ABSTRACT
Objective To explore the effects of vitamin C and niacinamide on the growth and differentiation of human primary cultured keratinocytes.Methods Normal human foreskin was used in this study.The epidermis was separated enzymatically from the dermis by thermolysin,and keratinocytes were isolated from the epidermis by digestion with trypsin plus EDTA.The single keratinocytes were cultured with undedying NIH-3T3 cells as feeder cells in a complete medium supplied with 50 mg/L (vitamin C group),niacinamide of 400 μmol/L(niacinamide group)or vehicle(control group).Immunocytochemistry and immunodot blot were performed using monoclonal antibodies directed against C-myc,cyclin D1,filaggrin and involucrin.Results The colony number was highest in vitamin C group,followed by the control group and niacinamide group,and the colony morphology in vitamin C group was similar to that in the control group,but distinct from that in the niacinamide group.A significant increase was noticed in the expression of C-myc,cyclin D1,filaggrin and involucrin in vitamin C-treated keratinocytes compared with the control keratinocytes(all P＜0.05);however,in niacinamide-treated keratinocytes,the expression of filaggrin was significantly enhanced(P＜0.01),that of involucrin remained unchanged(P＞0.05),while that of C-myc was depressed(P＜0.05).Conclusions These results demonstrate that vitamin C has a favorable effect on both the growth and differentiation of human keratinocytes,while niacinamide seems to only promote the differentiation but attenuate the growth of human keratinocytes.
ABSTRACT
Objective To compare the difference in quercetin against oxidative stress response in mouse and in NIH-3T3 cells before and after H2O2 treatment,to explore the underlying mechanism for the quercetin antioxidant.Methods The cultured NIH-3T3 cells were randomly divided into 4 groups: quercetin(Q) pre-protective group(Qb) firstly treated with quercetin for 24 h followed by incubation with H2O2 for 30 min;post-protective group(Qa) treated with H2O2 for 30 min followed by incubation with quercetin for 24 h;H2O2 group(H2O2) after exposure to H2O2 for 30 min,incubated with DMEM medium and the control group(C) only cultured with DMEM medium.The survival rate and apoptotic rate were detected respectively with MTT and TUNEL in NIH-3T3 cell sus-pension samples.The expression of cyclin D1,PTEN,NF-?B,HSP-70,BCl-2,BAX and caspase-3 were examined with immunocytochemistry and immunoblotting.Besides,20 Wistar rats were divided into control group and experimental group,the latter was given with quercetin in the doze of 0.13 mmol/kg.The levels of T-AOC,SOD,GSH-Px,GSH,MDA,NOS and NO2-/NO3-were detected both in the cleaved NIH-3T3 cells and in the plasma from both experimental and control animals prior to and post-1 h,2 h and after 24 h.Results When the Qb group was compared with H2O2 or Qa group,the survival rate was higher and the apoptotic rate was lower.When the H2O2 group was compared with C group,the expression of cyclin D1、PTEN or BCl-2 was down-regulated;while that of BAX、HSP-70、NF-?B or caspase-3 was up-regulated;the level of T-AOC,SOD,GSH-Px or GSH was decreased;that of NOS、NO2-/NO3-or MDA enhanced in the cleft NIH-3T3 cells.When the plasma level of the anti-oxidative enzyme system prior to-compared with post-1h and 2h-treatment with Q,the level of T-AOC,SOD,GSH-Px and GSH,especially the former two,were higher;MDA,lower;NOS or NO2-/NO3-promoted.However,the above parameters basically became normal 24 h after treatment with Q.Conclusion Quercetin down-regulates the promoted expression of HSP70,NOS,NO2-/NO3-and NF-?B etc.in H2O2-treatment NIH-3T3 cells.Qb could reverse the H2O2 damage effects more markedly.Moreover,the quercetin exerts anti-oxidant protective effect through modulating the anti-oxidative enzyme system both in vivo and in vitro.However,based on the cell heterogeneity in none-or pre/post-H2O2-treatment state,a difference in quercetin antioxidant response is noted.