Lysophosphatidylcholine의 혈관평활근세포에 대한 세포 독성
Cytotoxic Effects of Lysophosphatidylcholine on Vascular Smooth Muscle Cells
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초록
연구목적: 혈관평활근세포에 대한 lysophosphatidylcholine (lysoPC)의 세포 독성 효과를 조사하였다. 연구재료 및 방법: 쥐의 대동맥으로부터 혈관평활근세포를 얻어 배양한 후 lysoPC에 의해 세포사가 유도되는지의 여부를 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)와 lactic dehydrogenase (LDH) 측정 및 DNA 사다리 발현 양상 비교를 이용하여 관찰하였다. 세포자멸사의 변화를 propidium iodide (PI) 염색 및 FACS를 이용하여 조사하였고, 2’,7’-dichlorofluorescin diacetate (DCF-DA)를 이용하여 세포내 reactive oxygen species (ROS) 생성을 측정하였다. 또한 caspase, bcl-2 및 bax의 발현을 western blot 분석으로 측정하였다. 결과: MTT 측정 결과 25 μM 이상 농도의 lysoPC 처리 후 10시간이 경과하였을 때 50% 이상의 혈관평활근세포가 죽는 것이 관찰되었으며, LDH 수치는 변화가 없었다. 반면, DNA 사다리 양상 관찰 결과 세포자멸사에 특징적인 양상이 확인되었다. 항산화제인 비타민 E와 NF-kB 억제제인 caffeic acid phenylethyl ester (CAPE) 투여는 모두 lysoPC에 의한 세포자멸사를 억제하였다. Western blot 분석 결과 lysoPC 처리시 caspase-3 및 -8의 증가가 관찰되었다. 총 bcl-2 및 bax 단백 발현에는 변화가 없었으나, bax의 세포질에서 미토콘드리아 세포막으로의 위치 변화가 관찰되었다. 결론: LysoPC는 혈관평활근세포의 세포자멸사를 유도하며, 산화 기전 및 nuclear factor-kappaB (NF-kB) 경로가 관련되어 있다.
Abstract
Objectives: To investigate the cytotoxic effects of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoproteins (LDL), on vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from rat aorta. Cell death was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, lactic dehydrogenase (LDH) assay, and DNA fragmentation assay. Apoptosis was quantified by propidium iodide staining and fluorescent activated cell sorting (FACS) analysis, and intracellular free radical production was determined using 2’,7’-dichlorofluorescin diacetate (DCF-DA). In addition, the changes in caspases, bcl-2 and bax proteins were evaluated by western blot analysis. Results: LysoPC over 25 μM induced more than 50% of the cell death at 10 hours on MTT assay with no change in the level of LDH. The DNA ladder pattern showed that cell death induced by lysoPC was caused by apoptosis, which was associated with increased free radical production. Vitamin E, a potent antioxidant and caffeic acid phenylethyl ester (CAPE), an inhibitor of nuclear factor-kappaB (NF-kB), blocked apoptosis. The casepase-3 precursor decreased and the active form of caspase-8 increased. Total bcl-2 and bax proteins did not change with lysoPC treatment, but translocation of bax from cytosole to the mitochondria membrane was observed. Conclusion: LysoPC induces apoptosis in VSMCs via an oxidant mechanism, dependent on NF-kB.
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