Hubungan diet lemak tinggi, luka operasi dan Lipolisakarida Escherichia coli O111: B4 dalam Aterogenesis :: Suatu upaya mengungkap peranan C-reactive Protein dalam Aterosklerosis

ADJI, Dhirgo, Promotor Prof.drh. Hj. Hastari Wuryastuti, M.Sc.,Ph.D

2006 | Disertasi | S3 Ilmu Pertanian (Kedokteran Hewan)

Aterosklerosis , penyebab kematian yang utama di negara berkembang merupakan proses yang kompleks yang melibatkan faktor genetik dan faktor lingkungan, serta melibatkan berbagai tipe sel. Data terkini radang diduga mempunyai peranan penting dalam proses aterosklerosis. Tujuan dari penelitian ini adalah untuk mempelajari pengaruh diet tinggi lemak, endotoksin bakteri dan kerusakan jaringan setelah operasi terhadap perkembangan lesi aterosklerotik. Empat puluh ekor tikus Sprague Dawley jantan, umur 1,5 bulan digunakan sebagai hewan coba. Semua tikus diadaptasikan dalam 40 kandang tunggal percobaan selama 5 hari dengan diberi diet yang mengandung lemak normal dan air ad libitum. Tikus kemudian dibagi menjadi 2 kelompok (I dan II) masing-masing 20 ekor. Tikus dalam kelompok I diberi pakan diet basal (mengandung lemak normal) dan kelompok II diberi pakan yang mengandung lemak tinggi. Pada hari ke 59 setelah perlakuan pakan, masing-masing kelompok tikus dibagi menjadi 2 sub kelompok yaitu : I.1; I.2; II.1; dan II.2. Subkelompok I.1 and II.1 adalah hewan yang tidak dioperasi, sedangkan subkelompok I.2 dan II.2 adalah hewan yang dioperasi laparatomi. Masing-masing kelompok, baik yang tidak dioperasi maupun yang dioperasi kemudian masih dibagi lagi menjadi 2 Sub-sub kelompok yang lebih kecil, sehingga secara keseluruhan terdapat 8 Sub-sub kelompok (1.1.1; 1.1.2; I.2.1; I.2.2) dan (II.1.1; II.1.2; II.2.1; II.2.2). Sub-subkelompok (I.1.1; I.2.1; II.1.1; II.2.1) adalah sub -sub kelompok tikus yang hanya diberi plasebo (aquadestilata) secara intraperitoneal sedangkan empat Sub -subkelompok lainnya (I.1.2;I.2.2; II.1.2; II.2.2) adalah Sub-subkelompok yang diberi lipopolisakarida dari Escherichia coli O111:B4 juga secara intraperitoneal. Pada hari ke 60 (24 jam setelah operasi), sampel darah (serum dan plasma) dikoleksi dari vena orbitalis untuk analisa total kolesterol, malonedialdehida dan C-reactive protein, kemudian semua tikus dibunuh, jantung termasuk aorta diambil untuk pemeriksaan histopatologi dan imunohistokimia. Hasil analisis statistik terhadap data yang diperoleh menunjukkan bahwa konsentrasi kolesterol total dala m plasma dipengaruhi oleh diet yang diberikan (P<0,05), namun tidak dipengaruhi oleh operasi maupun pemberian Lipopolisakarida (P>0,05). Konsentrasi malonedialdehida plasma dipengaruhi oleh diet yang diberikan dan operasi