Pengaruh Pemberian Virgin Coconut Oil (VCO) Peroral Pada Pencegahan Peradangan Kolitis Ulseratif (Penilaian Gejala Klinis Dan Inflamasi Mukosa Kolon Mencit Model Kolitis Ulseratif)

Trismayanti, Ni Made Rika and Prof. Dian Handayani, SKM, M.Kes, PhD and Dr. dr. Umi Kalsum, M.Kes (2024) Pengaruh Pemberian Virgin Coconut Oil (VCO) Peroral Pada Pencegahan Peradangan Kolitis Ulseratif (Penilaian Gejala Klinis Dan Inflamasi Mukosa Kolon Mencit Model Kolitis Ulseratif). Doktor thesis, Universitas Brawijaya.

Abstract

Kolitis ulseratif (UC) adalah peradangan pada usus besar yang ditandai dengan diare disertai darah. Pada anak-anak, UC memiliki manifestasi lebih luas dan perjalanan penyakit yang lebih agresif. Jalur inflamasi NF-B, sitokin proinflamasi TNF-, IL-6, dan jumlah makrofag mukosa usus meningkat secara signifikan pada UC yang mengganggu fungsi pertahanan usus. Peroxisome proliferator-activated receptor gamma (PPAR-) yang diaktivasi oleh ligan menekan ekspresi sitokin proinflamasi dengan cara menghambat aktivitas faktor transkripsi NF-B, serta melemahkan ekspresi berbagai sitokin sel epitel kolon seperti IL-1β, IL-6, IL-8, TNF-, INF-, iNOS, COX-2, dan kemokin, serta mempengaruhi makrofag M1M2 yang menghambat jalur inflamasi. Terapi UC saat ini belum memberikan hasil yang memuaskan. Pembedahan direkomendasikan pada pasien yang tidak berespons terhadap pengobatan atau tidak lagi seefektif sebelumnya. Tindakan operasi berupa pengangkatan kolon dapat menjadi masalah tersendiri pada pasien. Hal tersebut menjadi dasar diperlukannya terapi preventif yang efektif, efisien dengan harga terjangkau guna mencegah peradangan usus UC sehingga dapat menurunkan angka morbiditas dan mortalitas UC anak. Virgin Coconut Oil (VCO) diyakini dapat mencegah manifestasi klinis UC dengan menghambat jalur inflamasi NF-B, melalui perannya sebagai ligan Peroxisome proliferator-activated receptor gamma (PPAR-). Penelitian ini bertujuan membuktikan VCO dapat mencegah timbulnya gejala klinis, inflamasi mukosa kolon mencit model UC melalui regulasi PPAR-, NF-B, sitokin inflamasi dan sel makrofag. Tujuan khusus penelitian adalah membuktikan VCO meningkatkan ekspresi PPAR-, menurunkan ekspresi NF-B p65, menurunkan sitokin proinflamasi (TNF-, IL-6), meningkatkan sitokin antiinflamasi IL-10, menurunkan ratio makrofag M1/M2, dan membuktikan mekanisme pertahanan mukosa kolon mencit model UC yang diberi VCO melalui jalur PPAR-. Penelitian ini adalah experimental study dengan menggunakan hewan coba mencit jantan galur BALB/c, yang dibagi menjadi 5 kelompok: (1) Kelompok kontrol negatif, diberi air mineral (KS); (2) Kelompok kontrol positif, diberi DSS 5% (KDSS); (3) Kelompok model UC1, diberi DDS 5% + VCO dosis 1000 mg/kgBB/hari (KL-VCO); (4) Kelompok model UC2, diberi DSS 5% + VCO dosis 3000 mgl/kgBB/hari (KM-VCO); (5) Kelompok model UC3, diberi DSS 5% + VCO dosis 9000 mg/kgBB/hari (KH-VCO), selama 5 hari. Gejala klinis dinilai menggunakan Disease Activity Index (DAI), sedangkan inflamasi mukosa kolon mencit menggunakan Mouse Colitis Histology Index (MCHI). Ekspresi PPAR-, NF-B p65 dan ratio makrofag M1/M2 diperiksa dengan Immunofluorescence. Konsentrasi sitokin proinflamasi (TNF-, IL-6), IL-10 diperiksa dengan Enzyme-linked Immunosorbent Assay (ELISA). Seluruh sampel pemeriksaan berasal dari kolon mencit. Hasil setiap kelompok dicatat dan dianalisis menggunakan uji normalitas data dan uji homogenitas varian, kemudian dilanjutkan dengan ANOVA tes, uji korelasi dan path analysis. Penelitian ini dilakukan di Laboratorium Penelitian dan Hewan Coba, Laboratorium Biomedik dan Laboratorium Patologi Anatomi Fakultas Kedokteran Universitas Brawijaya. Sedangkan pembuatan VCO dilakukan di Balai Penelitian Tanaman Palma (Balit Palma), Kementrian Pertanian, Manado dengan menggunakan metode Direct Micro Expelling (DME). Hasil: (1) DAI kelompok KS (0.00±0.00a) dan kelompok KDSS (7,33±1,21b) berbeda signifikan. DAI kelompok KL-VCO (4,00±2,53c), kelompok KM-VCO (2,50±1,87cd), kelompok KH-VCO (1,00±0,89ad) menurun, dan berbeda signifikan dengan kelompok KDSS. (2) MCHI kelompok KS (0.00±0.00a) dan kelompok KDSS (19.00±2.10b) berbeda signifikan. Rerata