Abstract
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. Key words: Diabetes ; H2S; Keratinocytes ; Methylglyoxal; Skin ulcer
Published Version
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