yang dilakukan (P<0,05), namun tidak dipengaruhi oleh pemberian lipopolisakarida (P>0,05). Hasil analisis tersebut juga membuktikan bahwa terdapat interaksi antara operasi dan lipopolisakarida serta diet, operasi dan lipopolisakarida dalam meningkatkan konsentrasi malonedialdehida plasma (P<0,05). Konsentrasi Creactive protein dalam serum tidak dipengaruhi oleh diet yang diberikan (P>0,05) namun dipengaruhi oleh operasi yang dilakukan dan lipopolisakarida yang diberikan (P<0,05), Hasil analisis tersebut juga membuktikan bahwa tidak ada interaksi antara masing-masing perlakuan dalam meningkatkan konsentrasi Creactive protein dalam serum (P>0,05). Secara histopatologik, aorta kelompok tikus yang diberi diet normal selama 2 bulan tidak menunjukkan adanya lesi aterosklerosis. Operasi dan lipopolisakarida tidak menyebabkan aterosklerosis, sejauh tidak disertai dengan diet lemak tinggi. Lipopolisakarida menyebabkan keradangan lokal dan sistemik. Analisis deskriptif dibawah mikroskop menunjukkan bahwa akumulasi sel radang ditemukan diantara serabut otot jantung (miokarditis ringan). Sebaliknya, operasi tidak menyebabkan miokarditis . Analisis Imunohistokimia menggunakan antibodi anti C-reactive protein terhadap jantung dan aorta menunjukkan bahwa 5 ekor tikus yang diberi diet normal, tanpa operasi dan lipopolisakarida negatif C-reactive protein; dua ekor tikus yang diberi diet normal lemak, operasi tetapi tidak diberi lipopolisakarida positif C-reactive protein; lima ekor tikus yang diberi diet normal lemak operasi dan lipopolisakarida positif C-reactive protein; tiga ekor tikus yang diberi diet tinggi lemak, tanpa operasi dan lipopolisakarida positif C-reactive protein; 5 ekor tikus yang diberi diet tinggi lemak, tanpa operasi namun diberi lipopolisakarida, positif C-reactive protein;5 ekor tikus yang diberi diet tinggi lemak , operasi, tanpa lipopolisakarida positif C-reactive protein; dan 5 ekor tikus yang diberi diet tinggi lemak, operasi dan lipopolisakarida positif C-reactive protein. Dari hasil dalam penelitian ini disimpulkan bahwa : 1. Diet lemak tinggi menyebabkan aterosklerosis, 2 Kerusakan jaringan akibat operasi tidak menyebabkan aterosklerosis, namun dapat menjadi faktor yang memperberat aterosklerosis, 3. Diet lemak tinggi dapat berinteraksi dengan luka operasi dalam menyebabkan aterosklerosis, 4. Lipopolisakarida tidak menginduksi aterosklerosis, 5. Diet lemak tinggi dapat berinteraksi dengan LPS dalam menyebabkan aterosklerosis, 6. C-reactive protein ikut berperan dalam pembentukan lesi aterosklerosis dan 7. Aplikasi deteksi C-reactive protein untuk diagnosis dini aterosklerosis masih perlu diteliti lebih lanjut

