LAPORAN HIBAH PENELITIAN STRATEGIS NASIONAL TAHUN ANGGARAN 2009
Judul
: EFEK NEUROPROTEKTIF EKSTRAK PEGAGAN (Centella asiatica) TERHADAP BDNF (BRAIN-DERIVED NEUROTROPIC FACTOR), TNFaR, NFkB DAN APOPTOSIS PADA KULTUR SEL SYARAF YANG DIINDUKSI LPS
Ketua Anggota
: :
Husnul Khotimah, SSi, MKes 1. Wibi Riawan, SSi 2. dr. Umi Kalsum, MKes
Dibiayai Oleh Direktorat Jenderal Pendidikan Tinggi, Departemen Pendidikan Nasional, Sesuai Dengan Surat Perjanjian Pelaksanaan Hibah Penelitian Strategis Nasional, Nomor : 0174.0/023-04.2/XV/2009, tanggal 31 Desember 2008 dan berdasarkan SK Rektor Nomor : 160/SK/2009, tanggal 7 Mei 2009.
UNIVERSITAS BRAWIJAYA NOPEMBER 2009
RINGKASAN Penyakit neurodegeneratif seperti Alzheimer’s dan Parkinson merupakan penyakit neurodegenerative yang paling sering dijumpai terutama di atas umur 40 tahun. Menurut WHO, pada tahun 2050 diperkirakan jumlah penderita Alzheimer akan meningkat sekitar 100 juta orang dan akan terus meningkat setiap tahunnya dan menyerang pada usia yang lebih muda. Penyebab penyakit ini masih belum dapat dipastikan dengan jelas. Namun diduga karena proses inflamasi yang terjadi pada sel-sel neuron maupun glia pada otak. Inflamasi merupakan suatu proses kompleks yang melibatkan banyak factor antara lain radikal bebas dan berbagai mediator inflamasi. Selain menyebabkan kematian sel-sel neuron, inflamasi juga dapat mengakibatkan memendek bahkan hilangnya dendrite pada sel-sel neuron dan menurunnya factor pertumbuhan yang mengakibatkan hilang dan berkurangnya kemampuan memori. Proses inflamasi menyebabkan dikeluarkannya mediator-mediator inflamasi seperti TNFα, berbagai interleukin yang pada akhirnya dapat menyebabkan neuronal loss dan kematian sel-sel neuron. Obat-obatan neurodegenerative selain membutuhkan jangka waktu yang panjang juga menimbulkan efek samping yang kurang menyenangkan. Untuk itu diperlukan suatu bahan yang efektif untuk mengatasi neurodegerasi namun dengan efek samping minimal. Salah satu bahan yang mempunyai potensi untuk dikembangkan adalah pegagan (Centella asiatica) yang memiliki bahan aktif antara lain antiinflamasi (Asiaticoside) dan antiaoksidan (madecasoside) dan berbagai bahan aktif lain mungkin berpotensi sebagai neurotonik. Penelitian ini bertujuan untuk mengetahui efek ekstrak etanol pegagan (Centella asiatica) terhadap BDNF, TNFα, NFkB, BCl2 dan apoptosis pada sel-sel neuron yang diinduksi LPS. Desain penelitian ini adalah eksperimental laboratories dengan menggunakan rancangan acak lengkap. Sampel yang digunakan dalam penelitian ini adalah kultur primer otak anak tikus (Rattus norvegicus) strain Wistar umur 1 hari. Sampel diambil dari otak tikus terutama bagian otak tengah kemudia ditriturasi dengan phosphat buffer saline (PBS) yang bebas kalsium dan magnesium yang ditambah antibiotic penisilin dan streptomycin. Sampel disentrifuge dan supernatant dibuang kemudian sel siap ditanam pada plate yang telah dilapisi dengan ply-D-lysine dengan medium MEM (minimum Essential medium), DMEM, yang diperkaya dengan l-glutamin dan FBS (fetal bovine serum) serta ditambah dengan antibiotic penisilin dan streptomycin. Sel ditumbuhkan dalam incubator CO2 5% suhu 37°C selama 7 hari untuk siap perlakuan. Ekstrak pegagan diperoleh dengan cara maserasi dengan pelarut etanol kemudian dievaporasi sehingga diperoleh ekstrak pegagan. Sebelum digunkan dalam perlakuan, ekstrak terlebih dahulu dievaporasi dalam incubator 37°C selama 30 menit untuk menghilangkan sisa etanol. Sel diberikan ekstrak pegagan dengan konsentrasi 50, 100 dan 200 µg/ml selama 3 jam kemudian dilanjutkan dengan pemaparan LPS 10 ng/ml selama 1 jam. Selanjutnya sel dipanen untuk kepentingan immunositokimia dengan parameter BCl2, NFkB dan apoptosis sel syaraf, sedangkan mediumnya digunakan untuk ELISA kadar BDNF dan TNFα. Hasil ELISA dan kuantifikasi immunositokimia dianalisa dengan ANOVA menggunakan SPSS 17 for windows.
