通过CD36受体摄取氧化脂蛋白可激活人视网膜色素上皮细胞中的NLRP3炎性小体。

Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells.

作者信息

Gnanaguru Gopalan, Choi Ariel R, Amarnani Dhanesh, D'Amore Patricia A

机构信息

Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.

Program in Liberal Medical Education, Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States.

出版信息

Invest Ophthalmol Vis Sci. 2016 Sep 1;57(11):4704-12. doi: 10.1167/iovs.15-18663.

DOI:10.1167/iovs.15-18663

PMID:27607416

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5024668/

Abstract

PURPOSE

Accumulation of oxidized phospholipids/lipoproteins with age is suggested to contribute to the pathogenesis of AMD. We investigated the effect of oxidized LDL (ox-LDL) on human RPE cells.

METHODS

Primary human fetal RPE (hf-RPE) and ARPE-19 cells were treated with different doses of LDL or ox-LDL. Assessment of cell death was measured by lactate dehydrogenase release into the conditioned media. Barrier function of RPE was assayed by measuring transepithelial resistance. Lysosomal accumulation of ox-LDL was determined by immunostaining. Expression of CD36 was determined by RT-PCR; protein blot and function was examined by receptor blocking. NLRP3 inflammasome activation was assessed by RT-PCR, protein blot, caspase-1 fluorescent probe assay, and inhibitor assays.

RESULTS

Treatment with ox-LDL, but not LDL, for 48 hours caused significant increase in hf-RPE and ARPE-19 (P < 0.001) cell death. Oxidized LDL treatment of hf-RPE cells resulted in a significant decrease in transepithelial resistance (P < 0.001 at 24 hours and P < 0.01 at 48 hours) relative to LDL-treated and control cells. Internalized ox-LDL was targeted to RPE lysosomes. Uptake of ox-LDL but not LDL significantly increased CD36 protein and mRNA levels by more than 2-fold. Reverse transcription PCR, protein blot, and caspase-1 fluorescent probe assay revealed that ox-LDL treatment induced NLRP3 inflammasome when compared with LDL treatment and control. Inhibition of NLRP3 activation using 10 μM isoliquiritigenin significantly (P < 0.001) inhibited ox-LDL induced cytotoxicity.

CONCLUSIONS

These data are consistent with the concept that ox-LDL play a role in the pathogenesis of AMD by NLRP3 inflammasome activation. Suppression of NLRP3 inflammasome activation could attenuate RPE degeneration and AMD progression.

摘要

目的

有研究表明，氧化磷脂/脂蛋白随年龄积累会促使年龄相关性黄斑变性（AMD）的发病。我们研究了氧化型低密度脂蛋白（ox-LDL）对人视网膜色素上皮（RPE）细胞的影响。

方法

用不同剂量的低密度脂蛋白（LDL）或氧化型低密度脂蛋白（ox-LDL）处理原代人胎儿RPE（hf-RPE）细胞和ARPE-19细胞。通过测定释放到条件培养基中的乳酸脱氢酶来评估细胞死亡情况。通过测量跨上皮电阻来检测RPE的屏障功能。通过免疫染色确定ox-LDL在溶酶体中的积累。通过逆转录聚合酶链反应（RT-PCR）测定CD36的表达；通过受体阻断检测蛋白印迹和功能。通过RT-PCR、蛋白印迹、半胱天冬酶-1荧光探针检测和抑制剂检测来评估NLRP3炎性小体的激活情况。

结果

用ox-LDL而非LDL处理48小时后，hf-RPE细胞和ARPE-19细胞死亡显著增加（P < 0.001）。与LDL处理组和对照组细胞相比，用氧化型低密度脂蛋白处理hf-RPE细胞导致跨上皮电阻显著降低（24小时时P < 0.001，48小时时P < 0.01）。内化的ox-LDL靶向RPE溶酶体。ox-LDL而非LDL的摄取使CD36蛋白和mRNA水平显著增加超过2倍。逆转录聚合酶链反应、蛋白印迹和半胱天冬酶-1荧光探针检测显示，与LDL处理组和对照组相比，ox-LDL处理可诱导NLRP3炎性小体激活。使用10 μM异甘草素抑制NLRP3激活可显著（P < 0.001）抑制ox-LDL诱导的细胞毒性。