Atherosclerosis, the most common cause of death in develop country is a complex process involving the interplay of genetic and environmental factors and the involvement of multiple cell types. Recent data suggest that inflammation may play an important part in the process of atherosclerosis. The aim of this study was to analyse the effect of high lipid diet, bacterial endotoxin and tissue injury after abdominal surgery in the development of atherosclerotic lesion, using C-reactive protein as indicator. Fourty male Sprague dawley rats, 1,5 months of age were used as experimental animals. All rats were adapted in 40 single cages for 5 days and given basal diet containing normal fat and water ad libitum. After adaptation, rats were divided into 2 groups (Group I and Group II) of 20 each. Group I was fed basal diet containing normal fat and Group II was fed diet containing high fat. After 59 days, each group was divided into 2 Subgroups (Subgroups I.1; I.2; II.1 and II.2). The rats in Subgroups I.1 and II.1 were not operated and the rats in subgroups I.2 and II.2 were operated. These Subgroups were attained to study the effect of surgery on atherogenesis. Before surgery was done, these Subgroups were then divided again into 2 Sub-subgroups (5 rats each subsubgroup). Those Sub -subgroups were: Subsubgroups I.1.1; I.1.2; I.2.1; I.2.2 and Subsubgroups II.1.1; II.1.2; II.2.1 and II.2.2. Four sub -subgroups I.1.1;I.2.1;II.1.1;II.2.1 were treated with plasebo (aquadest) intraperitoneally after surgery and the other four Sub -subgroups I.1.2; I.2.2; II.1.2; II.2.2 were treated with lipopolysacharide from Escherichia coli O111:B4 intraperitoneally after surgery. Twenty four hours after surgery, blood samples (serom and plasma) were collected from the orbital vein for total cholesterol, malonedialdehyde and C-reactive protein analyses. The rats were then killed, the heart including aorta were collected for histopathology and immunohistochemistry analyses. The Creactive protein in the hearts and aorta were then analyzed by immunohistochemistry (streptavidin-biotin) technique using antibody anti Creactive protein.Statistical analysis of the data showed that in general, total plasma cholesterol concentrations were affected by diet (P<0.05) but were not affected by surgery and lipopolysacharide (P>0.05). Statistical analyses of the data showed that in general, malonedialdehyde plasma concentrations were affected by diet and surgery (P<0.05) but were not affected by lipopolysacharide (P>0.05). The results of the present study also showed that there was an interaction between surgery and LPS, and among diet, surgery and lipopolysacharide (P<0.05). Statistical analyses of the data showed that in general, malonedialdehyde plasma concentrations were affected by diet and surgery (P<0.05) but were not effected by lipopolysacharide (P>0.05). The analysis also showed that there was an interaction between surgery and lipopolysacharide, and among diet, surgery and lipopolysacharide (P<0.05).Histopathologic analysis of aortas from rats that given normal fat diet for about 2 months showed that there was no atherosclerotic lesions. Base on the result, it can be proved that diet is a major factor for promoting the atherosclerotic lesion. Another evidence showed that surgery and LPS were only a minor factor. These factors would not promote atherosclerosis without high fat diet. Surgery and LPS was not directly induced atherosclerosis. In this present study, LPS was thought to play a crucial role in eliciting pathology changes culminating local inflammation and may be sistemic inflammation. Descriptive analyses under the mycroscope showed that there were accumulation of inflammation cells (neutrophyl and lymphocyte) found between cardiac muscles (mild myocarditis). From this study, 3 rats that given lipopolysacharide, without high fat diet and surgery suffered myocarditis; four rats that given lipopolysacharide with high fat diet but without surgery were also suffered mild myocarditis. This lesion appear also in rats that given lipopolysacharide, high fat diet and surgery. Five rats of this group suffered myocarditis. Surgery alone did not cause myocarditis. Immunohistochemistry analysis of hearts and aortas using antibody anti Creactive protein showed that 5 rats of group that given normal diet, without surgery and lipopolysacharide. The hearts and aortas all were C-reactive protein negatif. Two rats given normal fat diet, surgery, wi thout lipopolysacharide the hearts and aortas were C-reactive protein positive. Five rats given normal fat diet, operated and treated with lipopolysacharide, the hearts and aortas were Creactive protein positive. Three rats given high fat diet, non operated, without lipopolysacharide the hearts and aortas were C-reactive protein positive. Five rats given high fat diet, non operated but treated with lipopolysacharide, the hearts and aortas were C-reactive protein positive. Five rats given high fat diet, operated, without lipopolysacharide, the hearts and aortas were CRP positive and 5 rats given high fat diet, operated, treated with lipopolysacharide the hearts and aortas were C-reactive protein positive. Based on the result of this study could be concluded that : 1. High fat diet might cause atherosclerosis 2. Surgery did not cause atherosclerosis, 3.Surgery and LPS were supporter factors that could increase atherosclerosis, 4. Lipopolysacharide did not induce atherosclerosis, 5. High fat diet and LPS have an interaction effect to cause atherosclerosis, 6. C-reactive protein may involve in atherosclerosis, and 7. The detection of atherosclerosis using CRP still needs to be investigated.

Kata Kunci : Aterosklerosis, Diet Lemak Tinggi, Operasi, atherosclerosis, high fat diet, surgery, lipopolysacharide, C-reactive protein, plasma malonedialdehyde.