论文部分内容阅读
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。
This study examined the role of hyperlipidemic-mediated endoplasmic reticulum stress (ERS) in podocyte injury under inflammatory conditions and explored the role of intracellular cholesterol transporter type 1 Niemann-Pick Niemann-pick C1protein, NPC1) mediates the molecular mechanism of ERS induced by lipid disorders. The podocytes were divided into control group, low-density lipoprotein (LDL), hyperlipidemia + inflammatory cytokines (LDL + IL-1β), tunicamycin intervention group (TM) 4-PBA) intervention group and simvastatin intervention group (Statin). Flow cytometry was used to detect podocyte apoptosis. Oil red O staining was used to detect intracellular lipid deposition in podocytes. The expression of NPC1, glucose-regulated protein 78 (GRP78) and protein kinase R-like endoplasmic reticulum (PERK) and activating transcription factor 6 (ATF6) mRNA were detected by immunohistochemistry. The expression of GRP78 was analyzed by fluorescence immunoassay. The results showed that on the basis of high fat, with the increase of inflammatory cytokines concentration and prolongation of treatment time, the apoptosis rate of podocytes increased obviously. Intracellular lipid deposition and GRP78, PERK, ATF6, NPC1 m RNA expression in the group of high fat plus inflammatory factors increased significantly. Low-dose tunicamycin preconditioning and 4-PBA reduced the percentage of apoptotic cells, decreased the expressions of GRP78, PERK and ATF6 mRNA, and 4-PBA significantly attenuated the expression of GRP78. Simvastatin and 4-PBA reduced intracellular lipid deposition and NPC1 mRNA expression. The above results suggest that lipid may be over-transported to endoplasmic reticulum through NPC1 and cause podocyte injury induced by ERS in inflammatory state. Ease of endoplasmic reticulum stress and reduction of intracellular lipid deposition may reduce podocyte injury.