Uji Aktivitas Fraksi Medium Kalus Wortel (Daucus carota) Pada Sel Human Dermal Fibroblast Adult (HDFa) Yang Diinduksi Sinar UV-B dan Analisis Ekspresi Sitokin TNF-Alfa Dengan Metode imunotokimia

KRESENTIA HOESS VANDARINA SATYA DAMARI, Dr. Rumiyati, M.Si., Apt.

2021 | Skripsi | S1 FARMASI

Sel punca tanaman (Kalus) dapat berdiferensiasi membentuk jaringan sehingga beregenerasi dengan cepat. Penuaan memicu penyakit degeneratif seperti radikal bebas. Sumber radikal bebas seperti sinar UV memicu adanya peningkatan ekspresi pro-inflamasi TNF-alfa, sehingga dapat menyebabkan terjadinya inflammaging. Kalus tanaman dapat memperbaiki penuaan kulit dengan mencegah kerusakan sel akibat sinar UV. Fraksi medium kalus tomat diketahui mempunyai aktivitas sitoprotektif dan dapat menurunkan ekspresi sitokin TNF-alfa pada sel HGF. Ekstrak medium kultur suspensi sel (MKS) kalus wortel diduga mempunyai aktivitas sitoprotektif dari kandungan protein & eksosom. Penelitian ini bertujuan untuk mengetahui aktivitas pengaruh ekstrak MKS kalus wortel pada ekspresi sitokin TNF-alfa sel HDFa yang diinduksi dari paparan sinar UV-B. Ekstrak MKS kalus wortel diperoleh dengan metode acetone precipitation of proteins. Ekstrak MKS kalus wortel diuji pengaruhnya terhadap sel HDFa yang diinduksi paparan sinar UV-B kemudian diamati viabilitas selnya dengan metode MTT assay. Kadar ekstrak yang memberikan viabilitas sel tertinggi digunakan untuk menguji ekspresi TNF-alfa dengan metode imunositokimia. Hasil yang diperoleh dilakukan analisis statistik menggunakan SPSS dengan uji One-Way ANOVA dan Post Hoc Tukey LSD pada tingkat kepercayaan 95%. Hasil penelitian menunjukkan bahwa ekstrak MKS kalus wortel dengan kadar 20 mg/mL memberikan viabilitas sel HDFa sebesar 94,83% ± 8,69 dibandingkan kontrol sel dan meningkatkan secara signifikan viabilitas sel HDFa hingga 35,5% setelah diberi paparan UV-B dosis 200 mJ/cm2. Selain itu, sel HDFa yang diberi perlakuan ekstrak MKS kalus wortel mengekspresikan sitokin TNF-alfa sebesar 41,43% ± 0,44 dan menurunkan ekspresi TNF-alfa sebesar 20,17% pada sel HDFa yang diberi paparan sinar UV dibandingkan kontrol sel. Berdasarkan uji viabilitas sel HDFa dan ekspresi sitokin TNF-alfa, ekstrak MKS kalus wortel dapat melindungi sel HDFa dari paparan sinar UV.

Plant stem cells (calus) can differentiate to form tissues so that they regenerate quickly. Aging triggers degenerative diseases such as free radicals. Sources of free radicals such as UV light trigger an increase in the expression of pro-inflammatory TNF-Alpha, which can cause inflammation. Plant callus can improve skin aging by preventing cell damage caused by UV rays. Tomato callus medium fraction is known to have cytoprotective activity and can reduce TNF alpha cytokine expression in HGF cells. Carrot callus cell suspension culture medium (CCM) extract was suspected to have cytoprotective activity from protein & exosomes content. This study aimed to determine the effect of carrot callus MKS extract on TNF alpha cytokine expression in HDFa cells induced by exposure to UV-B light. Carrot callus CCM extract was obtained by acetone precipitation of proteins method. Carrot callus CCM extract was tested for its effect on HDFa cells induced by UV-B light exposure and then cell viability was observed using the MTT assay method. The extract content which gave the highest cell viability was used to test the expression of TNF-alpha by immunocytochemical method. The results obtained were statistically analyzed using SPSS with One-Way ANOVA and Post Hoc Tukey LSD tests at a 95% confidence level. The results showed that carrot callus CCM extract at concentration 20 mg/mL gave HDFa cell viability of 94.83% ± 8.69 compared to control cells and significantly increased HDFa cell viability up to 35.5% after exposure to UV-B dose of 200 mJ/cm2. In addition, HDFa cells treated with carrot callus MKS extract expressed TNF- cytokines by 41.43% ± 0.44 and decreased TNF-alpha expression by 20.17% in HDFa cells exposed to UV light compared to control cells. Based on the test of HDFa cell viability and TNF-alpha cytokine expression, carrot callus MKS extract can protect HDFa cells from UV light exposure.

Kata Kunci : ekstrak MKS kalus wortel, UV-B, viabilitas sel HDFa, ekspresi TNF-Alfa