Efek Fraksi Aktif Piper crocatum Ruiz & Pav terhadap Deposisi Kolagen dan Wound Closure melalui Regulasi Ekspresi p53, Alfa SMA, SOD1 dan E-cadherin pada Wounded Hyperglycemia Fibroblasts

ANDINA SETYAWATI, Piper crocatum Ruiz & Pav, diabetic wound healing, mechanism, p53, Alpha SMA, SOD-1, E-cadherin

2022 | Disertasi | DOKTOR ILMU KEDOKTERAN DAN KESEHATAN

Latar belakang: Piper crocatum Ruiz & Pav (P. crocatum) telah dilaporkan dapat mempercepat penyembuhan luka diabetik secara empiris. Beberapa studi menunjukkan manfaat P. crocatum dalam mengobati berbagai macam penyakit termasuk luka kronik. P. crocatum dilaporkan dapat menginduksi proliferasi fibroblas, namun studi lain melaporkan P. crocatum menghambat proliferasi. Oleh karena itu, pemisahan senyawa pada P. crocatum perlu dilakukan untuk mendapatkan efek yang konsisten. Tujuan penelitian: Penelitian ini bertujuan untuk mengidentifikasi efek fraksi aktif P. crocatum terhadap aktivitas in vitro pada wounded hyperglycemia fibroblasts (wHFs). Metode Penelitian: Penelitian ini merupakan penelitian in vitro dengan menggunakan fibroblas hiperglikemia (HFs) yang ditumbuhkan dari ditumbuhkan dari kultur primer jaringan sirkumsisi pada media basal dengan penambahan 17 mMol/L D-glucose. Identifikasi fraksi aktif dilakukan menggunakan prosedur Bioassay-guided fractionation dan didasarkan pada nilai IC50 tertinggi. Fraksi paling aktif kemudian diaplikasikan pada HFs yang dilukai dengan diagonal scratch (wHFs). Deposisi kolagen diukur dengan menggunakan Picro-Sirius Red dan wound closure diukur dengan menggunakan scratch assay. Ekspresi p53, Alfa SMA, SOD1 dan E-chaderin diukur menggunakan western blotting. Hasil Penelitian: Penelitian ini mengkonfirmasi bahwa fraksi IV (FIV) adalah fraksi aktif P. crocatum. Kelompok wHFs intervensi memiliki rerata deposisi kolagen (p<0.0001) dan wound closure rate (p<0.0001) lebih tinggi secara signifikan dibandingkan kelompok kontrol. Kelompok intervensi juga ditemukan memiliki ekspresi p53 lebih rendah secara signifikan dibandingkan dengan kelompok kontrol (p=0.001). Ekspresi Alfa SMA, SOD1 dan E-cadherin ditemukan lebih tinggi pada kelompok intervensi daripada kelompok kontrol (p=0.0001). Hasil uji korelasi menemukan deposisi kolagen dan wound closure berhubungan negatif dengan ekspresi p53 (p=0.01, r= -1.000), dan berhubungan positif dengan Alfa SMA, SOD1 dan E-chaderin (p=0.01, r=1.000). Profil KLT menunjukkan adanya kandungan senyawa polifenol glikosida. Kesimpulan: FIV P. crocatum berpotensi dapat mempercepat penyembuhan luka pada wHFs pada konsentrasi paling efektif 7.81 �µg/ml dengan meningkatkan deposisi kolagen dan kecepatan wound closure melalui penurunan p53 serta peningkatan Alfa SMA, SOD1, dan E-cadherin. Golongan senyawa polifenol glikosid diduga berkontribusi pada mekanisme tersebut.

Background: Piper crocatum Ruiz & Pav (P. crocatum) has been reported to accelerate the diabetic wound healing process empirically. Some studies showed the benefits of P. crocatum in treating various diseases. The effects of P. crocatum on fibroblasts have been reported to induce proliferation and vice versa. Hence, the separation of P. crocatum compounds needs to be done to get a consistent effect on diabetic wound healing. Aims: This study aimed to identify the effects of P. crocatum active fractions on in vitro activities of wounded hyperglycemia fibroblasts (wHFs). Methods: This study was an in vitro study using hyperglycemia fibroblasts (HFs) cultured from circumcision tissues and grown on basal media with the addition of 17mMol/L D-glucose. Active fraction was identified following bioassay-guided fractionation procedure and was determined based on the highest IC50 values. The most active fraction then was applied on wHFs which was diagonal scratched. Collagen deposition were measured using Picro-Sirius Red and wound closure were measured using scratch assay. The expressions of p53, Alpha SMA, SOD1, and E-cadherin were measured using western blotting and counted using Image J for Windows. The compound of FIV was determined using thin layer chromatography (TLC). Results: This study confirmed that fraction IV (FIV) is the most active fraction of P. crocatum. Further findings, the intervention group had significantly higher collagen deposition rates (p<0.0001) and wound closure rates (p<0.0001) than the control group. The treatment group was also found to have a significantly lower p53 relative expression than the control group (p=0.001). The expressions of Alpha SMA, SOD1 and E-cadherin were found to be higher in the intervention group than the control group (p=0.0001). The correlation tests showed a negative relationship between collagen deposition and wound closure with expressions of p53 (p=0.001, r= -1.000), and a positive relationship with Alpha SMA, SOD1, and E-cadherin (p=0.001, r=1.000) at FIV of 7.81 �µg/dl. The TLC profile showed the presence of glycoside polyphenols. Conclusion: FIV P. crocatum has the potential to accelerate wound healing in wHFs by increasing the rates of collagen deposition and wound closure. Regulations in p53 as well as Alpha SMA, SOD1, and E-cadherin are known to underlie these events at 7.81 �µg/dl the most optimal concentration of FIV. The glycoside polyphenols are supposed to contribute to these mechanisms.

Kata Kunci : Piper crocatum Ruiz & Pav, penyembuhan luka diabetik, mekanisme, p53, Alfa SMA, SOD1, E-cadherin