Pengaruh Pemberian Kuersetin Terhadap Ekspresi SMAD7, SMAD2/3 dan Sintesis Kolagen Tipe I melalui Penekanan Ekspresi p38 Terfosforilasi pada Kultur Sel Fibroblas Keloid (Studi In Vitro Pada Kultur Primer Fibroblas Keloid Manusia)

Widiatmoko, Arif and Prof.Dr.dr. Loeki Enggar Fitri, M.Kes, Sp.ParK and Prof. Agustina Tri Endharti, S.Si, Ph.D and Dr. dr. Sinta Murlistyarini, Sp.KK(K) (2024) Pengaruh Pemberian Kuersetin Terhadap Ekspresi SMAD7, SMAD2/3 dan Sintesis Kolagen Tipe I melalui Penekanan Ekspresi p38 Terfosforilasi pada Kultur Sel Fibroblas Keloid (Studi In Vitro Pada Kultur Primer Fibroblas Keloid Manusia). Doktor thesis, Universitas Brawijaya.

Abstract

Keloid merupakan pertumbuhan jaringan ikat kulit jinak akibat proses penyembuhan luka abnormal dan penumpukan matriks ekstraseluler berlebih. Profil pasien keloid di poliklinik kulit dan kelamin RSUP Prof.Dr. D. Kandou Manado tahun 2011 sampai 2015 menunjukkan insidensi keloid 1,68% dari semua kunjungan pasien kulit. Masalah yang timbul pada penderita keloid adalah gangguan kualitas hidup karena rasa nyeri, gatal, gangguan pergerakan tubuh dan tampilan kosmetik yang tidak baik. Angka kekambuhan terapi keloid masih tinggi. Terapi untuk menekan kekambuhan setelah pembedahan keloid dan mencegah terbentuknya keloid setelah luka perlu dikembangkan. Keloid terjadi akibat peningkatan sintesis dan penurunan degradasi kolagen sebagai pembentuk extracellular matrix (ECM) pada penyembuhan luka. Sintesis kolagen diaktifkan transforming growth factor-β (TGF-β) melalui berbagai sinyal transduksi yang salah satunya adalah jalur TGF-β/Smad. Ikatan TGF-β dengan reseptornya pada fibroblas menyebabkan fosforilasi Smad2/3 bergabung dengan Smad4 membentuk komplek Smad 2/3/4 dan translokasi ke nukleus memicu sintesis kolagen tipe I. Jalur TGF-β/Smad dihambat Smad7 sebagai Smad inhibitor melalui hambatan fosforilasi Smad2 dan Smad3. Ekspresi Smad7 pada fibroblas keloid dihambat p38 yang juga diaktifkan TGF-β melalui jalur Mitogen-Activated Protein Kinase (MAPK). Penelitian terhadap Smad7 sebagai target terapi hambatan sinyal TGF-β dan sintesis kolagen dikembangkan pada berbagai penyakit termasuk keloid. Upregulasi ekspresi Smad7 terjadi pada kultur fibroblas keloid yang mendapat terapi radiasi. Peningkatan ekspresi Smad7 ini terjadi melalui penekanan fosforilasi p38 dan menyebabkan penurunan sintesis kolagen. Ekspresi Smad7 pada kultur fibroblas meningkat setelah pemberian asiaticoside. Peningkatan ekspresi Smad7 ini menyebabkan penurunan ekspresi protein dan mRNA kolagen tipe 1. Kuersetin adalah flavonoid yang banyak ditemukan pada tanaman buah dan sayuran. Penelitian menunjukkan kuersetin menghambat jalur TGF-β/Smad melalui hambatan fosforilasi p38. Hambatan kuersetin pada fosforilasi p38 memberikan peluang kuersetin untuk meningkatkan Smad7 sebagai regulator negatif jalur TGF-β/Smad serta menekan fosforilasi Smad2/3 dan sintesis kolagen tipe I. Penelitian ini bertujuan untuk mengetahui pengaruh pemberian kuersetin terhadap ekspresi p38 terfosforilasi, Smad7, Smad2/3 dan sintesis kolagen tipe I pada kultur sel fibroblas keloid manusia. Desain penelitian adalah eksperimental murni post-test only control group. Sampel penelitian adalah kultur