黑碳暴露下人角膜上皮细胞中 NLRP3 抑制对 NLRP3 炎性小体激活的影响。
Effect of NLRP3 repression on NLRP3 inflammasome activation in human corneal epithelial cells with black carbon exposure.
机构信息
Department of Ophthalmology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing, China.
出版信息
Cutan Ocul Toxicol. 2022 Jun;41(2):107-112. doi: 10.1080/15569527.2022.2050746. Epub 2022 Mar 17.
PURPOSE
To investigate the inhibitory effects of NLRP3 siRNA on NLRP3 inflammasome activation in human corneal epithelial cells (HCECs) with fresh black carbon (FBC) particles and ozone-oxidized BC (OBC) particles treatment.
METHODS
HCECs were transfected with NLRP3 siRNA or control siRNA for 48 h, followed by 200 μg/ml FBC or OBC suspension for an additional 72 h. Untreated controls were cells with no siRNA transfection or BC treatment. RT-qPCR and Western blot were used to measure mRNA and protein levels of components of the NLRP3 inflammasome (NLRP3, ASC, and Caspase-1) and downstream cytokine (IL-1β), respectively.
RESULTS
Compared with untreated control cells, mRNA levels of NLRP3, ASC, Caspase-1, and IL-1β were significantly higher ( < 0.05) in control siRNA transfected cells with BC treatments. Compared with the control siRNA transfected cells, NLRP3 siRNA transfection reduced the expression of NLRP3 and ASC, whereas it had a limited effect on the expression of Caspase-1 and IL-1β with FBC or OBC exposures. Under FBC treatment, the reductions of NLRP3 and Caspase-1 mRNA levels were 53.5% ( < 0.001) and 34.2% ( < 0. 01), respectively, and NLRP3 and ASC protein levels were lowered by 58.2% ( < 0.001) and 45.4% ( < 0.001), respectively. Under OBC treatment, the reductions of NLRP3 and Caspase-1 mRNA levels were 39.8% ( < 0.001) and 25.6% ( < 0.05), respectively, and NLRP3 and ASC protein levels were lowered by 44.8% ( < 0.001) and 41.7% ( < 0.001), respectively. Moreover, mRNA levels of ASC and IL-1β, the protein levels of Caspase-1 and IL-1β showed a tendency to decrease in NLRP3 siRNA transfected cells, it was statistically insignificant ( > 0.05).
CONCLUSIONS
NLRP3 siRNA transfection could partially reverse the increased mRNA levels of NLRP3 and Caspase-1, the protein levels of NLRP3 and ASC in HCECs with BC treatment, whereas the reductions of protein levels of Caspase-1 and IL-1β were not significant, indicating that NLRP3 siRNA has a limited inhibitory effect on the activation of NLRP3 inflammasome triggered by BC.
目的
研究 NLRP3 siRNA 对新鲜黑碳 (FBC) 颗粒和臭氧氧化黑碳 (OBC) 颗粒处理人角膜上皮细胞 (HCEC) 中 NLRP3 炎性小体激活的抑制作用。
方法
用 NLRP3 siRNA 或对照 siRNA 转染 HCEC 48 小时，然后用 200μg/ml FBC 或 OBC 悬浮液再处理 72 小时。未处理的对照组为未进行 siRNA 转染或 BC 处理的细胞。分别用 RT-qPCR 和 Western blot 测定 NLRP3 炎性小体（NLRP3、ASC 和 Caspase-1）和下游细胞因子（IL-1β）成分的 mRNA 和蛋白水平。
结果
与未处理的对照组细胞相比，BC 处理的对照 siRNA 转染细胞中 NLRP3、ASC、Caspase-1 和 IL-1β 的 mRNA 水平明显升高（<0.05）。与对照 siRNA 转染细胞相比，NLRP3 siRNA 转染降低了 NLRP3 和 ASC 的表达，但对 FBC 或 OBC 暴露时 Caspase-1 和 IL-1β 的表达影响有限。在 FBC 处理下，NLRP3 和 Caspase-1 的 mRNA 水平分别降低了 53.5%（<0.001）和 34.2%（<0.01），NLRP3 和 ASC 蛋白水平分别降低了 58.2%（<0.001）和 45.4%（<0.001）。在 OBC 处理下，NLRP3 和 Caspase-1 的 mRNA 水平分别降低了 39.8%（<0.001）和 25.6%（<0.05），NLRP3 和 ASC 蛋白水平分别降低了 44.8%（<0.001）和 41.7%（<0.001）。此外，在 NLRP3 siRNA 转染细胞中，ASC 和 IL-1β 的 mRNA 水平以及 Caspase-1 和 IL-1β 的蛋白水平均呈下降趋势，但无统计学意义（>0.05）。
结论
NLRP3 siRNA 转染可部分逆转 BC 处理后 HCEC 中 NLRP3 和 Caspase-1 的 mRNA 水平以及 NLRP3 和 ASC 的蛋白水平升高，但 Caspase-1 和 IL-1β 的蛋白水平降低不显著，表明 NLRP3 siRNA 对 BC 触发的 NLRP3 炎性小体的激活抑制作用有限。
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