Study On Protective Effect Of Glutamine On Human Melanocyte Oxidative Stress Model

BackgroundVitiligo is a common primary depigmenting disorder that occurs locally or spreads throughout the skin with a 1%incidence worldwide,Studies show that the imbalance between oxidation and anti-oxidation in the local skin microenvironment,the melanocyte damage and apoptosis induced by the accumulation of oxidative stress factors are the important causes of vitiligo.Reactive oxygen species?ROS?are a group of oxidants and major indicators to evaluate oxidative stress within the body.Increased levels of ROS cause cellular oxidative stress,and cell injury or death subsequently.ROS levels are significantly increased in the blood and skin lesions of patients with vitiligo.Nuclear factor-E2-related factor 2?Nrf2?is the most important transcription factor that regulates the oxidative stress response.By following activated Nrf2-ARE signaling pathway,levels of Nrf2 mRNA and protein are increased,which then enhance the production of downstream antioxidant enzymes,such as GST,SOD,HO-1,Trx1,Prx-2 and HSP70.Nrf2 and several downstream genes of phase ?detoxicating enzymes and antioxidant enzymes are increased in vitiligo skin lesions,suggesting that Nrf2 and its downstream genes are involved in skin homeostasis in vitiligo.Therefore,Nrf2-ARE pathway plays an important role in protecting human melanocytes against oxidative stress.Melanocytes apoptosis due to various reasons is one of the initiating factors of vitiligo.Antioxidants can relieve H₂O₂-induced apoptosis in melanocytes.Activating Nrf2-ARE signaling pathway and enhance the production of downstream antioxidant enzymes have both anti-oxidation and anti-apoptosis effects.Glutamine is the most abundant free amino acid in the body,and participate in a series of metabolic processes.Glutamine has been a well-known antioxidant.Glutamine is the precursor for glutathione synthesis and remains glutathione in the reduction state.Glutamine can increase the level of antioxidant enzyme in vivo and reduce the production of oxidation metabolites.Glutamine can inhibit apoptosis and injury caused by oxidative stress.Moreover,glutamine can increase HSP expression and prevent cell apoptosis,thus reduce tissue damage under oxidative stress conditions.Glutamine exhibits antioxidant effects in a variety of diseases.However,the anti-oxidant role of glutamine in patients with vitiligo or in melanocytes has been unclear.Objective1.The purpose of this project is to establish oxidative stress model of melanocyte and to screen the appropriate concentration of glutamine.2.To verify the effect of Gin on antioxidant proteins and apoptotic factors in the oxidative stress model of melanocyte.3.To verify that Gin can activate Nrf2-ARE signaling pathway of melanocytes and exert both antioxidant and anti-apoptosis effects.Methods1.Establishment of oxidative stress model on melanocyteMelanocytes were cultured in vitro with M254,and use DOPA staining to identify cultured.Various concentrations of H₂O₂?0-2 mmol/L?were used to treat melanocytes and cell viability?CCK-8?was detected after treatment.Based on the detection results,appropriate concentration of H₂O₂ is used to build oxidative stress model on melanocyte.2.Glutamine dose screeningMelanocytes were randomly assigned into different groups:Glutamine group:M254 culture medium containing glutamine at 0.5-60 mmol/L was added at 24 h after cell seeding.Glutamine-H₂O₂ group:Cells were cultured with M254 medium containing glutamine of different concentrations?0.5-60mmol/L?for 24h,and then treated with 0.75mmol/L H₂O₂ for 24h.H₂O₂-Glutamine group:Melanocytes were first treated with 0.75mmol/L of H₂O₂ for 24h,and then cultured with M254 medium containing different concentrations of Gin?0.5-60mmol/L?for 24h.At 72 h after cell seeding,cell