The Study On The Mechanism Of Baicalein On Ferroptosis And Melanocyte Protection Under Oxidative Stress

Objective:(1)RSL3 was used to construct oxidative stress state in normal human melanocytes cultured in vitro.(2)To observe the morphological and biochemical changes of melanocytes under oxidative stress from the point of view of ferroptosis,in order to further improve the pathogenesis of vitiligo and and explore the target of baicalein in ferroptosis signal pathway.(3)Factors associated with ferroptosis and the onset of vitiligo were identified by network pharmacology.Methods:The experiment part:Normal human melanocytes were subcultured.Different concentrations of ferroptosis inducers RSL3,baicalein and ferric ammonium citrate(FAC)were set to act on melanocytes,and the appropriate concentrations were selected as the follow-up experimental concentrations.The experiment was divided into five groups as follows:control group,RSL3 group,RSL3+FAC group,baicalein+RSL3group and baicalein+RSL3+FAC group.Cells in baicalein group were preincubated with baicalein for 1 hour,then treated with RSL3 or RSL3 and FAC for 24 hours,with control cells as control.The viability,mitochondrial ultrastructure,mitochondrial membrane potential,intracellular ROS,lipid ROS,intracellular Fe²⁺,glutathione peroxidase 4(GPx4),transferrin receptor 1(TFR1)related protein and m RNA expression of melanocytes in the above five groups were observed by CCK-8 method,electron microscope,flow cytometry,Fe Rho Nox ^TM-1 probe,Western Blot and RT-q PCR.The network pharmacology part:The network of vitiligo and baicalein was constructed by using various databases and related software.Results:(1)The suitable concentration of RSL3 to cause oxidative stress in normal human melanocytes in vitro is 0.06?M(IC30).(2)Baicalein antagonizes melanocyte growth inhibition induced by RSL3 and FAC.(3)Baicalein can improve the mitochondrial morphology and dysfunction of melanocytes under oxidative stress.(4)Baicalein reduces RSL3-induced iron level and melanocyte ROS(intracellular,lipid).(5)Baicalein protects melanocytes by increasing the expression of GPx4 and decreasing the expression of TFR1.(6)P53 is involved in both the ferroptosis signaling pathway and the pathogenesis of vitiligo.Conclusion:(1)RSL3 can induce oxidative stress in normal human melanocytes in vitro.(2)Baicalein protects melanocytes under oxidative stress by reducing iron ion,reducing ROS(intracellular lipid),increasing GPx4 and reducing TFR1.(3)Baicalein can significantly reduce the level of ROS by interacting with iron ion,but the effect of directly reducing intracellular ROS is not obvious.(4)P53 is a potential target of vitiligo.