Toll 样受体4对氧化型低密度脂蛋白诱导巨噬细胞凋亡的影响及其机制研究

Effects of TLR4 on ox-LDL induced macrophage apoptosis and its mechanism

目的：探讨Toll样受体4（ TLR4）对氧化型低密度脂蛋白（ oxidized low density lipopro-tein，ox-LDL）诱导的巨噬细胞凋亡的影响及其机制研究。方法体外培养THP-1细胞，用PMA（丙二醇甲醚醋酸酯）诱导成巨噬细胞，分别设立正常对照组、ox-LDL组、ox-LDL＋LPS组、衣霉素组，用MTT和流式细胞术检测细胞存活率及凋亡情况、油红O染色观察细胞吞噬脂质情况，q-RT-PCR、Western blot检测内质网应激相关基因及葡萄糖调节蛋白78（glucose-regulated protein78， GRP78）和CCAAT／增强子结合蛋白同源蛋白（ CCAAT／enhancer-binding protein homologous protein ， CHOP ）的表达情况，并且用TLR4-siRNA抑制TLR4活性观察其对通路的影响。结果流式及MTT结果显示ox-LDL＋LPS组的凋亡细胞数较ox-LDL组明显增加（P＜0．01）， ox-LDL＋LPS组的内质网应激相关基因和蛋白GRP78、CHOP均较ox-LDL组增加，且两组都明显大于正常对照组（ P＜0．01），抑制TLR4活性后内质网应激程度明显减轻（ P＜0．05）。结论 TLR4加重ox-LDL诱导的巨噬细胞凋亡，其机制是加重内质网应激程度，增加CHOP表达，起到促凋亡作用。

Objective To study the effects of Toll-like receptor 4（TLR4） on oxidized low density lipoprotein （ ox-LDL） induced macrophage apoptosis and its possible mechanism .Methods THP-1 derived macrophages were divided into four groups including untreated control group , ox-LDL treated group , ox-LDL＋LPS treated group and tunicamycin treated group .MTT assay and flow cytometry analysis were performed to measure cell vitality and cell apoptosis , respectively .Oil red O staining was used to observe the phagocytosis of lipids by macrophages .The persistent and intense endoplasmic reticulum （ ER） stress markers were de-tected by analyzing the expression of glucose-regulated protein 78 （ GRP78 ） and CCAAT/enhancer-binding protein homologous protein （ CHOP） at mRNA and protein levels by q-RT-PCR and Western blot .Small in-terfering RNA （ siRNA） was used to silence the expression of TLR 4 to further elucidate its possible mecha-nism.Results Flow cytomotry and MTT assay showed that the number of apoptotic cells in ox-LDL＋LPS treated group were increased more significantly than that in ox-LDL treated group （P〈0.01）, and cell apop-tosis in both two groups were greater than that in control group （P〈0.01）.Compared with control group, the expression of GRP78 and CHOP at mRNA and protein levels were up-regulated in ox-LDL＋LPS treated group and ox-LDL treated group （P〈0.01）, and the expression of GRP78 and CHOP in ox-LDL＋LPS treated group was significantly higher than that in ox-LDL treated group （P〈0.01）.Silenced expression of TLR4 al-leviated the endoplasmic reticulum stress （P〈0.05）.Conclusion Increased expression of CHOP contribu-ted to cell apoptosis .TLR4 might promote ox-LDL induced macrophage apoptosis through accelerating endo-plasmic reticulum stress .