primer fibroblas keloid manusia metode skin explant sub kultur ke 3 dengan syarat sel monolayer, kepadatan minimal 105 sel/ml dan viabilitas minimal 80% jumlah sel. Variabel bebas adalah kuersetin dosis 5 μg/ml, 10 μg/ml, 20 μg/ml dan kontrol. Variabel tergantung adalah ekspresi p38 terfosforilasi, Smad7, Smad2/3 dan kadar kolagen tipe I. Metode Western Blot dilakukan untuk mengukur ekspresi p38 terfosforilasi, ekspresi Smad7 dan ekspresi Smad2/3. Kadar kolagen tipe I diukur dengan metode Enzyme-linked Immunosorbent Assay (ELISA). Uji perbedaan rata-rata variabel dependen dari berbagai kelompok perlakuan menggunakan uji one – way analysis of variance (ANOVA) diikuti uji post Hoc Tukey untuk ekspresi p38 terfosforilasi dan uji Kruskal-Wallis diikuti uji pairwise comparison untuk ekspresi Smad7, Smad2/3 sitoplasma, Smad2/3 nukleus dan kadar kolagen tipe I. Uji T independen dilakukan untuk membandingkan rata-rata Smad2/3 sitoplasma dengan nukleus pada kelompok kontrol, kuersetin 5 μg/ml dan 10 μg/ml. Uji Mann-Whitney dilakukan untuk membandingkan rata-rata Smad2/3 sitoplasma dengan nukleus pada kelompok kuersetin 20 μg/ml. Uji korelasi antar variabel dependen dalam satu kelompok perlakuan menggunakan uji non-parametrik Spearman. Nilai p<0,05 dianggap bermakna secara statistik. Analisis data dilakukan menggunakan program Statistical Package for the Social Sciences (SPSS) 27 for Windows. Hasil penelitian menunjukkan adanya pengaruh kuersetin terhadap ekspresi p38 terfosforilasi pada kultur fibroblas keloid. Kuersetin konsentrasi 10 μg/ml menyebabkan hambatan yang bermakna pada ekspresi p38 terfosforilasi bila dibandingkan dengan kelompok kontrol. Kuersetin konsentrasi 20 μg/ml memiliki kecenderungan meningkatkan ekspresi p38 terfosforilasi. Penelitian ini juga menunjukkan bahwa kuersetin menghambat ekspresi Smad7 kultur fibroblas keloid. Hal ini berlawanan dengan hipotesis penelitian. Pengaruh hambatan kuersetin terhadap ekspresi Smad7 tidak menguntungkan sebagai terapi keloid tetapi bisa bermanfaat untuk mencegah penuaan pada kulit. Penelitian ini juga menunjukkan pengaruh kuersetin terhadap ekspresi Smad2/3 baik di sitoplasma maupun nukleus fibroblas keloid. Kuersetin tidak memberikan pengaruh yang bermakna terhadap ekspresi Smad2/3 sitoplasma meskipun terdapat kecenderungan penurunan ekspresi pada kelompok kuersetin konsentrasi tertinggi. Kuersetin konsentrasi 20 μg/ml memberikan pengaruh hambatan bermakna terhadap ekspresi Smad2/3 nukleus fibroblas keloid meskipun konsentrasi kuersetin lainnya juga memiliki kecenderungan menghambat ekspresi Smad2/3 nukleus. Hasil ini menunjukkan bahwa kuersetin diduga memiliki potensi menghambat translokasi Smad2/3 ke inti fibroblas keloid. Hasil penelitian ini menunjukkan pemberian kuersetin tidak memberikan pengaruh yang bermakna terhadap kadar kolagen tipe I meskipun kuersetin konsentrasi 20 μg/ml memiliki kecenderungan menghambat kadar kolagen tipe I. Kesimpulan penelitian ini kuersetin masih memiliki potensi sebagai terapi keloid dengan menghambat ekspresi p38 terfosforilasi dan translokasi Smad2/3 ke inti fibroblas keloid. Penelitian ini menunjukkan kuersetin tidak dapat meningkatkan ekspresi Smad7 fibroblas keloid tetapi menekan ekspresinya.

