Bhattarai Niina, Piippo Niina, Ranta-Aho Sofia, Mysore Yashavanthi, Kaarniranta Kai, Kauppinen Anu
School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland.
Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, 70210 Kuopio, Finland.
Antioxidants (Basel). 2021 Jan 7;10(1):67. doi: 10.3390/antiox10010067.
Age-related macular degeneration (AMD) is an eye disease in which retinal pigment epithelium (RPE) cells play a crucial role in maintaining retinal homeostasis and photoreceptors' functionality. During disease progression, there is increased inflammation with nucleotide-binding domain, leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome activation, oxidative stress, and impaired autophagy in RPE cells. Previously, we have shown that the dietary supplement Resvega reduces reactive oxygen species (ROS) production and induces autophagy in RPE cells. Here, we investigated the ability of Resvega to prevent NLRP3 inflammasome activation with impaired protein clearance in human RPE cells. Cell viability was measured using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Enzyme-linked immunosorbent assays (ELISA) were utilized to determine the secretion of cytokines, NLRP3, and vascular endothelial growth factor (VEGF). Caspase-1 activity was measured with a fluorescent labeled inhibitor of caspase-1 (FLICA; FAM-YVAD-FMK) and detected microscopically. Resvega improved the cell membrane integrity, which was evident as reduced LDH leakage from cells. In addition, the caspase-1 activity and NLRP3 release were reduced, as was the secretion of two inflammatory cytokines, interleukin (IL)-1β and IL-8, in IL-1α-primed ARPE-19 cells. According to our results, Resvega can potentially reduce NLRP3 inflammasome-mediated inflammation in RPE cells with impaired protein clearance.
年龄相关性黄斑变性(AMD)是一种眼部疾病,其中视网膜色素上皮(RPE)细胞在维持视网膜内环境稳定和光感受器功能方面起着关键作用。在疾病进展过程中,核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体激活、氧化应激增加以及RPE细胞自噬受损,炎症反应加剧。此前,我们已经表明,膳食补充剂Resvega可减少活性氧(ROS)的产生并诱导RPE细胞自噬。在此,我们研究了Resvega在人RPE细胞中预防NLRP3炎性小体激活及蛋白质清除受损的能力。使用乳酸脱氢酶(LDH)和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐(MTT)法测定细胞活力。采用酶联免疫吸附测定(ELISA)法测定细胞因子、NLRP3和血管内皮生长因子(VEGF)的分泌。用荧光标记的半胱天冬酶-1抑制剂(FLICA;FAM-YVAD-FMK)测定半胱天冬酶-1活性,并通过显微镜检测。Resvega改善了细胞膜完整性,这表现为细胞LDH泄漏减少。此外,在白细胞介素-1α(IL-1α)预处理的ARPE-19细胞中,半胱天冬酶-1活性和NLRP3释放减少,两种炎性细胞因子白细胞介素(IL)-1β和IL-8的分泌也减少。根据我们的结果,Resvega可能会减少蛋白质清除受损的RPE细胞中NLRP3炎性小体介导的炎症反应。