MCHI kelompok KDSS (19.00±2.10b) lebih tinggi dibandingkan dengan rerata MCHI pada kelompok KL-VCO (15.58±1.59c), kelompok KM-VCO (12.42±1.20d), dan kelompok KH-VCO (5.08±4.08e). (3) Rerata ekspresi PPAR- antara kelompok KS (710±388a) dan kelompok KDSS) (568±310a) tidak berbeda signifikan. Rerata ekspresi PPAR- kelompok KDSS (568±310a) jauh lebih kecil dibandingkan dengan rerata ekspresi PPAR- pada kelompok KL-VCO (27014±4482b), kelompok KM-VCO (37040±9386b), dan kelompok KH-VCO (53318±8879c). (4) Rerata ekspresi NF-B p65 kelompok KS (200860±19499a) dan kelompok KDSS (833936±417675b) berbeda signifikan. Rerata ekspresi NF-B p65 kelompok KDSS (833936±417675b) jauh lebih besar dibandingkan dengan nilai rerata ekspresi NF-B p65 kelompok KL-VCO (225435±67988a), kelompok KM-VCO (153751±13411a), dan kelompok KH-VCO (108687±44623a). (5) Rerata konsentrasi TNF-∝ kelompok KS (1.87±0.27a pg/mL) dan kelompok KDSS (7.26±0.45b pg/mL) berbeda signifikan. Rerata konsentrasi TNF-∝ kelompok KDSS (7.26±0.45b pg/mL) lebih besar dibandingkan dengan rerata konsentrasi TNF-∝ kelompok KL-VCO (5.62±0.57c pg/mL), kelompok KM-VCO (3.65±0.31d pg/mL), dan kelompok KH-VCO (1.84±0.13a pg/mL). (6) Rerata konsentrasi IL-6 kelompok KS (46.06±12.30ac pg/mL) dan kelompok KDSS (72.58±4.98b pg/mL) berbeda signifikan. Rerata konsentrasi IL-6 kelompok KDSS (72.58±4.98b pg/mL) lebih besar dibandingkan dengan rerata konsentrasi IL-6 kelompok KL-VCO (53.88±17.17b pg/mL), kelompok KM-VCO (35.42±6.37cd) dan kelompok KH-VCO (24.53±3.43d pg/mL). (7) Rerata konsentrasi IL-10 kelompok KS (15.20±6.67a pg/mL) dan kelompok KDSS (1.72±0.52b pg/mL) berbeda signifikan. Rerata konsentrasi IL-10 kelompok KDSS (1.72±0.52b pg/mL) lebih kecil dibandingkan dengan rerata konsentrasi IL-10 kelompok KL-VCO (2.91±1.15b pg/mL), kelompok KM-VCO (5.93±0.74b pg/mL), kelompok KH-VCO (13.67±4.39a pg/mL). (8) Rerata ratio makrofag M1/M2 kelompok KS (0.66±0.05a) dan kelompok KDSS (0.82±0.02b) berbeda signifikan. Rerata ratio makrofag M1/M2 kelompok KDSS (0.82±0.02b) lebih kecil dibandingkan dengan rerata ratio makrofag M1/M2 kelompok KL-VCO (0.89±0.07b) dan kelompok KM-VCO (0.84±0.13b), namun lebih besar dibandingkan kelompok KH-VCO (0.76±0.10ab). (9) Analisa jalur menunjukkan ekspresi PPAR- berpengaruh langsung bermakna terhadap ekspresi NF-B p65 (p=0.000) dengan koefisien pengaruh -0.524. Pembahasan: Pemberian VCO yang mengandung PUFA, seperti asam oleat, asam linoleat, yang merupakan ligan PPAR- akan mengaktivasi PPAR-. PPAR- yang meningkat jumlahnya menjalankan fungsi transrepresi, blokade aktivitas faktor transkripsi Nuclear Factor-B melalui hambatan translokasi NF-B p65 ke nukleus sekaligus mempengaruhi polarisasi makrofag M1 dan makrofag M2. Penurunan sitokin proinflamasi seperti TNF- dan IL-6 akibat penekanan jalur inflamasi NF-B, meningkatkan IL-10 akibatnya inflamasi mukosa kolon menurun dan terjadi penekanan gejala klinis UC. Kesimpulan: Gejala klinis (DAI) mencit model UC yang diberi VCO lebih rendah dibandingkan mencit model UC, inflamasi mukosa kolon (MCHI) mencit model UC yang diberi VCO lebih rendah dibandingkan mencit model UC, ekspresi PPAR- kolon mencit model UC yang diberi VCO lebih tinggi dibandingkan mencit model UC, ekspresi NF-B p65 kolon mencit model UC yang diberi VCO lebih rendah dibandingkan mencit model UC, konsentrasi sitokin proinflamasi (TNF-, IL-6) kolon mencit model UC yang diberi VCO lebih rendah dibandingkan mencit model UC, konsentrasi IL-10 kolon mencit model UC yang diberi VCO lebih tinggi dibandingkan mencit model UC, ratio makrofag M1/M2 kolon mencit model UC yang diberi VCO tidak berbeda bermakna dibandingkan mencit model UC, mekanisme pertahanan mukosa kolon mencit model UC yang diberi VCO melalui peningkatan PPAR- dengan menghambat jalur NF-B. Saran: Pada penelitian sejenis di masa mendatang, sebaiknya kenaikan dosis VCO dibuat landai dengan interval seragam, untuk mengetahui dosis minimal aktivasi PPAR- yang memberi efek mendekati kelompok KS, perlu dilakukan penelitian lanjutan untuk mengetahui safe dosage range VCO yang aman dikonsumsi sehari-hari sehingga fungsi sebagai pangan fungsional dapat tercapai.