English Abstract

Keloids are benign growths of connective skin tissue resulting from abnormal wound healing processes and the buildup of excess extracellular matrix. Profiles of keloid patients at the skin and genital clinic at RSUP Prof.Dr. D. Kandou Manado from 2011 to 2015 showed a keloid incidence of 1.68% of all skin patient visits. The problems that arise in keloid sufferers are impaired quality of life due to pain, itching, impaired body movement and poor cosmetic appearance. The recurrence rate for keloid therapy is still high. Therapy to suppress recurrence after keloid surgery and prevent keloid formation after injury needs to be developed. Keloids occur due to increased synthesis and decreased degradation of collagen as a form of extracellular matrix (ECM) in wound healing. Collagen synthesis is activated by transforming growth factor-β (TGF-β) through various signal transductions, one of which is the TGF-β/Smad pathway. The binding of TGF-β to its receptor on fibroblasts causes phosphorylation of Smad2/3 to combine with Smad4 to form the Smad 2/3/4 complex and translocation to the cell nucleus triggering the synthesis of type I collagen. The TGF-β/Smad pathway is inhibited by Smad7 as a Smad inhibitor by inhibiting Smad2 and Smad3 phosphorylation. Smad7 expression in keloid fibroblasts is inhibited by p38 which is also activated by TGF-β through the Mitogen-Activated Protein Kinase (MAPK) pathway. Research on Smad7 as a therapeutic target for inhibiting TGF-β signaling and collagen synthesis has been developed in various diseases including keloids. Upregulation of Smad7 expression occurred in cultured keloid fibroblasts that received radiation therapy. This increase in Smad7 expression occurs through suppressing p38 phosphorylation and causing a decrease in collagen synthesis. Smad7 expression in fibroblast cultures increased after asiaticoside administration. This increase in Smad7 expression causes a decrease in type 1 collagen protein and mRNA expression. Quercetin is a flavonoid that is found in many fruit and vegetable plants. Research shows that quercetin inhibits the TGF-β/Smad pathway through inhibiting p38 phosphorylation. Quercetin's inhibition of p38 phosphorylation provides an opportunity for quercetin to increase Smad7 as a negative regulator of the TGF-β/Smad pathway and suppress Smad2/3 phosphorylation and type I collagen synthesis. This study aims to determine the effect of quercetin administration on the expression of phosphorylated p38, Smad7, Smad2/3 and type I collagen synthesis in human keloid fibroblast cell cultures. The research design was purely experimental, post-test only control group. The research sample was a primary culture of human keloid fibroblasts using the 3rd skin explant sub culture method with the requirements of monolayer cells, a minimum density of 105 cells/ml and a minimum viability of 80% of the number of cells. The independent variables were quercetin doses of 5 μg/ml, 10 μg/ml, 20 μg/ml and control. The dependent variables were phosphorylated p38 expression, Smad7 expression, Smad2/3 expression and type I collagen levels. The Western Blot method was carried out to measure phosphorylated p38 expression, Smad7 expression and Smad2/3 expression. Type I collagen levels were measured using the Enzyme-linked Immunosorbent Assay (ELISA) method. Mean difference of the dependent variable from various treatment groups analyzed by one-way analysis of variance (ANOVA) test for phosphorylated p38 expression followed by Tukey post Hoc analysis. The non-parametric Kruskal-Wallis test followed by pairwise comparison was performed for Smad7, cytoplasmic Smad2/3, nuclear Smad2/3 and type I collagen mean difference analyses. Mean difference between cytoplasmic and nuclear Smad2/3 expression in control, Quercetin 5 μg/ml and 10 μg/ml groups analyzed by Independent T test. Mean difference between cytoplasmic and nuclear Smad2/3 expression in Quercetin 20 μg/ml group analyzed by Mann-Whitney test. Correlation between dependent variables in one treatment group analyzed by Spearman test. Statistically significant is considered if p value <0.05. Data analysis was carried out using the Statistical Package for the Social Sciences (SPSS) 27 for Windows program. The results of the study showed the influence of quercetin on the expression of phosphorylated p38 in keloid fibroblast cultures. Quercetin concentration of 10 μg/ml caused a significant inhibition in the expression of phosphorylated p38 when compared with the control group. Quercetin concentration of 20 μg/ml has a tendency to increase the expression of phosphorylated p38. Publication of research on the effect of quercetin on the expression of phosphorylated p38 in keloid fibroblasts has not yet been found. This study also showed that quercetin inhibited the expression of Smad7 in cultured keloid fibroblasts. This is contrary to the research hypothesis. The inhibitory effect of quercetin on Smad7 expression is not beneficial for keloid therapy but could be useful for preventing skin aging. Publication of research on the effect of quercetin on Smad7 expression in keloid fibroblasts has not yet been found. This study also shows the effect of quercetin on Smad2/3 expression in both the cytoplasm and nucleus of keloid fibroblasts. Quercetin did not have a significant effect on cytoplasmic Smad2/3 expression although there was a tendency to decrease expression in the highest concentration quercetin group. Quercetin at a concentration of 20 μg/ml had a significant inhibitory effect on nuclear Smad2/3 expression in keloid fibroblasts, although other concentrations of quercetin also had a tendency to inhibit nuclear Smad2/3 expression. These results indicate that quercetin is thought to have the potential to inhibit Smad2/3 translocation to the keloid fibroblast nucleus. The results of this study show that administration of quercetin does not have a significant effect on type I collagen levels even though quercetin at a concentration of 20 μg/ml has a tendency to inhibit type I collagen levels. The conclusion of this study is that quercetin still has potential as a keloid therapy by inhibiting phosphorylated p38 expression and Smad2/translocation. 3 to keloid fibroblast nuclei. This study showed that quercetin cannot increase keloid fibroblast Smad7 expression but suppresses its expression.

Item Type:	Thesis (Doktor)
Identification Number:	0624060001
Divisions:	S2/S3 > Doktor Ilmu Kedokteran, Fakultas Kedokteran
Depositing User:	Unnamed user with email dini@ub.ac.id
Date Deposited:	01 Apr 2024 07:54
Last Modified:	01 Apr 2024 07:54
URI:	http://repository.ub.ac.id/id/eprint/217771

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