Pengaruh Minyak Kelapa Murni dan Hasil Hidrolisisnya Terhadap Parameter Luka pada Sel NIH 3T3 secara In Vitro

Abstract

Penyembuhan luka adalah suatu proses yang kompleks dengan melibatkan banyak sel, sitokin, growth factor, dan extracellular matrix (ECM). Minyak kelapa murni (Virgin Coconut Oil =VCO) dan hasil hidrolisisnya (Hydrolized Virgin Coconut Oil=HVCO) sudah diuji secara in vivo sebagai penyembuh luka. Penelitian ini bertujuan untuk mengevaluasi pengaruh VCO dan HVCO terhadap parameter penyembuhan luka secara invitro pada sel NIH 3T3. VCO dihidrolisis parsial menggunakan lipase dari Rhizomucor miehei (aktif pada posisi sn-1,3) untuk menghasilkan VCO terhidrolisis (HVCO) dan diukur bilangan asamnya. Sel NIH 3T3 ditanam di plate, kemudian setelah 24 jam diberi bahan uji dengan berbagai seri konsentrasi dimulai konsentrasi paling rendah 15,625 μg/mL hingga konsentrasi paling tinggi 1000 μg/mL lalu diinkubasi 24 jam dan diukur absorbansi menggunakan metode MTT [3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolium bromida] serta dihitung viabilitas sel. Konsentrasi dengan viabilitas sel paling tinggi digunakan untuk mengukur parameter penyembuhan luka yaitu aktivitas proliferasi sel, persen penutupan luka, ekspresi protein MMP-9, PDGF-BB, TGF-β1 dan ekspresi gen COX-2. Pengujian parameter penyembuhan luka dimulai dari sel NIH 3T3 ditanam di plate, kemudian setelah 24 jam diberi bahan uji lalu diinkubasi 24 jam serta dilanjutkan sesuai metode dalam parameter penyembuhan luka. Aktivitas proliferasi sel diukur dengan menghitung viabilitas sel yang diinkubasi 24 jam, 48 jam dan 72 jam. Persen penutupan luka diuji dengan metode scratch wound healing, ekspresi protein MMP-9, PDGF-BB, TGF-β1 diukur dengan metode imunositokimia dan ekspresi gen COX-2 ditentukan menggunakan RT-PCR. Sel yang diberi VCO dan HVCO, viabilitas nya meningkat pada konsenterasi 1000μg/mL sampai konsentrasi 62,5 μg/mL dan menurun dibawah konsentrasi 62,5 μg/mL. Proliferasi sel setelah inkubasi 24, 48, 72 jam dari kontrol sel, VCO, HVCO meningkat pada jam ke 48 tetapi menurun pada jam ke 72. Aktivitas proliferasi sel dari kelompok HVCO lebih besar daripada VCO dan kontrol sel. Persentase penutupan luka setelah inkubasi 24 dan 48 jam adalah kelompok kontrol sel dan VCO belum ada model luka yang tertutup sampai pada jam ke 48, tetapi kelompok HVCO model luka sudah menutup 100%. Hasil ekspresi protein MMP-9 meningkat dari 2,89% (kontrol sel) menjadi 28,16% (VCO), dan 55,40% (HVCO), persentase ekspresi protein PDGF-BB meningkat dari 28.11% (kontrol sel) menjadi 48,53% (VCO) dan 61,65% (HVCO), dan persentase ekspresi protein TGF-β1 meningkat dari 4,19% (kontrol sel) menjadi 18,41% (VCO) dan 36,35% (HVCO). Hasil ekspresi gen COX-2 dari kontrol sel, VCO 62,5 μg/mL dan HVCO 62,5 μg/mL berturut-turut adalah 1,00; 1,43; dan 1,93. Berdasarkan hasil pengujian invitro terhadap parameter penyembuhan luka maka dapat disimpulkan bahwa VCO dan HVCO meningkatkan proses penyembuhan luka.

Wound healing is a complex process involving many cells, cytokines, growth factors, proteases, and extracellular matrix (ECM). Minyak kelapa murni (Virgin Coconut Oil = VCO) and the results of its hydrolysis (Hydrolized Virgin Coconut Oil = HVCO) had been tested in vivo as wound healers. This study aimed to evaluate the effect of VCO and HVCO on invitro wound healing parameters in NIH 3T3 cells. VCO was partially hydrolyzed using lipase from Rhizomucor miehei (active at sn-1,3 position) to produce hydrolyzed VCO (HVCO) and its acid number was measured. NIH 3T3 cells were planted in a plate, then after 24 hours the test material was given with various concentrations with the lowest concentration of 15,625 μg/mL the highest concentration of 1000 μg / mL then incubated 24 hours and measured absorbance using the MTT method [3- (4, 5-dimethylthiazol-2-il) -2.5-diphenyl tetrazolium bromide] and cell viability calculated. The highest of cell viability was used to measure wound healing parameters, namely cell proliferation activity, percent wound closure, MMP-9 protein expression, PDGF-BB, TGF-β1 and COX-2 gene expression. Testing the wound healing parameters starting from NIH 3T3 cells was planted in a plate, then after 24 hours the test material was given and then incubated 24 hours and continued according to the method in the wound healing parameters. Cell proliferation activity was measured by calculating cell viability incubated 24 hours, 48 hours and 72 hours. The wound closure percentage was tested by the scratch wound healing method, the expression of MMP-9 protein, PDGF-BB, TGF-β1 was measured by the immunocytochemical method and COX-2 gene expression was determined using RT-PCR. Cells given VCO and HVCO, their viability increased at concentrations of 1000 μg / mL to concentrations of 62.5 μg / mL and decreased below the concentration of 62.5 μg / mL. Cell proliferation after incubation 24, 48, 72 hours from cell control, VCO, HVCO increased at 48 hours but decreased at 72 hours. Cell proliferation activity of the HVCO group was greater than VCO and cell control. The percentage of wound closure after 24 and 48 hours incubation was the cell control group and VCO had no closed wound model until the 48th hour, but the HVCO group wound model had closed 100%. The results of MMP-9 protein expression increased from 2.89% (cell control) to 28.16% (VCO), and 55.40% (HVCO), the percentage of PDGF-BB protein expression increased from 28.11% (cell control) to 48 , 53% (VCO) and 61.65% (HVCO), and the percentage of TGF-β1 protein expression increased from 4.19% (cell control) to 18.41% (VCO) and 36.35% (HVCO). The results of COX-2 gene expression from cell control, VCO 62.5 μg / mL and HVCO 62.5 μg / mL were 1.00; 1.43; and 1.93. Based on the results of invitro testing on wound healing parameters, it can be concluded that VCO and HVCO improve the wound healing process.