硫化氢抑制丙酮醛诱导的人皮肤角质形成细胞损伤

Hydrogen sulfide attenuates methylglyoxal-induced human skin keratinocytes injury

目的 观察硫化氢（H2S）对丙酮醛（MGO）诱导的人皮肤角质形成细胞（HaCaT）损伤的影响.方法 HaCaT细胞分为MGO组、对照组和MGO＋NSHD（H2S供体）组.MGO组用200、400、600 μmol/LMGO处理HaCaT细胞48 h造成细胞损伤;对照组给予等体积培养基;MGO＋NSHD组应用400 μmol/LMGO处理,48 h前用50、100、200μmol/LNSHD预处理1h.通过CCK-8检测各组细胞活性.HaCaT细胞经100 μmol/LNSHD预处理1h,用20 μmol/L的H2S荧光探针WSP-1染色结合荧光照相术检测NSHD预处理1h组和对照组培养基和细胞内的H2S含量.将HaCaT细胞分为MGO组、MGO＋NSHD组、NSHD组和对照组.MGO组仅用400 μmol/L MGO处理48 h;MGO＋NSHD组在400 μmol/L MGO处理48 h前用100 μmol/L NSHD预处理1 h;NSHD组的细胞仅用100 μmol/L NSHD处理1h;对照组则给予等体积培养基.Hoechst 33258核染色结合荧光照相术检测细胞凋亡.Rh123染色结合荧光照相术检测线粒体膜电位（MMP）.结果 HaCaT细胞经200、400和600 μmol/L MGO处理48 h,细胞活性依次为0.325±0.023、0.224±0.009和0.095±0.102,均低于对照组0.415±0.031（F=37.866,P＜0.05）,其中400 μmol/LMGO组细胞活性约为对照组的1/2,选用此浓度的MGO作为有效损伤浓度.100 μmol/L NSHD处理1h可使培养基和细胞内的H2S含量较对照组均增加.在400μmol/L MGO处理48 h前,分别用50、100和200 μnol/L NSHD预处理1h,细胞活性依次为0.235±0.028、0.314±0.017、0.346±0.020,均高于单独400μmol/L MGO处理组0.224±0.009（F=61.209,P＜0.05）.400 μmol/L MGO处理48 h后细胞凋亡率高于对照组和NSHD组[（32.6±3.5）％比（5.1±1.2）％、（3.4±0.8）％,均P＜0.05],MMP低于对照组和NSHD组（28.5±2.9比46.1±3.8、48.6±4.3,均P＜0.05）.MGO＋ NSHD组细胞凋亡率（18.3±2.6）％,低于MGO组但高于对照组和NSHD组（均P＜0.05）,MMP为38.9±3.2,高于MGO组但低于对照组和NSHD组（均P＜0.05）.结论 NSHD能通过释放H2S拮抗MGO诱导的皮肤细胞损伤.

Objective To investigate the effects of hydrogen sulfide （H2S） on methylglyoxal （MGO）-induced human skin keratinocytes （HaCaT cells） injury.Methods HaCaT cells were assigned to control group,MGO group and MGO＋NSHD（H2S donor） group.The MGO group was treated with 200,400 and 600 μmol/L MGO for 48 h to induce cell injury.Control group was treated with an aliquot of plain culture medium.In the MGO ＋NSHD group,before treatment with 400 μmol/L MGO for 48 h,the cells were preconditioned with 50,100 and 200 μmol/L NSHD for 1 h to examine the protective effects of H2S according to cell viability as measured by cell counting kit 8.After treatment with 100 μmol/L NSHD for 1 h,the levels of H2S in medium and cells of NSHD and control groups were assayed with 20 μmol/L WSP-1,the H2S fluorescent probe,for photofluorography.HaCaT cells were assigned to control group,MGO group,NSHD group and MGO＋NSHD group.The MGO group was treated with 400 μmol/L MGO for 48 h to induce cell injury.Control group was treated with an aliquot of plain culture medium.In the MGO＋NSHD group,before treatment with 400 μmol/L MGO for 48 h,the cells were preconditioned with 100 μmol/L NSHD,NSHD group was treated with only 100 μmol/L NSHD for 1 h.Cellular apoptosis and mitochondrial membrane potential （MMP） were observed by Hoechst 33258 and Rh123 staining followed by photofluorography respectively.Results Treatment with 200,400 and 600 μmol/L MGO for 48 h,were attenuated HaCaT cell viability 0.325±0.023,0.224±0.009 and 0.095±0.102 compared with control group 0.415±0.031 （F=37.866,P ＜ 0.05）.Both in medium and in cells of NSHD group,the content of H2S was enhanced after treatment with 100 μmol/L NSHD for 1 h compared with control group.The viability in 400 μmol/L MGO treatment for 48 h group was halved compared with control group,and therefore 400 μmol/L was deemed the optimal concentration for MGO treatment.Before treatment with 400 μtmol/L MGO for 48 h,the cells were preconditioned with 50,100 and 200 μmol/L NSHD for 1 h,with the viability being 0.235±0.028,0.314± 0.017 and 0.346±0.020 respectively,There were higher than that in the individual 400 μmol/L MGO group （F=61.209,P ＜ 0.05）.Compared with control and NSHD groups,The treatment with 400 μmol/L MGO for 48 h increased the apoptotic rate from （5.1±1.2）％,（3.4±0.8）％ to （32.6±3.5）％,and decreased the MMP from 46.1±3.8,48.6±4.3 to 28.5±2.9 （all P＜0.05）.Prior to treatment with 400 μmol/L MGO for 48 h in the presence of preconditioning with 100 μmol/L NSHD for 1 h,the apoptotic rate was decreased to （18.3±2.6）％ and the MMP was increased to 38.9±3.2.The apoptotic rate in MGO＋NSHD group was lower than that in MGO group but higher than those in control group and NSHD group.The MMP in MGO＋NSHD group was higher than that in MGO group but lower than those in control group and NSHD group （all P＜0.05）.Conclusion NSHD protects human skin keratinocytes from MGO-induced injury via release of H2S.