Hasil penelitian menunjukkan bahwa pemberian LPS menyebabkan peningkatan signifikan kadar TNFα. Hal ini membuktikan bahwa LPS merupakan pemicu terjadinya inflamasi. Pemberian ekstrak pegagan mampu menurunkan kadar TNFα seiring peningkatan konsentrasi ekstrak. Ekstrak pegagan konsentrasi 50 µg/ml menurunkan kadar TNFα namun tidak signifikan. Sedangkan pada konsentrasi 100 dan 200 µg/ml menurunkan kadar TNFα secara signifikan ( p = 0.001 dan p = 0.000) dan konsentrasi optimum dicapai pada konsentrasi 200 µg/ml. Hasil kuantifikasi ekspresi NFkB serupa dengan hasil pengukuran kadar TNFα. Ekstrak pegagan konsentrasi 50 µg/ml menurunkan kadar NFkB namun tidak signifikan. Sedangkan pada konsentrasi 100 dan 200 µg/ml menurunkan kadar NFkB secara signifikan (p = 0.045 dan p = 0.002) dan konsentrasi optimum dicapai pada konsentrasi 200 µg/ml. Hal ini diduga karena efek bahan aktif asiaticoside dan madecasoside yang memiliki efek antiinflamasi dan antioksidan. LPS menyebabkan peningkatan ekspresi NFkB melalui produksi radikal bebas yang selanjutnya menyebabkan peningkatan pengeluaran mediator-mediator inflamasi salah satunya TNFα. Asiaticoside terbukti mempunyai efek entiinflamasi dan madecasoside selain memiliki efek antiinflamasi juga berfungsi sebagai antioksidan. Hasil visualisasi DNA terfragmentasi menunjukkan pemberian ekstrak pegagan mampu menurunkan apoptosis. Hal ini dikonfirmasi dengan ekspresi BCL2 yang merupakan protein anti apoptosis yang menunjukkan bahwa ekspresi BCL2 meningkat pada kelompok yang diberikan ekstrak pegagan yang meningkat seiring peningkatan konsentrasi ekstrak. Peningkatan signifikan terjadi pada kelompok yang diberi ekstrak pegagan konsentrasi 200 µg/ml (p = 0.010). Keadaan ini berkaitan dengan menurunnya ekspresi NFkB yang diikuti menurunnya pelepasan TNFα sehingga kejadian apoptosis menurun karena TNFα dapat menyebabkan apoptosis. Kadar BDNF meningkat akibat pemberian ekstrak pegagan. Yang menarik di sini adalah bahwa Perbedaan signifikan didapatkan pada pemberian ekstrak pegagan konsentrasi 100 µg/ml ( p = 0.024) dan konsentrasi 200 µg/ml ( p = 0.015). pemberian ekstrak pegagan mampu mengembalikan kadar BDNF hampir mencapai normal pada sel neuron akibat induksi LPS pada konsentrasi 200 µg/ml (tidak berbeda bermakna dengan control, p=0.912). Peningkatan pelepasan BDNF ini dimungkinkan karena terdapat bahan aktif dalam ekstrak pegagan yang berfungsi sebagai aktivator ekspresi BDNF. Kemungkinan lain adalah juga karena efek antioksidan pegagan yang mampu meregulasi kadar kalsium intrasel karena salah satu factor yang mempengaruhi ekspresi CREB yang memediasi transkripsi BDNF yang merupakan Ca2+-dependent. Dari hasil-hasil diatas dapat disimpulkan bahwa pegagan mampu melindungi kerusakan sel-sel syaraf akibat inflamasi dengan menurunnya kadar TNFα dan NFkB, melalui mekanisme antioksidan dan antiinflamasi yang dimiliki oleh pegagan. Pegagan juga mampu menurunkan apoptosis dan meningkatkan ekspresi BCl2 karena proses inflamasi. Selain itu pegagan juga memberikan perlindungan terhadap fungsi kognitif melalui peningkatan kadar BDNF. Konsentrasi optimum ekstrak pegagan didap atkan pada konsentrasi 200 µg/ml.
