饱和脂肪酸对主动脉平滑肌细胞内二脂酰甘油合成和蛋白激酶C活性的影响

Effects of saturated fatty acids on diacylglycerol synthesis and protein kinase C activity in cultured aortic smooth muscle cells

目的:通过测定各种脂肪酸对体外培养的牛主动脉平滑肌细胞二脂酰甘油(DAG)和蛋白激酶C(PKC)水平的影响,探讨脂肪酸对动脉粥样硬化(AS)发生的可能作用机制。方法:分离牛主动脉平滑肌细胞在体外培养,分别用浓度为200μmol/L的饱和脂肪酸软脂酸和硬脂酸、不饱和脂肪酸油酸和花生四烯酸处理后,然后将细胞分别用分离液和细胞裂解液处理,通过液闪计数仪测定32P磷酸的cpm值(1cpm=6.17×108Bq),计算DAG和PKC。结果:饱和脂肪酸软脂酸和硬脂酸(200μmol/L)分别作用24h后,牛主动脉平滑肌细胞内的DAG水平分别为927.2和1533.3pmol/106个细胞,与对照组(190pmol/106个细胞)有显著性差异(P<0.01)。油酸增加细胞内DAG水平,为334.4pmol/106个细胞(P<0.01)。花生四烯酸对细胞内DAG含量无明显影响。酰基辅酶A抑制剂TriacsinC能够完全抑制软脂酸对DAG的刺激作用。此外,软脂酸和硬脂酸均能刺激细胞内PKC活性,分别为553.7和557.2cpm,对照组为394cpm(P<0.05)。结论:饱和脂肪酸可能通过其代谢产物酰基辅酶A促进主动脉平滑肌细胞内DAG的合成和提高PKC活性,这可能与AS有关。

Objective:To investigate the role of saturated free fatty acids (SFFAs) in the pathogenesis of atherosclerosis by determining the effects of FFAs on diacylglycerol (DAG) synthesis and protein kinase C (PKC) activity in cultured bovine aortic smooth muscle cells. Methods:Bovine aortic smooth muscle cells, cultured in vitro, were treated with 200 μmol/L SFFAs (including palmitate and stearate) and unsaturated FFAs (including oleate and arachidonate) separately for 24 h. Then the cells were lysed and the lipids were treated with lysis buffer and separation buffer. DAG level and PKC activity were calculated through measuring the radioactivity of 32P-PA by liquid scintillation counting (1 cpm=6.17×108 Bq). Results:Incubation of the cells with saturated FFAs(palmitate or stearate, 200 μmol/L, for 24 h) induced significant increase in DAG concentrations (927.2 and 1 533.3 pmol/106 cells by palmitate and stearate, respectively) compared with that of the control cells (190 pmol/106 cells,P<0.01). Unsaturated FFAs, oleate, also induced an increase in DAG concentration significantly(334.4 pmol/106 cells,P<0.01), while arachidonate had no effect on DAG. In addition, the stimulating effect of palmitate on DAG was completely inhibited by Triacsin C, an acyl-CoA synthetase inhibitor. Compared with PKC activity in the control group (394 cpm), palmitate and stearate significantly increased the PKC activity to 553.7 and 557.2 cpm, respectively(P<0.05). Conclusion: SFFAs, possibly through its metabolite acyl-CoA in cultured aortic smooth muscle cells, increase DAG synthesis and PKC activity, which may contribute to the pathogenesis of atherosclerosis.