| Vitiligo is acquired skin pigment and its main symptoms are skin pigment loss.The clinical manifestation of vitiligo is relatively simple, so it's easy to diagnose but difficult to treat. The incidence of vitiligo is in upward tendency for the past few years. The etiological factors and pathogenesis of vitiligo, which aren't yet understood,are considered to be concerned with immunity,endocrine,heredity,consciousness,microelement and free radicals. The pathomechanism of vitiligo aren't yet final conclusions. The research of anti-oxidant for vitiligo increase in recent years.More and more evidence prompt. The dysequilibrium of oxidationand and anti-oxidant lead to free radicals of microenvironment are abundantly accumulated, then induce oxidative stress and cell damage eventually,which become an important pathogenic factor of vitiligo. Mitochondrion is a master switch in apoptosis.At present,curative effect of Western medicine treatment is not very good, high recurrence and side effect. In contrast, curative effect of Chinese medicine treatment is good, with low recurrence and side effects. Chinese medicine et iology of vitiligo as wind stroke skin, blood disharmony, deficiency of liver and kidney. ZBGSF reinforces liver and kidney, nourishes blood,dispels wind, which is an effective prescription. This research is to explore the action target point of Chinese medicine for vitiligo by observing the protective effect of ZBGSF and peoniflorin in vitro formelanocyte apoptosis which can provide theoretical basis for Clinical application.The research include two parts.The first part is about research of ZBGSF's protective action for melanocyte oxidative damage. To observe the protective effect of ZBGSF conditioning against H2O2 induced damage on Mice B16 Melanocytes, the experiments consist of three parts. MTT was used to set up B16 melanocyte oxidative damage model:B16 Melanocytes were stimulated with lmm/1 H2O2 for half hour after pre-treatment with 10% different serum groups for 24h. MTT method was adopted to determine cellular activity. The activity of SOD, CAT, GSH-Px, MDA content and the inhibition of OH" were detected by biochemical methods. The results prompt that B16 cell activity reduce after H2O2 stimulating and model group (P<0.01) was lowest. Medcine groups improved the cell survival rates, increased SOD, CAT, GSH-Px activity(P<0.05), reduced OH- releasing and MDA content (P<0.05), compared with model group, and the effect of ZBGSF group was best. The B16 apoptosis rate was detected by flow cytometry, the B16 nucleus change was detected by Hoechst 33342 dyeing, the B16 mitochondrial membrane potential was detected by laser confocal microscopy, the activity of Caspase-3/9 were detected by substrate fluorescence method and the protein expression of Bcl-2,Cyt-c were detected by Western blot, the results display:①Compared with normal control group, the apoptosis rate of model group (29.50±3.49) was higher (P<0.01); we discovered that H2O2 caused nucleus enrich and sapphirine, emerged apoptotic body, mitochondrial membrane potential reducing, mitochondrion present dot shape or granular and gather toward nucleus, and H2O2 induced the activity of Caspase-3/9 increasing, increased the protein expression of Cyt-c and reduce Bcl-2.②Compared with model group, the apoptosis rate of ZBGSF groups was lower (P<0.01); we discovered that H2O2 caused only part cells of ZBGSF groups emerged nucleus enrich and sapphirine, emerged apoptotic body. After treatment of ZBGSF groups, mitochondrion still present line pipe or reticular and diffused distribution in cytoplasm. The membrane potential reducing was eased. The Caspase-3/9 activity of ZBGSF groups was induced compared with model group (P<0.01 separately). we discovered that ZBGSF groups reduced the protein expression of Cyt-c and increased the protein expression of Bcl-2 compared with model group (P<0.01).The effect of BGS group is best.The second part is about molecular mechanism research of peoniflorin's protective action for melanocyte oxidative damage.The method ibid. MTT was used to set up B16 melanocyte oxidative damage model:B16 Melanocytes were stimulated with lmm/1 H2O2 for four hours after pre-treatment with different peoniflorin groups for 24h. The results were:①Compared with normal control group, the apoptosis rate of model group (28.13±3.31) was higher (P<0.01); we discovered that H2O2 caused nucleus enrich and sapphirine, emerged apoptotic body, mitochondrial membrane potential reducing, mitochondrion present dot shape or granular and gather toward nucleus. H2O2 induced the activity of Caspase-3/9 increasing and increased the protein expression of Cyt-c and reduced Bcl-2.②Compared with model group, the apoptosis rates of High, Middle, low dose peoniflorin groups were lower than model group (P<0.01) and present dose dependent. We discovered that H2O2 caused only part cells of High, Middle, low dose peoniflorin groups emerge a nucleus enrich and sapphirine, emerged apoptotic body. After treatment of High, Middle, low dose peoniflorin groups, mitochondrion still present line pipe or reticular and diffused distribution in cytoplasm. The membrane potential reducing was eased. The Caspase-3/9 activity of High, Middle, low dose peoniflorin groups induced compared with model group (P<0.01 separately) and present dose dependent. High, Middle, low dose peoniflorin groups reduced the protein expression of Cyt-c and increased the protein expression of Bcl-2 compared with model group (P<0.01). The protective effect of peoniflorin for melanocytes present dose dependent.To sum up, The thesis invest igated from B16 melanocyte oxidat ive damage model to research, the inhibiting oxidative stress mechanism of ZBGSF groups and peoniflorin groups possibly works by elevating the protein expression of Bcl-2, inhibiting mitochondrial membrane permeability, preventing the Cyt-c releasing and activating caspase cascade reaction to inhibit apoptosis. In conclusion,The thesis revealed that the mitochondrial signaling pathway is the regulation mechanism that ZBGSF groups and peoniflorin groups interfere in B16 Melanocytes apoptosis by oxidative stress.
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