morphology was analyzed and photographed and the cell viability was measured using a CCK-8 kit.In the same way,both Glutamine-H₂O₂ group and H₂O₂-Glutamine group were treated,and the ROS levels of each group were detected.The Gin concentration of subsequent experiments was selected according to the experimental results.3.Effect of glutamine on levels of MDA and LDH release rate Melanocytes were randomly divided into different groups:groups 1 and 2 were normal control group and melanocyte oxidative stress model group,groups 3-7 were Gin?2,5,1 0,15,20 mmol/L?pretreated groups,and group 8 was post-treated group with Gin 15 mmol/L.72 h after cell seeding,MDA and LDH release rate were detected.4.Effect of glutamine on levels of antioxidant proteins Group as above.Cellular SOD activity and GST concentration were measured.Expressions of Nuclear Nrf2 and cellular HO-1,Trx1,Prx-2,HSP70 were detected using western blotting.5.Effect of glutamine on levels of apoptotic factors Group as above.72 h after seeding,Cellular Caspase-3?Bax?Bcl-2 were measured.6.Observation of the effects of ML-385 blocking Nrf2 on Cell viability,ROS,MDA and LDH release rateMelanocytes were randomly divided into the following 6 groups:control group,Gin group,.ML-385 group,H₂O₂ group,Gln+H₂O₂ group and Gln+ML-385+H₂O₂ group.Levels of CCK-8,ROS,MDA and LDH were measured.7.Observation of the effects of ML-385 blocking Nrf2 on levels of antioxidant proteinsGroup as above.Levels of SOD and GST,expressions of Nuclear Nrt2 and cellular HO-1,Trx1,Prx-2,HSP70 were detected.8.Observation of the effects of ML-385 blocking Nrf2 on levels of apoptotic factorsGroup as above.Cellular Caspase-3?Bax?Bcl-2 were measured.Results1.Establishment of oxidative stress model on melanocyte Various concentrations of H₂O₂?0-2 mmol/L?were used to treat melanocytes and CCK-8 was detected after treatment.The result showed that CCK-8 was reduced after H₂O₂ treatment in a dose-dependent manner and reached to 50%of normal level after 0.75 mmol/L H₂O₂ treatment.Therefore,0.75 mmol/L of H₂O₂ was selected as it not only effectively induced oxidative stress in melanocytes,but also maintained cells half functioning.2.Glutamine dose screeningGlutamine at concentrations lower than 30 mmol/L had no obvious effect on normal melanocytes.0.5-60 mmol/L of glutamine pre-H₂O₂-treatment and post-H₂O₂-treatment were performed:Compared to the control group,melanocyte cell bodies were crimped and round,the dendrites were shortened:.and disappeared after H₂O₂ treatment?0.75 mmol/L?for 24 h,ROS increased and CCK-8 was also reduced significantly.The above changes were rescued by glutamine 2-40 mmol/L pretreatment or 5-25 mmol/L post-treatment.In addition,within the concentration range of 10-40mmol/L;the effect of the same dose of Gin pretreatment was better than that of post-treatment.Therefore,we chose glutamine at concentrations of 2,5,10,15,20 mmol/L,respectively,to treat cells.3.Effect of glutamine on levels of MDA and LDH release rateROS,MDA and LDH release rate were significantly increased after H₂O₂?0.75 mmol/L?treatment for 24 h,compared to the control group?p<0.01?.Both glutamine 2-20mmol/L pre-treatment and glutamine 15 mmol/L post-treatment reduced MDA levels in melanocytes.Glutamine 2mmol/L pretreatment decreased LDH release rate in comparison with the control group?p<0.05?.Glutamine 5-20 mmol/L pre-treatment and glutamine 15mmol/L post-treatment significantly decreased LDH release rate of melanocytes?p<0.01?,compared to H₂O₂ group.Glutamine 15mmol/L pre-treatment showed the most effect result and decreased levels of MDA and LDH release rate?p<0.01?.4.Effect of glutamine on levels of antioxidant proteinsSOD levels were significantly decreased and GST,nuclear Nrf2,cellular HO-1,Trxl,Prx-2,HSP70 were increased after H₂O₂ treatment for 24 h,compared.to the control