Disertasi

PERUBAHAN EKSPRESI LIPOPROTEIN ASSOCIATED PHOSPHOLIPASE A DAN LYSOPHOSPHATIDYLCHOLINE DALAM PERKEMBANGAN AWAL ATEROSKLEROSIS: Studi In Vivo pada Tikus Model Diabetes Melitus 2 Tipe 2 dan Dislipidemia.

Cardiovascular disease (CVD) saat ini telah menjadi masalah kesehatan global yang serius dan telah menjadi penyebab kematian utama baik dinegara-negara maju maupun berkembang. Aterosklerosis merupakan penyebab paling umum CVD. Perkembangan aterosklerosis sangat berkaitan dengan gangguan metabolisme lipid dan inflamasi. OxLDL telah banyak dibuktikan berperan penting pada proses inflamasi vaskular dan sangat berhubungan dengan lipoprotein associated phospholipase A 2 (LpPLA2 ), sehingga sangat menarik disini untuk mengetahui lebih jauh tentang peran LpPLA2 Penelitian eksperimental laboratorik dikerjakan di Laboratorium Biosains Universitas Brawijaya Malang pada September 2014 hingga Agustus 2015 dengan disain post test with control group secara invivo, menggunakan 50 hewan coba tikus Sprague Dawley (SD) yang terbagi dalam 5 kelompok besar yaitu kelompok normal, kelompok dislipidemia (DL), DM tipe 2 (DMT2) dan kelompok DL serta DMT2 yang diberikan Darapladib (DP) (penyekat selektif Lp-PLA ) dengan dosis 30 mg/kg berat badan/hari. Untuk mengetahui perkembangan aterosklerosis, masing-masing kelompok terdiri dari 2 serial waktu perlakuan yaitu 8 dan 16 minggu. Parameter yang diukur dalam penelitian ini adalah ekspresi mRNA Lp-PLA 2 , protein Lp-PLA2 , kadar lysophosphatidylcholine (LisoPC), oxLDL di jaringan aorta dan plasma, ekspresi IL-6, TNF-α dan iNOS di jaringan aorta, profil lipid, KGD puasa, kadar insulin plasma, ketebalan PAT dan jumlah sel busa di jaringan aorta. Ekspresi mRNA Lp-PLA 2 di jaringan aorta dan darah diukur dengan metode RT-PCR, Ekspresi protein Lp-PLA2, IL-6, TNF-α dan iNOS di jaringan aorta menggunakan tekhnik immunoflouresence. Kadar LisoPC jaringan aorta dan darah diukur menggunakan metode LC-MS, kadar oxLDL jaringan aorta dan plasma, PAF serta insulin plasma diukur menggunakan ELISA. Jumlah sel busa dan ketebalan PAT diukur dengan pengecatan HE. Uji anova, manova, korelasi, regresi dan analisis jalur menunjukkan bahwa pada kondisi DL, ekspresi protein Lp-PLA2 di jaringan aorta berkorelasi bermakna dengan peningkatan jumlah sel busa. Pada kondisi DMT2, ekspresi relatif mRNA Lp-PLA2 darah berkorelasi bermakna dengan peningkatan jumlah sel busa. Pemberian DP menekan ekspresi protein Lp-PLA2 dan LisoPC di jaringan aorta baik pada kondisi DMT2 maupun DL. Akan tetapi DP tidak menekan ekspresi mRNA Lp-PLA2 jaringan aorta dan darah baik pada kondisi DMT2 maupun DL. Darapladib mampu menekan petanda inflamasi baik di jaringan aorta maupun plasma baik pada kondisi DL maupun DMT2. Profil ekspresi protein Lp-PLA2 jaringan aorta sesuai dengan profil perubahan kadar LisoPC jaringan aorta. Namun demikian, profil ekspresi mRNA Lp-PLA baik di jaringan aorta maupun darah tidak sesuai dengan profil perubahan kadar LisoPC jaringan aorta maupun plasma. Profil ekspresi mRNA dan protein Lp-PLA2 jaringan aorta berbeda pula dengan ekspresinya di darah sehingga tidak dapat menggambarkan ekspresi Lp-PLA2 yang sesungguhnya di jaringan aorta. Lipoprotein Associated Phospholipase A dan produk bioaktifnya (LisoPC) terbukti berperan penting pada aktifasi respons inflamasi dalam perkembangan awal aterosklerosis pada kondisi DMT2 dan DL. Terdapat perbedaan jalur patomekanisme Lp-PLA2 dalam mengaktivasi respons inflamasi diantara kondisi DMT2 dan DL dalam perkembangan awal aterosklerosis.
Kata Kunci : Aterosklerosis, Dislipidemia, DM Tipe 2, LP-PLA2 LisoPC.


