Sutjiati, Endang (2018) Pengaruh Asupan Rendah Metionin Terhadap Resiko Timbulnya Osteoartritis Melalui Penurunan Metilasi Dna Gen Il-1β Pada Kelinci New Zealand Induksi Anterior Cruciate Ligamen Transection (ACLT). Doctor thesis, Universitas Brawijaya.
Abstract
Melalui studi epigegenetik diketahui bahwa berbagai faktor risiko OA (seperti obesitas, keseimbangan zat gizi, cedera atau trauma mekanik ) dapat merubah fenotip kondrosit dan respon seluler. Metionin merupakan asam amino essensial yang mengandung gugus metil (CH3) memiliki efek kuat pada metilasi DNA dan ekspresi gen. Metionin sebagai substrat S-adenosylmetilmethionin (SAM) dan memodulasi aktifitas enzim DNA transferase yang penting dalam metilasi DNA. Defisiensi metionin menyebabkan hipometilasi DNA, menurunkan homosistein dan sintesis glikosaminoglikan (GAG) serta GSH menurun. Penelitian ini bertujuan untuk membuktikan apakah asupan rendah metionin dapat meningkatkan jumlah gen IL-1β tidak termetilasi, meningkatkan kadar IL-1β serum, meningkatkan ekspresi gen IL-1β dan ekspresi gen MMP-13 di jaringan rawan sendi lutut serta meningkatkan jumlah sel apoptosis dan derajat kerusakan rawan sendi lutut. Penelitian ini adalah True experimental dengan rancangan penelitian post tes only control group design. Penelitian dilakukan secara in vivo pada kelinci jenis silangan new zealand normal dan kelinci model kerusakan tulang rawan sendi dengan cara anterior cruciatet ligamen transcection (ACLT). Masing-masing kelompok dibagi 3 perlakuan yaitu Kelinci diberi formula DL-metionin 0.25 % per kg formula; kelinci diberi formula DLmetionin 0.15 % per kg formula; dan kelinci diberi formula rendah metionin. Pemeriksaan metilasi DNA promoter gen IL-1 β dengan metode Bisulfit PCR sequencing menggunakan EZDNA methylation GOLD "Kit (Katalog Nos.D5005 & D5006), Sequence Scanner 2 dari Thermo Fisher, KOD-Multi & epidemik Toyobo (katalog F1440K). Kadar IL-1β serum metode ELISA, ekspresi IL-1β dan ekspresi MMP-13 di jaringan rawan sendi lutut dan jumlah sel apoptosis serta derajat kerusakan rawan sendi secara imunohistokimia dengan antibodi spesifik yaitu antibodi primer poliklonal anti rabbit IL-1β kit Santa Cruz (Sc7884), anti bodi primer anti rabbit MMP-13 LSBic (LSC88504) dan TUNEL Biotin Labeled POD Enogene serta pewarnaan H&E. Keseluruhan data yang diperoleh diuji statistik uji t-test, uji anova one way dan uji jalur PLS (Partial Least Square) antar variabel menggunakan program SPSS Statistics Release 19.0 for Windows. Hasil penelitian menunjukkan bahwa rata-rata kelinci memiliki berat badan awal berbeda secara signifikan (p<0.05). Setelah pemberikan formula rendah metionin selama 35 hari rata-rata asupan makan per berat badan per minggu lebih tinggi dari kelinci yang diberi formula DL-metionin 0.25 % dan penambahan berat badan 3,27 % lebih rendah. Berbeda dengan kelinci ACLT pemberian formula rendah metionin rata-rata asupan makan per berat badan per minggu lebih tinggi dari kelinci yang diberi formula DL-metionin 0.25 % dan berat badannya turun 7,03 %. Sedangkan pemberian formula DL-metionin 0.25 % pada kelinci ACLT dapat menaikan berat badan sebesar 5,4 %. Kadar IL-1β serum kelompok kelinci normal dan kelinci ACLT ada perbedaan tapi tidak signifikan (p=0.12). Pemberian formula rendah metionin pada kelompok kelinci normal maupun kelinci ACLT kadar IL-1β serum sedikit lebih tinggi dari pada kelompok kelinci lain. Ada perbedaan signifikan antar kelompok (p=0.00) pada kelinci normal dan tidak ada perbedaan yang signifikan (p>0.05) antar kelompok pada kelinci ACLT. Gen IL-1β pada kelinci diketahui terletak pada kromosom 2 di lokasi 97.614851- 67.618656 reverse strand dan posisi 3589-3846. Daerah promotor gen IL-1 dengan panjang 258 bp didapat tujuh situs CpG gen IL-1β yaitu 25,48,81,116,118,142,153. Pemberian formula rendah metionin terdapat 3 (tiga) gen IL-1β yang tidak termetilasi, lebih banyak dari pada kelompok kelinci yang diberi formula DL-metionin 0.15 %. Kenaikkan gen IL-1β yang tidak termetilasi menyebabkan gen IL-1β akan terekspresikan di jaringan spesifik. Pada kelinci yang diberi formula rendah metionin ekspresi IL-1β di jaringan rawan sendi lutut lebih tinggi dari pada kelinci yang diberi ix formula DL metionin 0.25 %. Hal sama pada kelinci ACLT pasca trauma bersinergi dengan asupan rendah metionin meningkatkan ekspresi IL-1β lebih tinggi dari kelinci normal, secara statistik berbeda tapi tidak signifikan (p > 0.05). Peningkatan ekspresi IL-1β di jaringan rawan sendi secara autokrin menginduksi kondrosit untuk memproduksi MMP-13 lebih tinggi. Pemberian formula rendah metionin pada kelinci normal mampu menginduksi ekspresi MMP-13 di jaringan rawan sendi lutut 2 kali lipat lebih tinggi daripada kelinci yang diberi formula