group?HO-1 p<0.05,others p<0.01?.Glutamine 5 mmol/L pretreatment increased SOD and GST levels in comparison with the control group?p<0.05?.Glutamine 10-20 mmol/L pre-treatment and glutamine 15mmol/L post-treatment enhanced SOD and GST levels in melanocytes?p<0.01?.Glutamine.2-20 mmol/L pre-treatment and glutamine 15mmol/L post-treatment enhanced cellular HO-1,Trx1,Prx-2 and HSP70 expressions levels in melanocytes?p<0.01?,compared to H₂O₂ group.Glutamine 15mmol/L pre-treatment shows the highest increase in various indicators?p<0.01?.5.Effect of glutamine on levels of apoptotic factorsCaspase-3 and Bax levels were significantly increased,while Bcl-2 levels were significantly decreased after H₂O₂ treatment for 24 h,compared to the control group?p<0.01?.Glutamine 2-20 mmol/L pre-treatment and glutamine 15mmol/L post-treatment significantly decreased Caspase-3 and Bax levels and enhanced Bcl-2 levels in melanocytes?p<0.01?,compared to H₂O₂ group.The lowest Caspase-3 and Bax levels and the highest Bcl-2 levels were observed in glutamine 15mmol/L pre-treatment group?p<0.01?.-6.Effects of Nrf2 inhibitor ML-385 blocking Nrf2 on cell viability,ROS,MDA and LDHThe results showed that cell viability?CCK-8?,ROS levels,MDA levels and LDH release rates in Gln group and ML385 group were not significantly different from those in the normal control group.CCK-8 of H₂O₂ group was significantly lowerthan that of the normal control group?p<0.01?.While the levels of ROS,MDA and LDH release rates in H₂O₂ group were significantly higher than those in the normal control group?p<0.01?.CCK-8 level in Gln+H₂O₂ group was significantly higher than that in H₂O₂ group?p<0.01?.ROS level,MDA level and LDH release rate in Gln+H₂O₂ group were significantly lower than those in H₂O₂ group?p<0.01?.CCK-8 level in Gln+ML385+H₂O₂ group was significantly lower than that in Gln+H₂O₂ group?p<0.01?.The ROS level?p<0.01?,MDA level?p=0.01?and LDH release rate?p<0.01?of Gln+ML-385+H₂O₂ group were significantly higher than those of Gln+H₂O₂ group.7.Effects of ML-385 blocking Nrf2 on levels of antioxidant proteinsThe results showed that there was no significant difference in SOD activity,GST level,expressions of Nrf2,HO-1,Trx1,prx-2 and HSP70 between Gln group and ML385 group and normal control group.Compared with the normal control group,SOD level in H₂O₂ group was significantly reduced and GST,Nrf2,HO-1,Trxl,prx-2 and HSP70 level was significantly increased?p<0.01?.SOD activity,GST level,expression-of Nrt21,HO-1 Trxl,-prx-2.and HSP70-in-Gln+H₂O₂-group-were significantly higher than those in H₂O₂ group?p<0.01?.While various indicators in Gln+ML385+H₂O₂ group was significantly lower than that in Gln+H₂O₂ group?p<0.01?.8.Effects of ML-385 blocking N rf2 on levels of apoptotic factorsThe results showed that the levels of caspase-3,Bax and Bcl-2 in Gin group and ML385 group were not statistically different from those in the normal control group.The levels of caspase-3 and Bax in H₂O₂ group were significantly higher than those in the normal control group,while the levels of Bcl-2 were significantly lower?p<0.01?.The levels of caspase-3 and Bax in Gln+H₂O₂ group were significantly lower than those in H₂O₂ group,while Bcl-2 levels were significantly higher?p<0.01?.The caspase-3 and Bax levels in Gln+ML385+H₂O₂ group were significantly higher than those in Gln+H₂O₂ group,while Bcl-2 levels were significantly lower p<0.01).ConclusionsGlutamine can protects melanocytes from oxidative-stress-induced-damage by activating Nrf2-ARE signaling pathway,enhancing antioxidant enzymes downstream of the pathway,regulating the expression of apoptotic factors,reducing cell damage,increasing cell viability and maintain their normal morphology.The above effects showed a dose-related trend.Moreover,pre-treatment with glutamine at 15 mmol/L showed the best effect.