SUMMARY Neurodegenerative diseases like Alzheimer's and Parkinson's is a neurodegenerative disease most frequently encountered, especially in the age of 40 years. According to WHO, in 2050 estimated the number of people with Alzheimer's will rise by about 100 million people and will continue to increase every year and attacked at a younger age. The cause of this disease still has not been established clearly. However, allegedly due to inflammatory processes that occur in neuronal cells and glia in the brain. Inflammation is a complex process involving many factors including free radicals and various inflammatory mediators. In addition to causing the death of neuron cells, can also cause inflammation and even loss of dendrite retracts on neuron cells and a decrease in growth factors that result in lost and reduced memory abilities. Inflammatory process causes the issuance of a mediator-inflammatory mediators such as TNFα, various interleukins, which in turn can lead to neuronal loss and death of neuron cells. Neurodegenerative drugs in addition requires long-term side effects too unpleasant. One material that has the potential to be developed is Centella asiatica, which has active ingredients include antiinflammatory (Asiaticoside) and antioxidant (madecasoside) and various other active ingredients may be potentially as neurotonic. This study aims to determine the effect of ethanol extract of pegagan (Centella asiatica) for BDNF, TNFα, NFkB, apoptosis and BCl2 in LPS-induced neuronal cells. This research design is experimental laboratories using completely randomized design. The sample used in this study is the brain for primary culture of rat (Rattus norvegicus) Wistar strain aged 1 day. Samples taken from mouse brain, especially the midbrain and triturated with phosphat buffer saline (PBS) free calcium and magnesium are added antibiotics penicillin and streptomycin. Sample were centrifuge and the supernatant discarded then cells ready to be planted in plate that had been coated with poly-D-lysine in MEM medium (Minimum Essential medium), DMEM, enriched with L-glutamine and FBS (fetal bovine serum) and supplemented with penicillin and streptomycin as antibiotics. Cells grown for 7 days in 5% CO2 incubator temperature of 37°C. Centella asiatica extract obtained by maceration with ethanol solvent and thus obtained evaporated Centella. Before used for treatment extract was incubated in 40°C for 30 minutes, to eliminate the remaining ethanol. The Cells given Centella asiatica extracts with concentrations 50, 100 and 200 mcg / ml for 3 hours followed by exposure of LPS 10 ng / ml for 1 hour. Next, cells harvested for immunocytochemistry with parameters BCl2, NFkB and apoptosis of nerve cells, while the medium used for the ELISA of BDNF and TNFα levels. The result from ELISA assay and Immunocytochemistry quantification analyze using ANOVA from SPSS 17 for windows. The results showed that administration of LPS caused a significant increase in TNFα levels. This proves that LPS is a trigger of inflammation. Provision of Centella asiatica extract can reduce levels of TNFα with increased concentration of the extract. Centella asiatica extract concentration of 50 mcg / ml lower levels of TNFα but not significant. While the concentration of 100 and 200 mcg / ml TNFα levels decrease significantly (p=0.001 and p = 0.000) and the optimum concentration is reached at concentrations of
200 mcg / ml. NFkB expression quantification results are similar to the results of measurements TNFα levels. Centella asiatica extract concentration of 50 mcg / ml lower levels of NFkB, but not significantly. While the concentration of 100 and 200 mcg / ml decrease significantly NFkB levels (p = 0.045 and p = 0.002)and the optimum concentration is reached at concentrations of 200 mcg / ml. This is presumably because the effect of the active ingredients that asiaticoside and madecasoside has antiinflammatory and antioxidant effects. LPS causes increased expression of NFkB through the production of free radicals which in turn led to increased expenditure inflammatory mediator TNFα. Asiaticoside shown to have effects other than entiinflamasi and madecasoside have anti-inflammatory effects also function as antioxidants. Results showed fragmented DNA visualization giving Centella asiatica extracts to reduce apoptosis. Significantly different effect obtain in group that administered of Centella asiatica concentration 100 µg/ml ( p = 0.024) and Concentration 200 µg/ml ( p = 0.015). This is confirmed by the expression of BCL2 is an anti-apoptosis protein, indicating that increased expression of BCL2 in the group given the extract of Centella asiatica increase as increasing the concentration of the extract. This situation is associated with decreased expression of NFkB which followed the release of TNFα thus decreasing incidence decreased apoptosis due to TNFα can cause apoptosis. BDNF levels increased due to provision of Centella asiatica extract. What's interesting here is that the provision of Centella asiatica extract can restore BDNF levels almost reached normal neuronal cells by induction of LPS on the concentration of 200 mcg / ml (p=0.912). Increased BDNF release was made possible because there is an active ingredient in the extract of Centella asiatica that serves as the activator BDNF expression. Another possibility is also due to Centella asiatica antioxidant effects that can regulate intracellular calcium levels due to one factor that affects the expression of CREB that mediate transcription of BDNF, which is Ca2+-dependent. From the above results it can be concluded that Centella asiatica can protect damaged nerve cells from inflammatory with reduced levels of TNFα and NFkB, through antioxidant and antiinflammatory mechanisms are owned by Centella asiatica. Centella asiatica is also able to reduce apoptosis and increase expression of inflammatory BCl2 due process. In addition Centella asiatica also provides protection against cognitive function through increasing levels of BDNF. Centella asiatica extract the optimum concentration obtained at a concentration 200 mcg / ml.
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