地塞米松对脂多糖诱导人视网膜色素上皮细胞炎症损伤及NLRP3/Caspase-1通路的影响

Effects of dexamethasone on lipopolysaccharide-induced inflammatory damage of human retinal pigment epithelial cells and NLRP3/Caspase-1 pathway

目的探究地塞米松(Dex)对脂多糖(LPS)诱导人视网膜色素上皮(HRPE)细胞炎症损伤及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)通路的影响。方法体外培养ARPE-19细胞株,MMT法检测不同浓度Dex处理细胞24 h、48 h、74 h对LPS诱导ARPE-19细胞存活率的影响,确定Dex最佳处理浓度和时间。ARPE细胞随机分为:正常对照组(NC组)、LPS组(5 mg·L^-1 LPS)、Dex组(0.20 mol·L^-1 Dex)、Dex+LPS组(0.20 mol·L^-1 Dex+5 mg·L^-1 LPS),RT-qPCR检测各组细胞肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、NLRP3、Caspase-1 mRNA相对表达量,ELISA法测定TNF-α、IL-1β、IL-6蛋白表达水平,Western blot检测NLRP3、Caspase-1蛋白表达水平。结果同一时间点,与NC组比较,LPS组ARPE-19细胞存活率均显著降低(均为P<0.05),且随着Dex浓度增加,细胞存活率呈先升高后下降的趋势;0.20 mol·L^-1 Dex作用LPS诱导的ARPE-19细胞24 h时细胞存活率为(90.77±5.73)%,接近NC组,因此选用0.20 mol·L^-1 Dex作用24 h用于后续实验。NC组与Dex组间TNF-α、IL-1β、IL-6、NLRP3、Caspase-1 mRNA相对表达量差异均无统计学意义(均为P>0.05)。与NC组、Dex组比较,LPS组、Dex+LPS组TNF-α、IL-1β、IL-6、NLRP3、Caspase-1 mRNA及蛋白表达水平均显著升高,且Dex+LPS组TNF-α、IL-1β、IL-6、NLRP3、Caspase-1 mRNA及蛋白表达水平均显著低于LPS组(均为P<0.05)。结论Dex可能通过抑制NLRP3/Caspase-1通路减轻LPS诱导的HRPE炎症损伤。

Objective To investigate the effects of dexamethasone(Dex)on lipopolysaccharide(LPS)-induced inflammatory damage of human retinal pigment epithelial cells(HRPE)and the nucleotide-binding oligomerization domain like receptor protein 3/Caspase-1(NLRP3/Caspase-1)pathway.Methods ARPE-19 cell line was cultured in vitro,thiazole blue(MMT)was used to detect the effects of Dex at different concentrations on the activity of LPS-induced ARPE-19 cells to determine the optimum concentration and time.The ARPE-19 cells was divided into four groups:normal control group(NC group,normal growth ARPE-19 cells),LPS inflammatory injury group(LPS group,5 mg·L^-1 LPS treatment to cells),Dex group(0.20 mol·L^-1 Dex treatment to cells),Dex+LPS group(0.20 mol·L^-1 Dex+5 mg·L^-1 LPS treatment to cells).The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,NLRP3 and Caspase-1 mRNAs were detected by RT-qPCR,the levels of TNF-α,IL-1β,IL-6 were measured by enzyme-linked immunosorbent assay(ELISA),and Western blot was used to detect the expressions of NLRP3 and Caspase-1 proteins in ARPE-19 cells.Results At the same time,compared with NC group,the activity of ARPE-19 cells in LSP group decreased significantly(all P<0.05),and as the concentration of Dex increases,the cell survival rate increases first and then decreases.The cell survival rate of ARPE-19 cells induced by LPS by 0.20 mol·L^-1 Dex for 24 hours was(90.77±5.73)%,which was close to the NC group,and therefore,0.20 mol·L^-1 dex for 24 hours was chosen as the optimal concentration for the follow experiment.There was no significant difference in TNF-α,IL-1β,IL-6,NLRP3,Caspase-1 mRNA levels between NC group and Dex group(all P>0.05).Compared with NC group and Dex group,TNF-α,IL-1β,IL-6,NLRP3,Caspase-1 mRNA and protein levels were increased in LPS group and Dex+LPS group,and the levels of TNF-α,IL-1β,IL-6,NLRP3,Caspase1 mRNA and protein in Dex+LPS group were significantly lower than those in LPS group(all P<0.05).Conclusion Dex may reduce the lipopolysaccharide induced inflammatory damage of HRPE cells by inhibiting NLRP3/Caspase-1 pathway.