can perform the function for memory formation. Conclution The application Centella asiatica (L.) Urban ethanolic extract 300 mg/kg and 600 mg/kg following with chronis restraint stress 6 hours/day for 21st days can inhibit the decreased of cell proliferation in polymorphic layer of dentate gyrus of adult rat’s hippocampal formation.

Kata Kunci : Proliferasi sel – formasi hippocampus – stres kronis – ekstrak etanol pegagan (Centella asiatica (L.) Urban)