脂肪分化相关蛋白通过蛋白激酶C和酰基辅酶A:胆固醇酰基转移酶1促进THP-1巨噬细胞蓄积脂质

目的： 探讨脂肪分化相关蛋白促进THP-1巨噬细胞蓄积脂质的机制。方法： 使用高胆固醇饲料喂养新西兰兔12周，复制动脉粥样硬化动物模型。然后用Western blotting和免疫组织化学染色观察兔主动脉脂肪分化相关蛋白及蛋白激酶C的表达。THP-1巨噬细胞与氧化低密度脂蛋白孵育不同的时间，使细胞负荷胆固醇酯后，用RT-PCR的方法观察脂肪分化相关蛋白及酰基辅酶A:胆固醇酰基转移酶1的表达，用PepTagassay琼脂糖凝胶电泳及分光光度法观察蛋白激酶C的活性。在负荷胆固醇酯的THP-1巨噬细胞中分别加入蛋白激酶C的激动剂佛波酯和抑制剂钙磷酸结合蛋白，观察脂肪分化相关蛋白的表达，同时使用油红O染色和高效液相色谱法测定细胞内脂质的变化。瞬时转染pcDNA3.1-HA-adi表达载体到THP-1巨噬细胞，复制高表达脂肪分化相关蛋白的细胞模型。在负荷、不负荷胆固醇酯或ACAT抑制剂存在的状态下，观察酰基辅酶A:胆固醇酰基转移酶1表达及细胞内胆固醇酯的改变。结果： 与对照组相比，兔主动脉病变处脂肪分化相关蛋白及蛋白激酶C表达均显著增加。负荷胆固醇酯的THP-1巨噬细胞中脂肪分化相关蛋白及酰基辅酶A:胆固醇酰基转移酶1表达上调，蛋白激酶C的活性升高。荷脂细胞与蛋白激酶C激动剂共孵育后，可以协同增强上调脂肪分化相关蛋白的效应，细胞内脂质增多；如果荷脂细胞同时与蛋白激酶C抑制剂共孵育，可以有效抑制荷脂所致的脂肪分化相关蛋白高表达，细胞内脂质减少。高表达脂肪分化相关蛋白的THP-1巨噬细胞能够使酰基辅酶A:胆固醇酰基转移酶1表达增加，促进细胞内胆固醇酯蓄积。加入酰基辅酶A:胆固醇酰基转移酶抑制剂后，这些作用被减弱。结论： 脂肪分化相关蛋白可能是通过蛋白激酶C活性的改变影响酰基辅酶A:胆固醇酰基转移酶1的活性，从而影响细胞内脂质的蓄积。

Adipophilin accumulates cellular cholesteryl ester by PKC and ACAT1 in THP-1 macrophages

AIM: To investigate the mechanism of adipophilin accumulating cellular cholesteryl ester in THP-1 macrophages. METHODS: New Zealand rabbit atherosclerotic model was made with high cholesterol diet for 12 weeks. The expressions of adipophilin and PKCα were determined by Western blotting and immunochemical staining in aortic arteries. Cholesteryl ester-loading cells (CE-loading cells) were made from THP-1 macrophages incubated with oxidized low density lipoprotein. In CE-loading cells, expressions of adipophilin and ACAT1 were analyzed by RT-PCR, and the activity of PKC was determined by PepTag assay and spectrophotometry. When the CE-loading cells were incubated with PKC activator PMA and inhibitor calphostin C, expression of adipophilin was observed with RT-PCR, and cellular lipid was measured with oil red O staining and HPLC. The pcDNA3.1-HA-adi vector was transfected to THP-1 macrophage for making adipophilin over expression cells. After the CE-loading adipophilin over expression cells were incubated with or without ACAT inhibitor, the ACAT1 expression and cellular cholesteryl ester were analyzed. RESULTS: Compared with control, both adipophilin and PKCα expression increased in aortic arteries of atherosclerotic animal. In CE-loading THP-1 macrophages, adipophilin and ACAT1 highly were expressed and PKC activity was augmented also. PMA enhanced the high expression of adipophilin and cellular cholesteryl ester in CE-loading THP-1 macrophages, but calphostin C inhibited the effect. ACAT1 expression and cellular cholesteryl ester increased in adipophilin over expression cells, the effect was impaired by incubating with ACAT inhibitor. CONCLUSION: The results suggest that adipophilin increases ACAT1 activity through enhancing PKC activity, resulting in cellular cholesteryl ester accumulation in THP-1 macrophages.