Differential Response Of NHEKs And HaCaT Cells To Hydrogen Peroxide-induced Oxidative Stress And The Defense Mechanism Study Of Against Oxidative Stress-induced Senescence In NHSFs

Part One Differential Response of Normal Human Epidermal Keratinocytes and HaCaT Cells to Hydrogen Peroxide-induced Oxidative StressObjective To investigate the different response of Normal Human Epidermal Keratinocytes and HaCaT cells to hydrogen peroxide-induced oxidative stress.Methods Normal Human epidermal keratinocytes (NHEKs) were obtained from children’s foreskins and HaCaT cells were cultured. We examined changes of cell viability, intracellular reactive oxygen species (ROS) and superoxide dismutase (SOD), caspase-3/7levels, cellular apoptosis, the percent of cells arrested in G1phase, senescence associated β-galactosidase (SA-β-Gal) positive cells and the senescence-related protein klotho in NHEKs and HaCaT cells after H2O2treatment. Statistical analysis was performed using SPSS13.0software.Results The viability of NHEKs and HaCaT cells decreased in a concentration-dependent and time-dependent manner after exposure to H2O2. The inhibitory effect of150uM H2O2on cell viability was greater in HaCaT cells than in NHEKs (p<0.05). Intracellular ROS, the percent of cells arrested in G1phase and caspase-3/7levels increased more significantly in HaCaT cells than in NHEKs, while intracellular SOD increased more significantly in NHEKs than in HaCaT cells after exposure to150uM H2O2(P<0.05). After H2O2treatment, cells became bigger and uneven in shape, which represents a senescence phenotype. SA β-Gal positive cells increased significantly in NHEKs after treatment with H2O2(P<0.05). Klotho was significantly downregulated in both NHEKs and HaCaT cells after H2O2treatment. However, we didn’t observe SA-β-Gal positive HaCaT cells even after treatment with H2O2.Conclusions NHEKs are more resistant to H2O2-induced oxidative stress than HaCaT cells. HaCaT cells have senescence phenotypes, but do not express p-galactosidase. Part Two Effects of CD147on Oxidative Stress-induced Senescence in Human Skin FibroblastsObjectiveTo investigate the effects of CD147on senescence of oxidative Stress induced by hydrogen peroxide in human skin fibroblasts.MethodsNormal human skin fibroblasts (NHSFs) were obtained from children’s foreskins. We use lentivirus encoding CD147siRNA to silence the CD147expression in NHSFs. U6-vshRNA-CMV-PUR lentivirus encoding CD147shRNA was constructed. NHSFs were infected with empty lentivirus or lentivirus encoding CD147shRNA, respectively. Cells were treated with100uM H2O2, we examined changes of cell viability with the MTT assay, senescence associated β-galactosidase (SA-β-Gal) positive cells, caspase-3/7levels, cellular apoptosis and the percent of cells arrested in G1phase in NHSFs-virus and NHSFs-CD147shRNA cells and after H2O2treatment or un-treatment. Statistical analysis was performed using SPSS13.0software.ResultsWe observed the mild morphological changes related to senescence and associated with a mild increase in the number of SAβ-Gal positive cells in NHSFs-CD147shRNA cells without H2O2exposure, after H2O2treatment, NHSFs-virus cells became bigger and uneven in shape and the percentage of SA P-Gal positive cells increased moderately, representing a senescence phenotype, which was further amplified in NHSFs-CD147shRNA treated cells. The viability of NHSFs-virus treated cells decreased moderately at12,24,36,48h after H2O2treatment, which was further expanded in NHSFs-CD147shRNA cells with H2O2treatment. However, we found that NHSFs-CD147shRNA cells without H2O2exposure can also result in a mild decrease in cell viability. The cells arrested in G1phase resulted in a mild increase in NHSFs-CD147shRNA cells without H2O2exposure, after H2O2treatment, the percentage of cells arrested in G1phase was increased moderately in NHSFs-virus cells, which was further potentiated in NHSFs-CD147shRNA cells. NHSFs-CD147shRNA without H2O2exposure could induce in a mild increase in the caspase-3/7activity, after H2O2treatment, the caspase-3/7activity and the apoptotic rate was increased moderately in NHSFs-virus treated cells, which was further intensified in NHSFs-CD147shRNA cells.ConclusionsOur results provide a new understanding that CD147may play important roles in protecting NHSFs from senescence under both normal conditions and oxidative stress. The results presented in this study are consistent with the speculation that CD147also plays an regulate-skin aging function. Part Three The Defense Mechanism Study of against Oxidative Stress-induced Senescence in Human Skin FibroblastsObjectiveTo investigate the defense mechanism of CD147on senescence of oxidative Stress induced by hydrogen peroxide in human skin fibroblastsMethodsNormal human skin fibroblasts (NHSFs) were obtained from children’s foreskins. We use lentivirus encoding CD147siRNA to silence the CD147expression in NHSFs. U6-vshRNA-CMV-PUR lentivirus encoding CD147shRNA was constructed. NHSFs were infected with empty lentivirus or lentivirus encoding CD147shRNA, respectively. Cells were treated with100uM H2O2, we examined changes of intracellular ROS levels, SOD activity and the senescence-related protein klotho in NHSFs-virus and NHSFs-CD147shRNA cells and after H2O2treatment or un-treatment. Statistical analysis was performed using SPSS13.0software.ResultsWe observed the intracellular ROS level (Geo Mean) in NHSFs-virus was increased moderately after H2O2treatment (NHSFs-virus+H2O2:17.62±1.12, NHSFs-virus:6.87±0.42, P<0.05), which was further enlarged in NHSFs-CD147shRNA (NHSFs-CD147shRNA+H2O2:28.91±1.33, P<0.05). Whereas NHSFs-CD147shRNA without H2O2exposure can only enhance the intracellular ROS level slightly (NHSFs-CD147shRNA:7.30±0.58, P>0.05). In addition, after H2O2treatment, intracellular SOD level increased moderately in NHSFs-CD147shRNA treated cells (NHSFs-CD147shRNA+H2O2:0.88±0.06, NHSFs-virus:0.28±0.06), which was further augmented in NHSFs-virus cells (NHSFs-virus+H2O2:2.18±0.17, P<0.05). In contrast, CD147suppression alone produced no effect in NHSFs-CD147shRNA cells without H2O2exposure (NHSFs-CD147shRNA:0.26±0.03, P>0.05). However, klotho expression was not detected before or after treatment with H2O2in NHSFs-CD147shRNA.ConclusionsCD147can exacerbate the oxidative stress-induced senescence induced by H2O2by increasing ROS accumulation and destroying the intrinsic antioxidant defenses in NHSFs. This process may be related to klotho protein. CD147might play a crucial role in anti-oxidation and anti-aging via modulating klotho in NHSFs. We believe that the relationship of CD147and klotho and the exact mechanisms of CD147in senescence of NHSFs might be a novel and potent way to study skin aging.