内质网应激在乙酰化低密度脂蛋白引起的人单核巨噬细胞凋亡中的作用

目的 观察在脂质引起佛波酯（PMA）促分化后人单核细胞源性巨噬细胞（THP-1）凋亡中有无内质网（ER）应激，探讨巨噬细胞凋亡在慢性肾脏疾病并发加速进展的动脉粥样硬化和心血管事件中的作用。 方法 密度梯度法提取低密度脂蛋白，制备乙酰化低密度脂蛋白（ACLDL）。Hoechst染色定量计数凋亡细胞比例。caspase3,7活性测定检测凋亡。油红O染色观察细胞内脂滴变化。酶学反应法测定细胞内游离胆固醇（FC）和胆固醇酯（CE）的含量。Western印迹和实时定量PCR测定细胞乙酰基辅酶A:胆固醇酰基转移酶（ACAT）、生长停滞及DNA损伤基因（CHOP）和Bcl-2的表达。 结果 ACLDL呈剂量依赖和时间依赖促进细胞凋亡。100 mg/L ACLDL与THP-1巨噬细胞作用24 h，油红O染色下细胞内脂滴显著增多，细胞内FC和CE含量显著增加（P < 0.01）。Western印迹显示，ACLDL 100 mg/L作用3～24 h，ACAT1、CHOP表达较作用前持续升高，Bcl-2表达持续降低。实时定量PCR显示，ACLDL作用12～24 h，较作用前促进CHOP mRNA表达（P < 0.01），抑制Bcl-2 mRNA表达（P < 0.01）；ACLDL作用24 h点，较无ACLDL作用组促进CHOP mRNA表达（P < 0.01），抑制Bcl-2 mRNA表达（P < 0.01），但对ACAT1表达没有影响。 结论 在ACLDL引起的THP-1巨噬细胞凋亡中存在ER应激。

Endoplasmic reticulum stress is involved in acetylated low density lipoprotein-induced apoptosis in THP-1 differentiated macrophages

Objective To clarify why accelarated atheroslcerosis is complicated in chronic kidney disease patients, and to investigate whether endoplasmic reticulum (ER) stress can be observed in acetylated low density lipoprotein (ACLDL)-induced apoptosis in THP-1 macrophages differentiated by 160 nmol/L PMA for five days. Methods Hoechst 33258 stain and caspase 3,7 assay were used to detect apoptosis. Oil red O was used to examine the lipid droplet. High performance liquid chromatography was used to measure intracellular free cholesterol(FC) and cholesterol ester(CE). Western blot was applied to demonstrate the protein level of acyl-coenzyme A cholesterol acyltransferase(ACAT)1, growth arrest and DNA damage(CHOP) and Bcl-2. Real-time PCR was used to detect the changes of mRNAs. Results ACLDL could induce THP-1 macrophages apoptosis in time-and dose-dependent manner. After exposure to 100 mg/L ACLDL for 24 hours, the level of free cholesterol and cholesterol ester mass had a significant increment by 1.5- and 2.4-fold respectively (P<0.01). CHOP increased and Bcl-2 decreased both in protein and mRNA levels. ACLDL loading also resulted in an increase of ACAT1 protein without any change in mRNA level. Conclusion In THP-1 macrophages foam cell, apoptosis can be induced by ACLDL accompanied by ER stress pathway activation.