热休克转录因子1对视网膜色素上皮细胞抗氧化和抗衰老的调控作用

Regulatory role of heat shock transcription factor 1 in antioxidant and anti-aging function of the retinal pigment epithelial cells

目的探讨热休克转录因子1(HSF1)对人视网膜色素上皮细胞(ARPE-19)抗氧化和抗衰老的作用。方法利用成簇规律间隔短回文重复序列及相关蛋白9(CRISPR/Cas9)基因编辑技术敲除人ARPE-19细胞系中HSF 1基因,构建并获得2种HSF1缺失ARPE细胞株(ARPE/Hsf1-/-),分别命名为H8、H9细胞株。取野生型、H8和H9细胞株,应用DHE探针染色结合流式分析技术测定细胞内活性氧簇(ROS)含量,应用流式细胞分析方法测定细胞周期;采用细胞计数试剂盒-8(CCK-8)法测定不同培养时间点细胞活力值;采用结晶紫染色实验测定细胞相对存活率;采用β-半乳糖苷酶(SA-β-gal)染色实验检测衰老细胞比率;采用Western blot法检测各细胞株中热休克蛋白(HSP)70、HSP27、聚集素(CLU)、p53、p21和白细胞介素(IL)-1β蛋白表达水平;采用实时荧光定量PCR法测定各细胞株中p53、p21、IL-6、IL-8、IL-1β、单核细胞趋化蛋白1(MCP1)mRNA表达水平。比较各细胞株在不同热休克处理条件下和HSP90抑制剂IPI504处理下热休克反应相关蛋白相对表达量,比较各细胞株不同浓度H_(2)O_(2)处理后相对存活率,比较各细胞株经或未经ROS清除剂N-乙酰半胱氨酸(NAC)处理后p21蛋白相对表达量。结果基因测序显示H8和H9细胞株成功携带突变基因,Western blot检测结果显示H8、H9细胞株不表达HSF1蛋白,HSF1在ARPE-19细胞中被成功敲除。与野生型细胞株相比,H8、H9细胞株中的HSP70、HSP27、CLU蛋白相对表达水平显著降低,差异均有统计学意义(均P<0.05);各细胞株HSP90蛋白相对表达水平总体比较,差异无统计学意义(F=0.29,P>0.05)。在不同热休克刺激和IPI504诱导下野生型细胞株中HSP70、HSP27、CLU蛋白相对表达水平显著升高,差异均有统计学意义(均P<0.05);与野生型细胞株相比,H8和H9细胞株的HSP70、HSP27、CLU蛋白相对表达水平显著低于相应处理的野生型细胞,差异均有统计学意义(均P<0.05)。与野生型细胞株相比,H8和H9细胞株培养24、48、72和96 h的细胞活力均显著降低,差异均有统计学意义(均P<0.05)。与野生型细胞株比较,H8和H9细胞株G1期细胞百分比显著升高,细胞周期抑制因子p53、p21的mRNA和蛋白相对表达水平显著升高,差异均有统计学意义(均P<0.05),SA-β-gal染色阳性细胞比率显著增加,差异均有统计学意义(均P<0.001);衰老相关炎症因子IL-6、IL-8、IL-1β、MCP1 mRNA相对表达量显著下降,差异均有统计学意义(均P<0.001)。H8和H9细胞株ROS含量明显高于野生型细胞株,差异均有统计学意义(均P<0.001);H8和H9细胞株经NAC处理后p21蛋白相对表达量较未经NAC处理明显降低,差异均有统计学意义(P<0.05)。H8和H9细胞株200、400、600、800μmol/L H 2O 2处理条件下细胞相对存活率明显低于野生型细胞,差异均有统计学意义(均P<0.05)。结论敲除HSF1可下调HSP的表达,激活ROS/P53/P21通路,诱导RPE细胞衰老,并增加RPE对氧化应激刺激的敏感性。HSF1在RPE细胞中可能具有抗衰老和抗氧化调控作用。

Objective To investigate the anti-aging and antioxidant effect of the heat shock transcription factor 1(HSF1)on human retinal pigment epithelial cells.Methods Two HSF1-deficient ARPE cells(ARPE/Hsf1-/-)were constructed by using the clustered regularly interspaced short palindromic repeat and associated protein 9(CRISPR/Cas9)gene editing system and named H8,H9 konckout cell strains.Experiments were operated on the 3 cell strains:wild-type,H8 and H9 cells.The content of reactive oxygen species in ARPE-19 cell was measured by DHE probe staining combined with flow cytometry technology,and the cell cycle was measured by flow cytometry technology.The cell viability at different time points was measured using cell counting kit-8(CCK-8).Crystal violet staining assay was used to measure the relative ratio of cell survival.SA-β-gal staining assay was used to detect the ratio of ARPE-19 senescent cells.The expressions of HSP70,HSP27,clusterin(CLU),p53,p21 and interleukin(IL)-1βproteins were measured by Western blot technology.The expressions of p53,p21,IL-6,IL-8,IL-1βand monocyte chemoattractant protein 1(MCP1)mRNA were measured by quantitative real-time PCR technology.Relative expression of heat shock response protein under different heat shock treatment conditions and HSP90 inhibitor IPI504,relative survival with different concentrations of H_(2)O_(2),relative expression of p21 protein after treatment with or without ROS scavenger N-acetylcysteine(NAC)were compared in each cell strain.Results Gene sequencing showed that H8 and H9 cell strains successfully carried mutated genes.Western blot experiment results showed that H8 and H9 cell strains did not express HSF1 protein,and HSF1 was successfully knocked out in ARPE-19 cells.Compared with wild-type cell,the expression levels of HSP70,HSP27 and CLU proteins in H8 and H9 cell strains significantly decreased,with statistically significant differences(all at P<0.05),and no significant difference was found in the relative HSP90 protein expression level(F=0.29,P>0.05).Under different heat shock stimulation and IPI504 induction,the HSP70,HSP27,and CLU protein levels significantly increased in wild-type cells compared with before treatment,and the HSP70,HSP27,and CLU protein levels were significantly lower in H8 and H9 cell strains than in corresponding treated wild-type cells(all at P<0.05).Compared with wild-type cell strains,cell viability significantly decreased in H8 and H9 cell strains at 24,48,72,and 96 hours(all at P<0.05).Compared with wild-type cell strains,the percentage of cells in G1 phase was significantly higher and the mRNA and protein levels of the cell cycle inhibitors p53 and p21 significantly increased in H8 and H9 strains,showing statistically significant differences(all at P<0.05),and the ratio of positive cells for SA-β-gal staining significantly increased,showing statistically significant differences(all at P<0.001).The relative expression of aging-related inflammatory factors IL-6,IL-8,IL-1β,and MCP1 mRNA decreased,and the differences were statistically significant(all at P<0.001).In addition,compared with wild-type cell strains,the content of reactive oxygen species(ROS)was higher in H8 and H9 cell strains,and the differences were statistically significant(all at P<0.001).The expression of p21 protein in H8 and H9 cell strains wtih NAC treatment decreased significantly compared with non-NAC treatment cells(both at P<0.05).Compared with wild-type cell strains,H8 and H9 cell viability decreased at 200,400,600,and 800μmol/L H_(2)O_(2) treatment conditions,and the differences were statistically significant(all at P<0.05).Conclusions Knockdown of HSF1 can downregulate the expression of heat shock proteins,activate the ROS/P53/P21 pathway,induce senescence in RPE cells,and increase the sensitivity of RPE to oxidative stress stimuli.HSF1 may have anti-senescence and anti-oxidant regulatory effects in RPE cells.