Investigation Of Stress Disorder In Patients With Vitiligo And The Effect Of Stress Regulating Drugs On Melanocyte In Vitro

Vitiligo is an idiopathic depigmentary skin disorder characterized by selective destruction of melanocytes. An autoimmune theory has been proposed to explain the pathogenesis of vitiligo although the exact cause is still unknown. The latest progress focus on the imbalance of T cell subsets especially reduced serum regulatory T cells. On the contrary, research related to neuropsychiatric impairment, one of pathogenic factors of vitiligo, is a weak point. Drug research and development in this field still lie in a blank state.From the angle of the relationsip between mental stress and the onset of vitiligo, this research focused on the main axis of stress injuries--neuroendocrine abnormalities--stress disorder regulator impacting on proliferation and function of melanocyte in order to explore the neuroimmune pathogenesis of vitiligo. Clue to the functional and physical abnormalities in neuroendocrine system, targeted drug screening and mechanism of drug action were studied to provide a new train of thought and direction to search for new therapy for vitiligo.Part I Investigation of stress events&stress injuries in patients with vitiligoObjectives:1. To investigate the stress events before onset or recurrence of disease and the stress injuries in patients with vitiligo.2. To evaluate and analyze the incidence and the severity of the common stress disorders such as anxiety or depression.Methods:Case-control study was carried out in55patients as case group and118healthys as control group. Yangdesen’s Life Event Scale (LES) was applied for stress events investigation including occurence,kinds, properties, number, intensity and duration of life events. Hamilton Anxiety Scale (HAMA,14item version) was applied for anxiety evaluation as well as Hamilton Depression Scale (HAMD,24item version) for depression. According to the score, the incidence and the severity of anxiety and depression were evaluated. Results:1.The number of stress events One year before onset or reccurrence, there was no significant difference between the case group and the control group in the number of total events and total negative events, the number of total events and total negative events about working&learning, the number of total family events and total family negative events (P>0.05) while the number of total events and total negative events about sociality&others in the case group was fewer compared with that in the control group (P<0.05)2. The score of stress events Before onset or recurrence, the score of working&learning total events and negative events, the mean score of total events and negative events, the mean score of working&learning total events and negative events in case group increase significantly compared with that in control group (P<0.05)3. Accompanied stress disorders The incidence and the score of anxiety or depression in case group also increase significantly compared with that in control group (P<0.01)Conclusion:1. Stress events in the case group were no more than that in the control group, but some types of life events had greater influence and brought larger injury in the case group. It indicates that there may be stress injury susceptibility gene in vitiligo and stress injury plays an important role on onset and reccurence of vitiligo.2. Incidence of accompanied stress disorders is higher in vitiligo and stress disorders closely related to patients’onset.Part Ⅱ Detection of serum levels of some important stress associated hormones and nerve protective factors in vitiligoObjectives:1. To reveal the neuroendocrine imbalance appeared in vitiligo by detecting serum levels of some important stress associated hormones and nerve protective factors in vitiligo patients.2. To analyze the correlation between stress disorders and the neuroendocrine abnormity.Methods:ELISA was applied to detect serum levels of NPY, ACTH, CORT, MLT and BDNF both in90patiens and in87healthys. Pearson’s correlation analysis was used for the correlation between variables.Results:1. serum levels of some important stress associated hormones and nerve protective factors Serum levels of NPY and ACTH were higher in the case group than that in the control group (P<0.01) while serum levels of CORT, MLT and BDNF were lower in the case group than that in the control group (P<.01)2. The correlation between serum levels of CORT and other stress associated hormones or nerve protective factors Further analysis in90cases showed that there was positive correlation between serum levels of CORT and BDNF(r=0.447, P<0.001), but no significant correlation between serum levels of CORT with NPY、ACTH、MLT, respectively (P>0.05).3. The correlation between stress disorders and the neuroendocrine abnormity In55cases, both the serum level of CORT and BDNF negatively correlated to the score of anxiety as well as the score of depression(P<0.001), but there was no significant correlation between serum levels of MLT with the score of anxiety or the score of depression (P>0.05)Conclusion:1. Abnormity of some important stress