4种化学物质对HaCaT细胞氧化应激相关基因表达的影响

目的: 探讨过氧化氢、姜黄素、槲皮素及叔丁基对苯二酚对84种氧化应激相关基因表达的影响。方法: 取对数生长期的HaCaT细胞,采用不同浓度的过氧化氢、姜黄素、槲皮素及叔丁基对苯二酚,用四甲基噻唑蓝(MTT)法评价受试物的细胞毒性。将不同浓度的过氧化氢(0.5和1.25 mmol/L)、姜黄素(5和20μmol/L)、槲皮素(200和500μmol/L)及叔丁基对苯二酚(10和50μmol/L)分别处理HaCaT细胞4 h及24 h后,提取RNA,对RNA质量进行评价,然后采用实时荧光定量PCR(qPCR)方法检测HaCaT细胞中84种氧化应激相关基因的表达水平。结果: MTT试验显示,过氧化氢、姜黄素和叔丁基对苯二酚浓度分别在>1 mmol/L、>10μmol/L和>50μmol/L时,HaCaT细胞的存活率在80%以下;而槲皮素在浓度为1 000μmol/L仍未显示出明显的细胞毒性。qPCR结果显示,上述4种化学物质在不同浓度不同时间对HaCaT细胞氧化应激相关基因的表达有一定的影响。过氧化氢1.25 mmol/L作用4 h能引起HMOX1、SPINK1等基因表达上调而1.25 mmol/L作用24 h时CCL5表达下调;姜黄素20μmol/L作用4 h能引起HMOX1、ALB等基因表达上调以及GSR等基因表达下调;槲皮素500μmol/L作用4 h能引起ALB、HMOX1等基因表达上调以及SEPP1等基因表达下调;叔丁基对苯二酚10μmol/L作用4 h能引起基因EPX、HMOX1等基因表达上调,而10μmol/L作用24 h时AOX1等基因表达下调。结论: 过氧化氢、槲皮素、姜黄素及叔丁基对苯二酚,这4种化学物质均可引起氧化应激相关基因发生改变,均能引起基因HMOX1表达上调,而且高浓度的化学物质引起的基因表达变化高于低剂量,为抗氧化剂的添加提供了一定的理论依据。

Effects of four chemicals on the expression of oxidative stress-related genes in HaCaT cells

OBJECTIVE: To investigate effects of hydrogen peroxide,curcumin,quercetin and tert-butyl hydroquinone(tBHQ) on expressions of 84 oxidative stress-related genes. METHODS: HaCaT cells in logarithmic growth phase were used,and different concentrations of hydrogen peroxide,curcumin,quercetin and tert-butyl hydroquinone were applied.The concentrations were:hydrogen peroxide (0.5 and 1.25 mmol/L),curcumin (5 and 20μmol/L),quercetin (200 and 500μmol/L) and tert-butyl hydroquinone (10 and 50μmol/L) for 4 h and 24 h,respectively.Cytotoxicity of the test substances were evaluated by tetramethylthiazole blue (MTT).RNA samples were extracted and their qualities were evaluated.Then,expression levels of 84 oxidative stress-related genes in HaCaT cells were detected by quantitative real-time PCR (qPCR). RESULTS: MTT assay showed that the survival rate of HaCaT cells was less than 80% when the concentrations of hydrogen peroxide,curcumin and tert-butyl hydroquinone were>1 mmol/L,>10μmol/L and>50μmol/L,respectively.Quercetin did not show obvious cytotoxicity at the concentration of 1 000μmol/L.Resultsof qPCR showed that the four chemicals affected expressions of oxidative stress-related genes in HaCaT cells at different concentrations and at different times.The expression levels of some oxidative stress-related genes were increased,and otherswere decreased.Hydrogen peroxide increased expression of HMOX1,SPINK1 (1.25 mmol/L,4 h) but reduced that of CCL5 (1.25 mmol/L,24 h).Curcumin increased HMOX1,ALB (20μmol/L,4 h)expression but reduce that of GSR (20μmol/L,4 h) and other genes.Quercetin increased ALB,HMOX1 (500μmol/L,4 h) expressions but reduced expression of SEPP1 (500μmol/L,4 h).tert-butyl hydroquinone increased expression of EPX,HMOX1 (10μmol/L,4 h) but reduced that of AOX1 (10μmol/L,24 h),etc. CONCLUSION: Exposure to hydrogen peroxide,curcumin,quercetin and tert-butyl hydroquinone caused changes in expression of oxidative stress-related genes,especially increased expression the HMOX1 gene.Moreover,higher concentrations of the chemicals caused more changes than lower doses.