| Part I Ultrastructural investigations of mitochondria in perilesional vitiligo skin by transmission electron microscopeBackground and objective: Vitiligo is an acquired pigmentary disorder of unknown etiology.Evidences show mitochondrial biochemical dysfunctions in peripheral blood monouclear cells of patients with vitiligo,as well as the mitochondrial damage of keratinocytes,but the reports of the mitochondrial ultrastructural alterations of melanocytes in situ have not been reported.In this part,we want to evaluate the mitochondrial ultrastructure in melanocytes from the edge of lesion in patients with vitiligo.Methods: We have performed an ultrastructural study on normal and perilesional skin from 10 healthy volunteers and 10 patients with active or stable disease by observing the morphology of the melanocytes with transmission electron microscope(TEM).Besides,stereological parameters of mitochondria such as volume density,surface density and numerical density were measured.Results: 1.There were a large number of melanosomes(28.57±3.21)in normal group mainly by stage Ⅲand Ⅳ.Mitochondria were abundant with normal structure and cristae mitochondriales concentrated.Autophagosomes were seen in some cells;2.Melanosomes decreased significantly in vitiligo group,especially the stage Ⅲmelanosomes(p<0.05).The number of melanosomes in patients with active stage was 22±6.16 and that of stable phase was 17.43±6.24.Mitochondria,displayed various shapes and sizes,were much more less than those in control group.Meanwhile,mitochondrial swelling,cristae disappearance as well as vacuolization were also observed.However,we did not see any mitophagy in patients compared with the normal ones;3.The stereology results of the three groups were significantly different.In the control group,the Nv,Vv and Sv of mitochondria were 7.194±1.434 μm-2,4.8±1.2 %,2.42±0.86 ‰,compared with 4.055±0.906 μm-2,7.4±2.1 %,3.58±1.15 ‰ in active phase.Also those in stable stage of vitiligo were markedly changed.Nv was 5.311±0.873 μm-2,Vv was 6.5±1.4 % and Sv was 2.82±0.94 ‰.Remarkable differences were noted among the groups(p<0.05).Conclusions: Mitochondria were injured in melanocytes of perilesional vitiligo skin.The damage degree of patients in active phase were more serious than that in stable stage.Part II The effects of calcipotriol on the dendritic morphology of human melanocytes under oxidative stress and a possible mechanism: Is it a mitochondrial protector?Backgroud and objective:Vitiligo is an acquired pigmentary disorder of unknown etiology that is clinically characterized by the development of white macules related to the selective loss of melanocytes in the skin.Evidence shows that mitochondria might be an unifying target of cytokine production,catecholamine release and/or alteration of Ca2+ metabolism that leads to melanocyte loss.Methods: In this study,we investigated mitochondrial protection by calcipotriol under oxidative stress by observing the retraction velocity of dendrites,the detection of malondialdehyde(MDA)and superoxide dismutase(SOD),the mitochondrial membrane potential with JC-1 and the measurement of intercellular calcium concentration.In addition,the ultrastructural alteration of mitochondria in melanocytes was observed by transmission electron microscopy.Results: 1.Calcipotriol,from 20 n M to 80 n M,decreases the levels of MDA,increases the activity of SOD,hampers the retraction velocity of melanocyte dendrites,suppresses the reduction of MMP and recovers Ca2+ homeostasis by reducing [Ca2+]i in a concentration-dependent manner;2.Electron microscopic observations showed that melanocytes were injured under oxidative stress compared with the control group,especially in the non-calcipotriol group,and disruption of the cytomembrane,a decreased quantity of cristae as well as swelling and vacuolization of mitochondria could be observed.Further,in the calcipotriol-treated groups,autophagosomes were seen occasionally;3.Stereological analysis showed that in 20 to 80 n M calcipotriol group,mitochondrial Vv(7.78 ± 0.95,7.48 ± 0.88,6.99 ± 0.94)and Sv(4.50 ± 0.99,3.95 ± 0.86,3.61 ± 0.95)were significantly lower than those in H2O2 group(Vv was 8.72 ± 0.99,and Sv was 5.38 ± 0.78);Nv(4.71 ± 0.91,5.21 ± 1.03,6.11 ± 0.94)was remarkably higher compared with H2O2 group(3.38 ± 0.95).Besides,there was a significant difference between each calcipotriol group in a concentration-dependent manner(p <0.05);4.Acridine orange staining showed 20-80 n M calcipotriol induced autophagy in melanocytes.The rates were 19.51 ± 3.56 %,32.34 ± 3.52 % and 48.05 ± 4.12 % respectively;Western blotting results showed 20-80 n M calcipotriol up-regulated autophagy protein expression levels.Gray value of beclin 1 were 320.18±32.57,520.05 ± 35.19,823.0 ± 24.40,and LC3 B were 522.5 ± 28.58,657.61 ± 35.65,858.80 ± 16.18,which were significantly higher than those in H2O2 group(Beclin 1 was 115.42 ± 23.23 and LC3 B was 169.16 ± 16.81).(p <0.05)Conclusions: Calcipotriol is potentially a protector via inducing autophagy to maintain the normal structure and function of mitochondria.It could be a promising drug delivery strategy to protect melanocytes against oxidative damage in the skin of patients with vitiligo. |
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