DL-metionin 0.25 %. Peningkatan ekspresi MMP-13 di jaringan rawan sendi lutut kelinci selain distimuli oleh IL-1 β juga stres mekanik akibat incisi ACLT. Hasil gambaran histologi ekspresi MMP-13 di jaringan rawan sendi lutut kelinci ACLT lebih banyak pada kelinci yang diberi formula rendah metionin. Apoptosis penting dalam mempertahankan homeostasis jaringan normal dan hasil analisis uji anova one way, terdapat perbedaan yang signifikan (p=0.00) antar kelompok. Pemberian formula rendah metionin dapat menginduksi kondrosit apoptosis lebih tinggi 4 kali dari kelinci yang mendapat formula DL-metionin 0.25 %. Hasil gambaran histologi tampak kondrosit apoptosis lebih banyak pada jaringan rawan sendi lutut kelinci yang diberi formula rendah metionin, hal ini menunjukkan cedera mekanik akibat trauma ACLT menginduksi kondrosit apoptosis. Derajat kerusakan tulang rawan sendi lutut kelinci berdasarkan penilaian modifikasi skore Mankin terhadap struktur, terdapat perbedaan signifikan (p=0.01) antar kelompok kelinci normal maupun kelinci ACLT. Jumlah fisure pada permukaan rawan sendi sampai zona tengah lebih banyak pada kelinci normal yang diberi formula rendah metionin dan kelinci ACLT yang diberi formula rendah metionin terdapat fisure pada permukaa rawan sendi sampai zona dalam. Berdasarkan analisis jalur PLS diperoleh hasil bahwa jalur yang berpengaruh dari asupan rendah metionin terhadap kerusakan tulang rawan sendi melalui peningkatan ekspresi IL-1β di jaringan rawan sendi lutut yang disebabkan oleh penurunan metilasi DNA gen IL-1β. Peningkatan ekspresi IL-1 di jaringan rawan sendi dapat meningkatkan ekspresi MMP-13 sebagai enzim utama dalam degradasi tulang rawan sendi dan meningkatkan kondrosit apoptosis sehingga menghambat sintesis matrik rawan sendi. Dapat disimpulkan dalam penelitian ini bahwa asupan rendah metionin meningkatkan resiko kerusakan tulang rawan sendi osteoartritis melalui penurunan metilasi DNA gen IL-1β yang menyebabkan peningkatan ekspresi gen IL-1β di jaringan rawan sendi yang dapat meningkatkan ekspresi MMp-13 dan kondrosit apoptosis.
English Abstract
Epigegenetic studies show that various risk factors for OA (such as obesity, nutritional balance, injury or mechanical trauma) can change the chondrocyte phenotype and cellular response. Methionine is an essential amino acid containing a methyl group (CH3) that has a strong effect on DNA methylation and gene expression. Methionine as a substrate of S-adenosylmetilmethionin (SAM) and modulates the activity of the enzyme DNA transferase which is important in DNA methylation. Methionine deficiency causes hypomethylation of DNA, decreases homocysteine and the synthesis of Glycosaminoglycans (GAG) and decreased GSH. This study aims to prove whether low intake of methionine can increase the number of IL-1β genes which are not methylated, increase serum IL-1β levels, increase IL-1β gene expression and MMP-13 gene expression in knee joints cartilage tissues and increase the number of apoptotic cells and cartilage damage to knee joints. This study was a True experimental study with a post test only control group design. The study was carried out in vivo on normal new zealand crossing rabbits and rabbit models with joint cartilage damage by means of the anterior cruciate ligament transcection (ACLT). Each group was divided into 3 treatments, namely rabbits that were given with DL-methionine formula 0.25% per kg formula; rabbits with DL-methionine formula 0.15% per kg formula; and rabbits that were were given with a low methionine formula. Examination of IL-1 β gene promoter DNA methylation using Bisulfite PCR sequencing method using EZDNA methylation GOLD "Kit (Catalog Nos.D5005 & D5006), Thermo Fisher Sequence Scanner 2, KOD-Multi & Toyobo epidemic (catalog F1440K). Serum 1β of ELISA method, expression of IL-1β and expression of MMP-13 in prone tissue of knee joints and number of apoptotic cells and immunohistochemical susceptibility to joint damage with specific antibodies, namely Santa Cruz polyclonal anti-rabbit primary antibody kit (Sc7884), anti rabbit primary body MMP-13 LSBic (LSC88504) and TUNEL Biotin Labeled POD Enogene and H & E staining. All data obtained were tested statistically by t-test, one way ANOVA test and PLS (Partial Least Square) path test between variables using the SPSS program Statistics Release 19.0 for Windows. The results showed that the average rabbit had a different initial weight (p <0.05). After giving a low methionine formula for 35 days the average food intake per weight per week was higher than rabbits which were given a DL-methionine formula of 0.25% and weight gain of 3.27% lower. In contrast to the rabbit with ACLT the administration of a low methionine formula had a higher