炎症状态下高脂通过NPC1介导内质网应激致足细胞损伤机制研究

High-lipid Induced Endoplasmic Reticulum Stress and Inflammatory Factor Caused the Podocyte Injure through NPC1 and Its Mechanism

该研究观察了炎症状态下高脂介导的内质网应激(endoplasmic reticulum stress,ERS)在足细胞损伤中的作用,并探讨了胞内胆固醇转运蛋白C型1类尼曼-匹克蛋(Niemann-pick C1protein,NPC1)介导脂质紊乱致ERS的分子机制。将足细胞分为对照组、高脂组(low-density lipoprotein,LDL)、高脂+炎症因子组(LDL+IL-1β)、衣霉素干预组(TM)、4-苯基丁酸(4-PBA)干预组、辛伐他汀干预组(Statin)。流式细胞术检测足细胞凋亡,油红O染色检测足细胞胞内脂质沉积,荧光定量PCR检测NPC1、葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、蛋白激酶R样内质网激酶(protein kinase R-like endoplasmic reticulum kinase,PERK)和活化转录因子6(activating transcription factor 6,ATF6)m RNA水平;荧光免疫技术分析GRP78的表达。结果表明,在高脂基础上,随炎症因子浓度增大和处理时间延长,足细胞的凋亡率明显上升。胞内脂质沉积以及GRP78、PERK、ATF6、NPC1 m RNA的表达在高脂加炎症因子组明显上升。与高脂加炎症因子组比较,小剂量衣霉素预处理和4-PBA能降低细胞凋亡比例,降低GRP78、PERK和ATF6 m RNA的表达,且4-PBA明显减弱GRP78荧光表达。辛伐他汀和4-PBA可以降低胞内脂质沉积和NPC1 m RNA的表达。以上结果说明,炎症状态下脂质可能通过NPC1过度转运到内质网并诱发ERS引起足细胞损伤,缓解内质网应激和降低胞内脂质沉积能减轻足细胞损伤。

The goals of this study is to investigate the role of lipid-induced endoplasmic reticulum stress （ERS） in podocyte injury under inflammatory condition and to explore whether it is related to intracellular cholesterol transporters Niemann-pick C 1 protein （NPC 1）. Podocytes were cultured and divided into control group, low-density lipoprotein （LDL） group, IL-1β＋LDL group, tunicamycin group （TM）, 4-phenyl butyric acid （4-PBA） group and statin group. The apoptosis of podocytes was measured by flow cytometry. The accumulation of lipid droplet in the cells was detected by oil red O staining. The mRNA leves of NPC1, glucose-regulated protein78 （GRP78）, protein kinase R-like endoplasmic reticulum kinase （PERK） and activating transcription factor 6 （ATF6） were determined by real-time PCR. GRP78 protein level was detected by immunofluorescence assay. We found that inflammation increased the rate of lipid-induced apoptosis in a time and dose dependent manners. Co-treatment of cells with high levels of lipid and IL-1β significantly increased the intracellular lipid accumulation and the levels of GRP78, PERK, ATF6, NPC1 mRNA. Pretreatment of cells with low concentrations of TM and 4-PBA significantly decreased apoptosis rate and down-regulated GRP78, PERK, ATF6 expression, while statin and 4-PBA treatment can reduce NPC1 expression and intracellular lipid accumulation. These results suggested that lipid might be transported to ER by NPC1 and its accumulation in ER promotes podocyte injury through activating ERS under inflammatory conditions. Reduction of ERS and the intacellular accumulation of lipid droplet can alleviate the damages on podocytes.