Cardiovascular disease (CVD) has now become a serious global health problem and has be become the leading cause of death in both developed and developing countries. The development of atherosclerosis is highly associated with lipid metabolic disorders and inflammation. Oxidized LDL (OxLDL) has been widely demonstrate to have an important role in vascular inflammation and is closely associated with lipoprotein-associated phospholipase A2 (Lp-PLA2 ), so it is very interesting to know more about the real role of Lp-PLA2. This study was done in Laboratory of Bioscience, Laboratory of Biological Life Sciences- Brawijaya University Malang on September 2014 until August 2015. This was true experimental laboratory using posttest with invivo control group design. Fifty Spraque Dowley rats were divided into 5 main groups including normal, dyslipidemia (DL), type 2 diabetes mellitus (DMT2), DL and DMT2 with Darapladib (DP) administration 30 mg/kg body weight daily. To find out atherosclerosis development, each group consisted of 2 serials treatment time, which are 8-weeks and 16-weeks treatment groups. The parameters measured in this study were mRNA Lp-PLA2 and protein Lp-PLA2 expresions, lysophosphatidylcholine (LysoPC) and oxLDL levels in aorta tissue and plasma, IL-6, TNF-α and iNOS expressions, perivascular adipose tissue (PAT) thickness in aortic tissue, lipid profile, glucose and insulin plasma levels and the number of foam cells in aorta. mRNA of Lp-PLA2 in blood and aorta was measured using the RT-PCR method. The measurement of Lp-PLA2 protein, IL-6, TNF-α and iNOS expressions in aorta tissue used staining immunoflouresence. Lysophoshatidylcholine level in aorta and blood were measured using LC-MS/MS method. The oxLDL level, PAF and insulin plasma level were measured by ELISA method. The number of foam cells and PAT thickness in aorta were measured using HE staining. The statistical analysis results using ANOVA, MANOVA, regression, correlation and pathway analysis showed that in DL condition Lp-PLA2 protein expression in aortic tissue correlate significantly with the increase of foam cells, while in DMT2 condition mRNA Lp-PLA2 blood expression correlate significantly with the increase of foam cells. Darapladib decreases Lp-PLA2 protein expression and LysoPC in aortic tissue both in DL and DMT2 condition. Darapladib failed to decrease blood and aorta tissue mRNA Lp-PLA2 expressions in DMT2 and DL. Darapladib is able to decrease inflammation marker both in aortic tissue and plasma both in DMT2 and DL condition. The pattern of Lp-PLA2 protein expression in aorta is similar to LysoPC level. However, mRNA Lp-PLA2 expression pattern both in aorta and plasma is different from lisoPC level pattern both in aorta and plasma. mRNA Lp-PLA2 and Lp-PLA2 protein expressions in aorta is different from mRNA Lp-PLA2 and Lp-PLA2 protein expressions in blood. Therefore, Lp-PLA2 expression in blood does not represent the expression of Lp-PLA2 in the aorta. Lipoprotein associated phospholipase A and its bioactive products (LysoPC) is proved to have important role in inflammation activation during early stage of atherosclerosis development in DMT2 and DL conditions. There is a different pattern of Lp-PLA2 pathomechanism in activating inflammation response between DMT2 and DL conditions.
Key Word : Atherosclerosis, Dyslipidemia, Type 2 DM, LP-PLA2, LysoPC.