Investigation Of Human Melanocyte Mitochondrial Dysfunction Induced By Hydrogen Peroxide And The Amelioration Of Strengthening Liver And Tonifying Kidney Traditional Chinese Medicine

Part I:Effects of traditional Chinese medicine of the tonifying the kidney and strengthening liver on human melanocyte melanogenesis and migrationObjective:To investigation of the extracts of Polygoni multiflori radix praeparata, Ecliptae herba, Rehmanniae radix praeparata, which are high frequently used for the treatment of vitiligo, on the proliferation, tyrosinase activity, melanogenesis, migration and MITF protein expression on human melanocyte.Methods:1、Culture the normal human epidermal melanocyte in vitro.2、After treatment of melanocytes with the extracts of Polygoni multiflori radix praeparata, Ecliptae herba, Rehmanniae radix praeparata, MTT assays are applied to determine proliferation, the tyrosinase activitives are measured by dopa oxidase assay, the melanin contents are detected by NaOH assay. The effects of those herbs on the migration of human melanocytes were determined using a modified transwell assay. The expression of MITF was measured by western blot.Results:1、The dosages of 25、50、100、200ug/mL extracts of Polygoni multiflori radix praeparata have no stimulationary effects on proliferation,but 400ug/mL show a positive result. A variety of different concentrations of extracts of Ecliptae herba showed no effect. The dosages of 25、50、100、200ug/mL extracts of Rehmanniae radix praeparata have no stimulationary effects on proliferation,but 400ug/mL promoted.2、A variety of different concentrations of extracts of Polygoni multiflori radix praeparata and Rehmanniae radix praeparata showed no effect on tyrosinase activitives. Treatment with extracts of Ecliptae herba (at 50、100、200、400ug/mL) significantly increased tyrosinase activitives.3、A variety of different concentrations of extracts of Polygoni multiflori radix praeparata have no stimulationary effects on melanogenesis. Treatment with extracts of Ecliptae herba and Rehmanniae radix praeparata (at 50、100、200、400ug/mL) significantly increased melanogenesis.4、The exposure of melanoctyes to 100 ug/mL Polygoni multiflori radix praeparata and Ecliptae herba promoted the migration activity by 263% and 172%, respectively. In contrast, Rehmanniae radix praeparata has no effect.5、Treatment with Polygoni multiflori radix praeparata, Ecliptae herba and Rehmanniae radix praeparata at 100 ug/mL significantly increased MITF expression levels by 140%,145% and 143, respectively.Conclusion:1-. Aqueous extract of Polygoni multiflori radix praeparata exhibit significant stimulating effect on migration and MITF protein expression. Rehmanniae radix praeparata exhibit significant stimulating effect on melanogenesis and MITF protein expression.2、 Ecliptae herba has a synergistic effect on up-regulating tyrosinase activity, enhancing melanin synthesis, promoting cell migration as well as elevating protein expression of Mitf.Part Ⅱ Effect of mitochondrial dysfunction caused by hydrogen peroxide on human melanocyte migration and the intervention influence of Ecliptae herbaObjective:To establish oxidative stress model of normal human melanocyte and study the effect of hydrogen peroxide on migration, cytoskeleton, mitochondrial function in normal epidermal human melanocytes and the amlirate influences of pretreatment with different concentration extract of Ecliptae herba.Methods:MTT assays were used to determine the proliferation in different concentration and time of hydrogen peroxide. Melanocytes were divided into five groups, as followed:the control group, the hydrogen peroxide treatment group and 50ug/mL、100ug/mL、200ug/mL extract of Ecliptae herba and hydrogen peroxide treatment groups. The migration of melanocyte was detected by the transwell assay. Cellular fibrous actin were immunostained with rhodamine phalloidin and then cells were scaned with the laser scanning confocal microscope. The ultrastructure changes of mitochondria were detected by transmission electron microscope. The mitochondrial membrane potential was examined by JC-1 staining and the adenosine triphosphate content was measured by using a luciferase-based luminescence assay kit.Results:The establishment of the model of hydrogen peroxide oxidative stress in melanocyte in vitro. The ability of migration and actin polymerization induced by hydrogen peroxide in normal human epidermal melanocyte was inhibited. The ultrastructure pretreated with hydrogen peroxide show that mitochondria were swelled and the ridges were dilated, ambiguous or even disappear. Downregulation of mitochondrial membrane potential and Adenosine triphosphate levels by hydrogen peroxide. Extracts of Ecliptae herba ameliorated effects on melanocytes on its suppressed migrating ability by hydrogen peroxide. At the meantime, the shrinkage of dendrites, and disrupted cytoskeleton caused by hydrogen peroxide were restored by Ecliptae Herba, possibly through its mitigating effect on the depressed mitochondrial membrane potential and reduced production of Adenosine triphosphate induced by hydrogen peroxide.Conclusion:1、Treatment with 0.1 mM of hydrogen peroxide for 30 minutes induced oxidative stress in melanocyte.2、Mitochondrial dysfunction induced by hydrogen peroxide might mediate the decreased ability of melanocyte migration.3、Ecliptae herba might protect against hydrogen peroxide induced downregulation of melanocyte migration by regulating mitochondria function.Part Ⅲ The screening of relevant signaling molecules of mitochondrial dysfunction caused by hydrogen peroxide in human melanocyte and the amliorate effect of Ecliptae herbaObjective:To study the relevant signaling molecules of mitochondrial dysfunction caused by hydrogen peroxide in human melanocyte and the protection effects of pretreatment with different concentration extract of Ecliptae herba.Methods:1、Microarrays were used to screen the relevant signaling pathways of mitochondrial dysfunction caused by hydrogen peroxide on human melanocyte. The selected genes were followed by quantitative RT-PCR analysis.2、Melanocytes were divided into five groups, as followed:the control group, the hydrogen peroxide treatment group and 50ug/mL、100ug/mL、 200ug/mL extract of Ecliptae herba and hydrogen peroxide treatment groups.The expressions of filtered genes were determined by RT-PCR and western blot.Results:1、With a Fold change≥2 for the standard, the results show that a total of 1880 genes differentially expression,763 genes up-regulation,1117 genes down-regulaton. After quantitative RT-PCR analysis, the expressions genes of NADH dehydrogenase 2, cytochrome c oxidase 1 and cytochrome c oxidase 3 were significantly suppressed by hydrogen peroxide in melanocytes.2、The down-regulated effects of hydrogen peroxide on gene expression of NADH dehydrogenase 2, cytochrome c oxidase 1 and cytochrome c oxidase 3 can be reversed by Ecliptae Herba.Conclusion:The inhibitory effect of hydrogen peroxide on melanocytic migration is a cascade events. Hydrogen peroxide down regulated the expression of NADH dehydrogenase 2, cytochrome c oxidase 1 and cytochrome c oxidase 3, interfere mitochondrial function of respiratory chain and oxidative phosphorylation, and then decreased adenosine triphosphate production that ultimately inhibited the cell migration. Ecliptae herba is possibly protect against hydrogen peroxide induced downregulation of melanocyte migration by up-regulating these genes expressions.