English Abstract

Ulcerative colitis (UC) is inflammation of the large intestine characterized by diarhea accompanied by blood. UC has a wider range of manifestations and a more aggressive course of disease in children. In UC, the inflammatory pathway NF-B, proinflammatory cytokines TNF-, IL-6, and the number of intestinal mucosal macrophages are significantly increased which disrupts intestinal defense function. Peroxisome proliferator-activated receptor gamma (PPAR-) activated by ligands suppresses the expression of proinflammatory cytokines by inhibiting the activity of transcription factor NF-B, as well as weakening the expression of various cytokines of colonic epithelial cells such as IL-1β, IL-6, IL-8, TNF-, INF-, iNOS, COX-2, and chemokines, and affects M1/M2 macrophages that inhibit inflammatory pathways. Current therapies have not yielded satisfactory results. Surgery may be recommended for patients who have stopped responding to their medication or if their medication is no longer as effective as it once was, but the operation could give other problems to the patients. This is the basis for the need for effective, efficient and affordable preventive therapy to reduce the morbidity and mortality of UC in children. Virgin Coconut Oil (VCO) is believed to prevent ulcerative colitis by inhibiting the inflammatory pathway NF-B and its role as a ligand for Peroxisome proliferator-activated receptor gamma (PPAR-). This study aims to prove that VCO can prevent the occurrence of clinical symptoms, inflammation of the colonic mucosa of mice UC model through the regulation of PPAR-, NF-B, inflammatory cytokines and macrophage cells. The specific objectives of the study were to prove that VCO increases PPAR- expression, decreases NF-B p65 expression, decreases proinflammatory cytokines (TNF-, IL-6), increases antiinflammatory cytokine IL-10, macrophage ratio M1/M2, and proves the defense mechanism of colonic mucosa of UC mice model administered by VCO via PPAR- pathway. This research was an experimental study using BALB/c strain male mice, which were divided into 5 groups: (1) Negative control group, given mineral water (KS); (2) Positive control group, given DSS 5% (KDSS); (3) UC1 model group, given DDS 5% + VCO at a dose of 1000 mg/kgBW/day (KL-VCO); (4) UC2 model group, given DSS + VCO at a dose of 3000 mgl/kg BW/day(KM-VCO); (5) The UC3 model group, given DSS + VCO at a dose of 9000 mg/kg/day (KH-VCO), for 5 days. Clinical symptoms were assessed using the Disease Activity Index (DAI), while inflammation of the colonic mucosa of mice used the Mouse Colitis Histology Index (MCHI). Expression of PPAR-, NF-B p65, and macrophage ratio M1/M2 were examined by Immunofluorescence. Levels of proinflammatory cytokines (TNF-, IL-6), IL-10 were examined by Enzyme-linked Immunosorbent Assay (ELISA). All examination samples came from the mouse colon. The results of each group were recorded and analyzed using the data normality test and variance homogeneity test, then continued with the ANOVA test, correlation test and path analysis. This research was conducted at the Experimental Animal and Research Laboratory, Biomedical Laboratory and Anatomical Pathology Laboratory, Faculty of Medicine, University of Brawijaya. While the production of VCO is carried out at the Palma Plant Research Institute (Balit Palma), Ministry of Agriculture, Manado using the Direct Micro Expelling (DME) method. The results: (1) DAI group KS (0.00±0.00a) and group KDSS (7,33±1,21b) significantly different, DAI group KL-VCO (4,00±2,53c), KM-VCO (2,50±1,87cd), KH-VCO (1,00±0,89ad) decreased and significantly different from group KDSS; (2) MCHI group KS (0.00±0.00a) and group KDSS (19.00±2.10b) significantly different, MCHI group KDSS higher than MCHI group KL-VCO (15.58±1.59c), KM-VCO (12.42±1.20d), KH-VCO (5.08±4.08e); (3) the expression of PPAR- group KS (710±388a) and KDSS (568±310a) was not significantly different, expression of PPAR- group KDSS lower than the expression of PPAR- group KL-VCO (27014±4482b), KM-VCO(37040±9386b), KH-VCO (53318±8879c); (4) The expression of NF-B p65 group KS (200860±19499a) and the KDSS (833936±417675b) was significantly different, the expression of NF-B p65 group KDSS higher than the expression of NF-B p65 group KL-VCO (225435±67988a), KM-VCO (153751±13411a), KH-VCO (108687±44623a); (5) The levels of TNF- group KS (1.87±0.27a pg/mL) and group KDSS (7.26±0.45b pg/mL) were significantly different, the levels of TNF- group KDSS higher than the levels of TNF- group KL-VCO (5.62±0.57c pg/mL), KM-VCO (3.65±0.31d pg/mL), KH-VCO (1.84±0.13a pg/mL); (6) The levels of IL-6 group KS (46.06±12.30ac pg/mL) and group KDSS (72.58±4.98b pg/mL) were significantly different, the levels of IL-6 group KDSS higher than the levels of IL-6 group KL-VCO (53.88±17.17b pg/mL), KM-VCO (35.42±6.37cd), KH-VCO (24.53±3.43d pg/mL); (7) The levels of IL-10 group KS (15.20±6.67a pg/mL) and group KDSS (1.72±0.52b pg/mL) were significantly different, the levels of IL-10 group KDSS lower than the levels of IL-10 group KL-VCO (2.91±1.15b pg/mL), KM-VCO (5.93±0.74b pg/mL), KH-VCO (13.67±4.39a pg/mL); (8) The macrophages ratio M1/M2 group KS (0.66±0.05a) and group KDSS (0.82±0.02b) ) were significantly different, The macrophages ratio M1/M2 group KDSS lower than The macrophages ratio M1/M2 group KL-VCO (0.89±0.07b), KM-VCO (0.84±0.13b), and higher than group KH-VCO (0.76±0.10ab); (9) Path analysis showed that PPAR- expression had a significant direct effect on NF-B expression p65 (p=0.000) with an influence coefficient of -0.524. Discussion: VCO containing PUFA, such as oleic acid, linoleic acid, which are PPAR- ligands will activate PPAR-. The increasing number of PPAR- performs a transpression function, blockade of the activity of the transcription factor Nuclear Factor-B through the inhibition of translocation of NF-B p65 to the nucleus while affecting the polarization of M1 macrophages and M2 macrophages. Decreased proinflammatory cytokines such as TNF- and IL-6 due to suppression of the NF-B inflammatory pathway, increased IL-10 as a result of which colonic mucosal inflammation decreases and suppression of UC clinical symptoms. Conclusion: Clinical symptoms of UC mice given VCO were lower than UC mice model, inflammation of the colonic mucosa of UC mice given VCO was lower than UC mice model, PPAR- expression of UC model mice given VCO was higher than UC mice model, NF-B p65 expression of colon UC mice given VCO was lower than UC mice model, colonic levels of proinflammatory cytokines (TNF-, IL-6) in UC mice given VCO were lower than UC mice model, IL-10 levels of UC mice given VCO were higher than UC mice model, the ratio of M1/M2 macrophages of colon UC mice given VCO was not significantly different from UC mice model, colonic mucosal defense mechanism of UC model mice given VCO through the PPAR- pathway and blockade NF-B pathway. Suggestion: in similar studies in the future, it is better to increase the dose of VCO to be made ramps at uniform intervals, to determine the minimum dose of PPAR- activation that has an effect close to the KS group, further research needs to be carried out to find out the safe dosage range of VCO that is safe for daily consumption so that its function as functional food can be achieved.

Item Type:	Thesis (Doktor)
Identification Number:	0624060005
Divisions:	S2/S3 > Magister Ilmu Biomedis, Fakultas Kedokteran
Depositing User:	Unnamed user with email dini@ub.ac.id
Date Deposited:	02 Apr 2024 04:13
Last Modified:	02 Apr 2024 04:14
URI:	http://repository.ub.ac.id/id/eprint/217834

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