average of food intake per body weight per week than rabbits that were given with DL-methionine formula 0.25% and their weight dropped by 7.03%. While giving DL-methionine formula of 0.25% in ACLT rabbits it can increase body weight by 5.4%. The serum IL-1β levels in the normal rabbit group and the ACLT rabbit were different but not significant (p = 0.12). Giving a low methionine formula in the normal rabbit group and the ACLT rabbit serum IL-1β level was slightly higher than the other rabbit groups. There were significant differences between groups (p = 0.00) in normal rabbits and there were no significant differences (p> 0.05) between groups in ACLT rabbits. The IL-1β gene in rabbits is known to be located on chromosome 2 at location 97.614851-67.618656 reverse strand and position 3589-3846. The 258 bp IL-1 gene promoter area obtained seven sites of CpG IL-1β gene, namely 25, 48, 81, 116, 118,142, ,153. The administration of a low methionine formula contained 3 (three) IL-1β genes which were not methylated, more than the rabbit group given the DL-methionine formula of 0.15%. Increasing the non-methylated IL-1β gene site causes the IL-1β gene to be expressed in specific tissues. In rabbits given with a low formula methionine, the xi expression of IL-1β in cartilage tissue of the knee joint was higher than that of rabbits given the DL methionine formula of 0.25%. The same thing occurs in post-traumatic ACLT rabbits synergizing with low intake of methionine increased IL-1β expression higher than normal rabbits, statistically different but not significant (p> 0.05). Increased expression of IL-1β in cartilage tissue joint autocrine induces chondrocytes to produce higher MMP-13. Giving a low methionine formula in normal rabbits was able to induce MMP-13 expression in 2-fold higher tissue in knee joints than rabbits given with DL-methionine formula of 0.25%. Increased expression of MMP-13 in rabbit cartilage tissue knee joint aside from being stimulated by IL-1β is also a mechanical stress due to ACLT incision. The results of the histology of MMP-13 expression in cartilage tissue of ACLT rabbit knee joints were greater in rabbits given the low methionine formula. Apoptosis is important in maintaining normal tissue homeostasis. The results of one way ANOVA test analysis shows that there are significant differences (p = 0.00) between groups. The administration of a low formula methionine can induce chondrocyte apoptosis 4 times higher than rabbits which 0.25% DL-methionine formula. The results of histology of chondrocytes apoptosis appear more in cartilage tissue of rabbit knee given with low methionine formula, this shows that mechanical injury due to ACLT trauma induces chondrocyte apoptosis. The degree of damage to rabbit knee cartilage based on the assessment of Mankin scoring modifications to the structure shows that there are significant differences (p = 0.01) between normal rabbit groups and ACLT rabbits. The number of fissures on the cartilage surface of the joint to the middle zone is greater in normal rabbits given the low methionine formula and the ACLT rabbit which is given a low methionine formula with fissure on the cartilage surface of joints to the inner zone. Based on the PLS path analysis, results showed that the pathway that affected the low intake of methionine against joint cartilage damage through increased expression of IL-1β in tissue cartilage to knee joints caused by decreased methylation of IL-1β gene DNA. Increased expression of IL-1 in joint-prone tissues can increase the expression of MMP-13 as the main enzyme in the degradation of joint cartilage and increase chondrocyte apoptosis thereby inhibiting joint-cartilage matric synthesis. It can be concluded in this study that a low intake of methionine increases the risk of osteoarthritis cartilage damage through a reduction in methylation of IL-1β gene DNA which causes an increase in IL-1β gene expression In joint cartilage tissue knee can increase the expression of MMP-13 and chondrocytes apoptosis.
Other obstract
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| Item Type: | Thesis (Doctor) |
|---|---|
| Identification Number: | DIS/612.015 756/SUT/p/2018/061901121 |
| Uncontrolled Keywords: | AMINO ACIDS HUMAN |
| Subjects: | 600 Technology (Applied sciences) > 612 Human Physiology > 612.01 Biophysics and biochemistry > 612.015 28 Pigments > 612.015 75 Proteins > 612.015 756 Components of proteins |
| Divisions: | S2/S3 > Doktor Ilmu Kedokteran, Fakultas Kedokteran |
| Depositing User: | Endang Susworini |
| Date Deposited: | 19 Feb 2021 03:35 |
| Last Modified: | 19 Feb 2021 03:43 |
| URI: | http://repository.ub.ac.id/id/eprint/183431 |
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