棕榈酸钠暴露不同时间对肾小管上皮细胞损伤及对NLRP3炎症小体激活的影响

Effects of palmitic acid sodium exposure for different time on renal tubular epithelial cells injury and NLRP3 inflammasome activation

目的 探究棕榈酸钠(PA)暴露不同时间对肾小管上皮细胞(HK-2)的损伤作用和机制。方法 CCK-8法检测PA作用不同时间HK-2细胞活力;油红染色法检测PA作用不同时间脂质沉积情况;Annexin-Ⅴ-FITC/PI凋亡试剂盒检测PA作用不同时间HK-2细胞凋亡情况;活性氧试剂盒检测PA作用不同时间活性氧(ROS)生成情况;ELISA检测细胞中谷胱甘肽(GSH)、超氧化物歧化酶(SOD)活性以及上清液中白细胞介素(IL)-1β和IL-18的水平;Western blot测定HK-2细胞中核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、半胱天冬酶1(Caspase-1)、NADPH氧化酶4(NOX4)蛋白表达情况。结果 CCK-8法结果显示,PA(6、9、12、24 h)组HK-2细胞活力下降;油红染色结果显示,PA(6、9、12、24 h)组脂质沉积增加;Annexin-Ⅴ-FITC/PI凋亡试剂盒结果显示,细胞凋亡率增加;活性氧试剂盒结果显示,PA(6、9、12、24 h)组ROS的生成显著增加;ELISA结果显示,PA(6、9、12、24 h)组细胞中GSH和SOD的活性降低,细胞上清液中IL-1β和IL-18的水平增加;Western blot结果显示,PA(6、9、12、24 h)组炎症相关蛋白NLRP3、Caspase-1、ASC及NOX4表达水平升高。结论 PA诱导能导致HK-2细胞脂质沉积和损伤,其机制可能与棕榈酸钠刺激后细胞内NLRP3炎性小体激活介导的炎症反应有关。

Objective To explore the effect and mechanism of palmitic acid sodium(PA)exposure for different time on HK-2 cells injury.Methods CCK-8 assay was used to detect the activity of HK-2 cells treated with PA at different times;oil red staining was used to detect lipid deposition at different time of PA treatment;Annexin-Ⅴ-FITC/PI apoptosis kit was used to detect the apoptosis of HK-2 cells treated with PA at different times;reactive oxygen species(ROS)production was detected by ROS kit at different time of PA treatment;ELISA was used to detect GSH and SOD in cells as well as the levels of IL-1βand IL-18 in the cell supernatant;Western blot was used to determine the expressions of NLRP3,ASC,Caspase-1 and NOX4 in HK-2 cells.Results CCK-8 assay showed that HK-2 cell viability decreased in PA(6,9,12,24 h)groups;oil red staining showed that lipid deposition increased in PA(6,9,12,24 h)groups;Annexin-Ⅴ-FITC/PI apoptosis kit results showed that the apoptosis rate increased;ROS kit results showed that ROS production significantly increased in PA(6,9,12,24 h)groups;ELISA results showed that the activities of GSH and SOD decreased in PA(6,9,12,24 h)groups,while the levels of IL-1βand IL-18 in supernatant increased;Western blot results showed that the expression levels of inflammatory related proteins NLRP3,Caspase-1,ASC and NOX4 in PA(6,9,12 and 24 h)increased.Conclusion PA exposure can induce lipid deposition and HK-2 cells damage,and the mechanism may be related to PA-induced inflammatory response by activating NLRP3 inflammasome in HK-2 cells.