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目的观察热休克转录因子1(HSF1)与热休克蛋白70(HSP70)对紫外线A(UVA)诱导HaCaT细胞凋亡的保护作用及其机制。方法建立8mJ/cm2UVA辐射损伤HaCaT细胞的病理模型。将细胞随机分为对照组、8mJ/cm2UVA照射组、HSP70转录抑制剂组(50μmol/L槲皮素)。Honechst 33258荧光染色观察细胞凋亡;蛋白质印迹法检测UVA辐射HaCaT细胞后p-HSF1和HSP70蛋白的经时变化及UVA辐射后孵育6h JNK(c-Jun氨基末端激酶)、p-JNK的蛋白表达;Real-Time PCR检测HSP70 mRNA的表达。结果 UVA辐射后HaCaT细胞内p-HSF1、HSP70蛋白表达量均出现先增加后减少的时间依赖性趋势,其中p-HSF1于1h开始增加,3h达高峰,HSP70于6h达高峰,24h基本恢复原始水平;UVA辐射前预先加入HSP70转录抑制剂槲皮素能显著抑制HSP70 mRNA的表达,增加p-JNK的表达量,同时Honechst 33258荧光染色观察其与UVA辐射组比较凋亡率明显升高。结论 8mJ/cm2UVA辐射HaCaT细胞在一定时间内可使HSF1活化致HSP70表达增加。HSF1/HSP70通路对UVA诱导的HaCaT细胞凋亡具有保护作用,其机制与HSP70大量表达后抑制JNK的活化有关。
Objective To observe the protective effect of heat shock transcription factor 1 (HSF1) and heat shock protein 70 (HSP70) on the apoptosis of HaCaT cells induced by ultraviolet A (UVA) and its mechanism. Methods The pathological model of HaCaT cells damaged by 8mJ / cm2 UVA radiation was established. The cells were randomly divided into control group, 8mJ / cm2 UVA irradiation group and HSP70 transcription inhibitor group (50μmol / L quercetin). Honechst 33258 fluorescence staining was used to observe the apoptosis. The expression of p-JNK and protein of JNK (c-Jun N-terminal kinase) were detected by western blotting after the UVA radiation HaCaT cells were incubated with UVA for 6h Real-time PCR was used to detect the expression of HSP70 mRNA. Results The expression of p-HSF1 and HSP70 in HaCaT cells increased first and then decreased in a time-dependent manner. UV-irradiated radiation increased the expression of p-HSF1 at 1h, reached the peak at 3h, peaked at 6h and returned to its original value at 24h Pretreatment with quercetin, a transcription inhibitor of HSP70 before UVA irradiation, significantly inhibited the expression of HSP70 mRNA and increased the expression of p-JNK. Meanwhile, the apoptosis rate of HUVECs was significantly increased by Honechst 33258 fluorescence staining. Conclusion HaCaT cells irradiated by 8mJ / cm2UVA can activate HSF1 to increase the expression of HSP70 in a certain period of time. HSF1 / HSP70 pathway has a protective effect on UVA-induced HaCaT cell apoptosis, and its mechanism is related to the suppression of JNK activation after HSP70 is overexpressed.