associated hormones exist in vitiligo. It demonstrates neuroencrine pathway may play an important role on pathogenic process of vitiligo and provides the experimental evidence for glucocorticoid therapy.2. Because of the reduced serum levels of MLT and BDNF in vitiligo, there may be a new therapy for vitiligo by increasing the serum levels of MLT and BDNF.3. Both the serum level of CORT and BDNF negatively correlated to the stress disorders.Part Ⅲ Effect of some stress regulating compounds on melanocyte proliferation and the melanin biosynthesis in vitroObjectives:1. To investigate the impact of some stress disorder regulators on the melanocyte proliferation, the tyrosinase activity and the melanin biosynthesis in vitro.2. To investigate the role of Dex, MLT and NGF in regulating Treg cell differentiation in vitro.Methods:Cck-8assay was applied to determine cell proliferation, the tyrosinase activity was detected by measuring the oxidation rate of L-DOPA and the melanin biosynthesis was measured by NaOH-assay. CD4+CD25T cells from peripheral blood in the healthy were sorted by MACS and the percent of Treg cell among CD4+T cells was determined by FACS.Results:1. The respective effect of Dex、MLT and NGF on melanin biosynthesis and Treg cell differentiation in vitro1.1The respective effect of Dex、MLT and NGF applied aloneCompared with the blank control, both Dex and NGF promoted the melanocyte proliferation, the tyrosinase activity and the melanin biosynthesis (P<0.05) while MLT had no significant influence in MC in vitro (P>0.05)1.2The respective effect of Dex、MLT and NGF together with patients’ serum before treatmentAt first screening for serum concentration was conducted and the result showed the serum(2%,5%,10%,respectively) promoted the melanocyte proliferation compared with the blank control while the serum(15%,20%,respectively) inhibited the melanocyte proliferation, therefore15%serum was selected. Compared with the healthys,15%serum from the patients promoted the melanocyte proliferation and that from the patients after treatment enhanced the melanocyte proliferation significantly compared with that from the patients before treatment (P<0.05)Together with the patients’ serum, NGF promoted all of the melanocyte proliferation, the tyrosinase activity and the melanin biosynthesis while MLT had no significant influence in MC. It also indicate Dex might increase the melanin biosynthesis only though the pathway of promoting the melanocyte proliferation.1.3The role of Dex, MLT and NGF on regulating Treg cell differentiationCompared with the blank control, NGF promoted Treg cell differentiation (P<0.05) while Dex reduced the population of Treg cell(P<0.05), and MLT had no significant influence in differentiation of Treg cell (P>0.05)2. The effect of Chinese herbal medicine on melanocyte in vitro2.1Intervention of Chinese herbs aloneCompared with the blank control, the melanin biosynthesis increased by Yiqi-huoxue mixture mainly though promotion of the melanocyte proliferation and by Compound1or Compound2mainly though enhancement of the tyrosinase activity.Further study on every component of Compound1and Compound2showed the melanocyte proliferation was promoted by astragalus or millettiae while weakened by uncaria rhynchophylla, the tyrosinase activity was reinforced by either salvia miltiorrhiza or semen ziziphi jujubae. In the end, the melanin biosynthesis increased in the presence of astragalus, salvia miltiorrhiza or semen ziziphi jujubae, respevtively (P<0.05) and had significant change in the presence of neither millettiae nor uncaria rhynchophylla (P>0.05)2.2The effect of Chinese herbs in the presence of patients’ serum before treatmentTogether with the patients’ serum before treatment, the melanocyte proliferation, the tyrosinase activity and the melanin biosynthesis were all promoted in the presence of either Compound1or Compound2. Yiqi-huoxue mixture only enhanced the melanocyte proliferation but had no significant influence in the tyrosinase activity or the melanin biosynthesis. It also indicate Compound2promoted both the tyrosinase activity and the melanin biosynthesis compared with Yiqi-huoxue mixture.Conclusion:1. NGF may have a better prospects of clinical application because of not only its quite good effect on the melanocyte proliferation and function but also its promotion of Treg cell differentiation.2. The melanin biosynthesis can be enhanced though the pathway of promoting the melanocyte proliferation and/or reinforcing the tyrosinase activity.3. The efficacy of Compound1or Compound2on promotion of the melanin biosynthesis may be better in vivo.4. Compared with Yiqi-huoxue mixture, Compound2containing uncaria rhynchophylla presents enhancement of the melanin synthesis.5. Compared with the serum from the healthys, that from the patients can promote the melanocyte proliferation. It suggests that in patient’s serum there are not only some pathogenic factors but also many regulatory factors that may play regulatory role on keeping healthy. All of these elements interact to uniform a sophisticated regulatory network affecting the development and prognosis of illness.